Holliday Junction Resolvases

Four-way DNA intermediates, called Holliday junctions (HJs), can form during meiotic and mitotic recombination, and their removal is crucial for chromosome segregation. A group of ubiquitous and highly specialized structure-selective endonucleases catalyze the cleavage of HJs into two disconnected DNA duplexes in a reaction called HJ resolution. These enzymes, called HJ resolvases, have been identified in bacteria and their bacteriophages, archaea, and eukaryotes. In this review, we discuss fundamental aspects of the HJ structure and their interaction with junction-resolving enzymes. This is followed by a brief discussion of the eubacterial RuvABC enzymes, which provide the paradigm for HJ resolvases in other organisms. Finally, we review the biochemical and structural properties of some well-characterized resolvases from archaea, bacteriophage, and eukaryotes.Homologous recombination (HR) is an essential process that promotes genetic diversity during meiosis (see Lam and Keeney 2014; Zickler and Kleckner 2014). However, in somatic cells, HR plays a key role in conserving genetic information by facilitating DNA repair, thereby ensuring faithful genome duplication and limiting the divergence of repetitive DNA sequences (see Mehta and Haber 2014). As shown in Figure 1, HR is initiated by a DNA double-strand break, the ends of which are resected to produce single-stranded (ss) 3′-overhangs (see Symington 2014). Homologous strand invasion by one of the 3′ overhangs (e.g., one catalyzed by Escherichia coli RecA or human RAD51) leads to the formation of a displacement loop (D-loop) (see Morrical 2014). The invading 3′ end of the D-loop can then be extended by a DNA polymerase, which uses the homologous strand as a template for DNA synthesis. Recombination then proceeds in one of several different ways, some of which involve second-end capture, such that the other resected 3′ end anneals to the displaced strand of the D-loop (Szostak et al. 1983). In the resulting recombination intermediate, the two interacting DNAs are linked by nicked Holliday junctions (HJs). Additional DNA synthesis and nick ligation lead to the formation of a double Holliday junction (dHJ) intermediate. In eukaryotes, dHJs are removed primarily by “dissolution” (Fig. 1, bottom left) (see Bizard and Hickson 2014). This pathway involves the combined activities of a DNA helicase and a type IA topoisomerase, which catalyze branch migration and decatenation of the dHJ into noncrossover products (Manthei and Keck 2014). In somatic cells, this is essential for the avoidance of sister-chromatid exchanges (SCEs) and loss of heterozygosity. Alternatively, dHJs can be processed by “resolution” in reactions mediated by canonical or noncanonical mechanisms of endonuclease-mediated cleavage into either crossover or noncrossover products (Fig. 1, bottom middle and right).Open in a separate windowFigure 1.Pathways for the formation and processing of Holliday junctions. Resected DNA double-strand breaks invade homologous duplex DNA to create a joint molecule, or displacement-loop structure. The invading 3′ end then serves as a primer for DNA synthesis, leading to second end capture and the formation of a double Holliday junction. In eukaryotes, these structures are removed by “dissolution” (bottom left panel) or “resolution” (bottom middle and right panels). Canonical Holliday junction resolvases introduce a pair of symmetrical and coordinated nicks across one of the helical axes (bottom middle panel) to generate nicked DNA duplexes that can be directly ligated. Alternatively, noncanonical resolvases cleave Holliday junctions with asymmetric nicks to produce gapped and flapped DNA duplexes that require further processing prior to ligation (bottom right panel). *Mitochondrial Holliday junction resolvase.     

Human Primpol1: a novel guardian of stalled replication forks

Huang and colleagues identify a human primase-polymerase that is required for stalled replication fork restart and the maintenance of genome integrity.EMBO reports (2013) 14 12, 1104–1112 doi:10.1038/embor.2013.159The successful duplication of genomic DNA during S phase is essential for the proper transmission of genetic information to the next generation of cells. Perturbation of normal DNA replication by extrinsic stimuli or intrinsic stress can result in stalled replication forks, ultimately leading to abnormal chromatin structures and activation of the DNA damage response. On formation of stalled replication forks, many DNA repair and recombination pathway proteins are recruited to resolve the stalled fork and resume proper DNA synthesis. Initiation of replication at sites of stalled forks differs from traditional replication and, therefore, requires specialized proteins to reactivate DNA synthesis. In this issue of EMBO reports, Wan et al [1] introduce human primase-polymerase 1 (hPrimpol1)/CCDC111, a novel factor that is essential for the restart of stalled replication forks. This article is the first, to our knowledge, to ascertain the function of human Primpol enzymes, which were originally identified as members of the archaeao-eukaryotic primase (AEP) family [2].Single-stranded DNA (ssDNA) forms at stalled replication forks because of uncoupling of the DNA helicase from the polymerase, and is coated by replication protein A (RPA) for stabilization and recruitment of proteins involved in DNA repair and restart of replication. To identify novel factors playing important roles in the resolution of stalled replication forks, Wan and colleagues [1] used mass spectrometry to identify RPA-binding partners. Among the proteins identified were those already known to be located at replication forks, including SMARCAL1/HARP, BLM and TIMELESS. In addition they found a novel interactor, the 560aa protein CCDC111. This protein interacts with the carboxyl terminus of RPA1 through its own C-terminal region, and localizes with RPA foci in cells after hydroxyurea or DNA damage induced by ionizing irradiation. Owing to the presence of AEP and zinc-ribbon-like domains at the amino-terminal and C-terminal regions, respectively [2], CCDC111 was predicted to have both primase and polymerase enzymatic activities, which was confirmed with in vitro assays, leading to the name hPrimpol1 for this unique enzyme.The most outstanding discovery in this article is that hPrimpol1 is required for the restart of DNA synthesis from a stalled replication fork (Fig 1). With use of a single DNA fibre assay, knock down of hPrimpol1 had no effect on normal replication-fork progression or the firing of new origins in the presence of replication stress. After removal of replication stress, however, the restart of stalled forks was significantly impaired. Furthermore, the authors observed that hPrimpol1 depletion enhanced the toxicity of replication stress to human cells. Together, these data suggest that hPrimpol1 is a novel guardian protein that ensures the proper re-initiation of DNA replication by control of the repriming and repolymerization of newly synthesized DNA.Open in a separate windowFigure 1The role of hPrimpol1 in stalled replication fork restart. (A) Under normal conditions, the replicative helicase unwinds parental DNA, generating ssDNA that is coated by RPA and serves as a template for leading and lagging strand synthesis. Aside from interacting with RPA bound to the short stretches of ssDNA, the role of hPrimpol1 in normal progression of replication forks is unknown. (B) Following repair of a stalled replication fork, (1) hPrimpol1 rapidly resumes DNA synthesis of long stretches of RPA-coated ssDNA located at the stalled fork site. Later, the leading-strand polymerase (2) or lagging-strand primase and polymerase (3) replace hPrimpol1 to complete replication of genomic DNA. RPA, replication protein A; ssDNA, single-stranded DNA.Eukaryotic DNA replication is initiated at specific sites, called origins, through the help of various proteins, including ORC, CDC6, CDT1 and the MCM helicase complex [3]. On unwinding of the parental duplexed DNA, lagging strand ssDNA is coated by the RPA complex and used as a template for newly synthesized daughter DNA. DNA primase, a type of RNA polymerase, catalyses short RNA primers on the RPA-coated ssDNA that facilitate further DNA synthesis by DNA polymerase. While the use of a short RNA primer is occasionally necessary to restart leading-strand replication, such as in the case of a stalled DNA polymerase, it is primarily utilized in lagging-strand synthesis for the continuous production of Okazaki fragments. The lagging-strand DNA polymerase must efficiently coordinate its action with DNA primase and other replication factors, including DNA helicase and RPA [4]. Cooperation between DNA polymerase and primase is disturbed after DNA damage, ultimately resulting in the collapse of stalled replication forks. Until now, it was believed that DNA primase and DNA polymerase performed separate and catalytically unique functions in replication-fork progression in human cells, but this report provides the first example, to our knowledge, of a single enzyme performing both primase and polymerase functions to restart DNA synthesis at stalled replication forks after DNA damage (Fig 1).… this report provides the first example of a single enzyme performing both primase and polymerase function to restart DNA synthesis at stalled replication forksA stalled replication fork, if not properly resolved, can be extremely detrimental to a cell, causing permanent cell-cycle arrest and, ultimately, death. Therefore, eukaryotic cells have developed many pathways for the identification, repair and restart of stalled forks [5]. RPA recognizes ssDNA at stalled forks and activates the intra-S-phase checkpoint pathway, which involves various signalling proteins, including ATR, ATRIP and CHK1 [6]. This checkpoint pathway halts cell-cycle progression until the stalled forks are properly repaired and restarted. Compared with the recognition and repair of stalled forks, the mechanism of fork restart is relatively elusive. Studies have, however, begun to shed light on this process. For instance, RPA-directed SMARCAL1 has been discovered to be important for restart of DNA replication in bacteria and humans [7]. Together with the identification of hPrimpol1, these findings have helped to expand the knowledge of the mechanism of restarting DNA replication. Furthermore, both reports raise many questions regarding the cooperative mechanism of hPrimpol1 and SMARCAL1 with RPA at stalled forks to ensure genomic stability and proper fork restart [7].First, these findings raise the question of why cells need the specialized hPrimpol1 to restart DNA replication at stalled forks rather than using the already present DNA primase and polymerase. One possibility is that other DNA polymerases are functionally inhibited due to the response of the cell to DNA damage. Although the cells are ready to restart replication, the impaired polymerases might require additional time to recover after DNA damage, necessitating the use of hPrimpol1. In support of this idea, we found that the p12 subunit of DNA polymerase δ is degraded by CRL4^CDT2 E3 ligase after ultraviolet damage [8]. As a result, alternative polymerases, such as hPrimpol1, could compensate for temporarily non-functioning traditional polymerases. A second explanation is that the polymerase and helicase uncoupling after stalling of a fork results in long stretches of ssDNA that are coated with RPA. To restart DNA synthesis, cells must quickly reprime and polymerize large stretches of ssDNA to prevent renewed fork collapse. By its constant interaction with RPA1, hPrimpol1 is present on the ssDNA and can rapidly synthesize the new strand of DNA after the recovery of stalled forks. Third, the authors found that the association of hPrimpol1 with RPA1 is independent of its functional AEP and zinc-ribbon-like domains and occurs in the absence of DNA damage. These results might indicate a role for hPrimpol1 in normal replication fork progression, but further work is necessary to determine whether that is true.The discovery of hPrimpol1 is also important in an evolutionary contextSeveral questions remain. First, what is the fidelity of the polymerase activity? Other specialized polymerases that act at DNA damage sites sometimes have the ability to misincorporate a nucleotide across from a site of damage, for example pol-eta and -zeta [9]. It will be interesting to know whether hPrimpol1 is a high-fidelity polymerase or an error-prone polymerase. Second, is the polymerase only brought into action after fork stalling? If hPrimpol1 is an error-prone polymerase, one could envision other types of DNA damage that can be bypassed by hPrimpol1. Third, is the primase selective for ribonucleotides, or can it also incorporate deoxynucleotides? The requirement of the same domain—AEP—for primase and polymerase activities raises the possibility that NTPs or dNTPs could be used for primase or polymerase activities.The discovery of hPrimpol1 is also important in an evolutionary context. In 2003, an enzyme with catalytic activities like that of hPrimpol1 was discovered in a thermophilic archeaon and in Gram-positive bacteria [10]. This protein had several catalytic activities in vitro, including ATPase, primase and polymerase. In contrast to these Primpol enzymes, those capable of primase and polymerase functions had not been found in higher eukaryotes, which suggested that evolutionary pressures forced a split of these dual-function enzymes. Huang et al''s report suggests, however, that human cells do in fact retain enzymes similar to Primpol. In summary, the role of hPrimpol1 at stalled forks broadens our knowledge of the restart of DNA replication in human cells after fork stalling, allowing for proper duplication of genomic DNA, and provides insight into the evolution of primases in eukaryotes.     

The Block of DNA Polymerase �� Strand Displacement Activity by an Abasic Site Can Be Rescued by the Concerted Action of DNA Polymerase �� and Flap Endonuclease 1

Abasic (AP) sites are very frequent and dangerous DNA lesions. Their ability to block the advancement of a replication fork has been always viewed as a consequence of their inhibitory effect on the DNA synthetic activity of replicative DNA polymerases (DNA pols). Here we show that AP sites can also affect the strand displacement activity of the lagging strand DNA pol δ, thus preventing proper Okazaki fragment maturation. This block can be overcome through a polymerase switch, involving the combined physical and functional interaction of DNA pol β and Flap endonuclease 1. Our data identify a previously unnoticed deleterious effect of the AP site lesion on normal cell metabolism and suggest the existence of a novel repair pathway that might be important in preventing replication fork stalling.Loss of purine and pyrimidine bases is a significant source of DNA damage in prokaryotic and eukaryotic organisms. Abasic (apurinic and apyrimidinic) lesions occur spontaneously in DNA; in eukaryotes it has been estimated that about 10⁴ depurination and 10² depyrimidation events occur per genome per day. An equally important source of abasic DNA lesions results from the action of DNA glycosylases, such as uracil glycosylase, which excises uracil arising primarily from spontaneous deamination of cytosines (1). Although most AP sites are removed by the base excision repair (BER)⁵ pathway, a small fraction of lesions persists, and DNA with AP lesions presents a strong block to DNA synthesis by replicative DNA polymerases (DNA pols) (2, 3). Several studies have been performed to address the effects of AP sites on the template DNA strand on the synthetic activity of a variety of DNA pols. The major replicative enzyme of eukaryotic cells, DNA pol δ, was shown to be able to bypass an AP lesion, but only in the presence of the auxiliary factor proliferating cell nuclear antigen (PCNA) and at a very reduced catalytic efficiency if compared with an undamaged DNA template (4). On the other hand, the family X DNA pols β and λ were shown to bypass an AP site but in a very mutagenic way (5). Recent genetic evidence in Saccharomyces cerevisiae cells showed that DNA pol δ is the enzyme replicating the lagging strand (6). According to the current model for Okazaki fragment synthesis (7–9), the action of DNA pol δ is not only critical for the extension of the newly synthesized Okazaki fragment but also for the displacement of an RNA/DNA segment of about 30 nucleotides on the pre-existing downstream Okazaki fragment to create an intermediate Flap structure that is the target for the subsequent action of the Dna2 endonuclease and the Flap endonuclease 1 (Fen-1). This process has the advantage of removing the entire RNA/DNA hybrid fragment synthesized by the DNA pol α/primase, potentially containing nucleotide misincorporations caused by the lack of a proofreading exonuclease activity of DNA pol α/primase. This results in a more accurate copy synthesized by DNA pol δ. The intrinsic strand displacement activity of DNA pol δ, in conjunction with Fen-1, PCNA, and replication protein A (RP-A), has been also proposed to be essential for the S phase-specific long patch BER pathway (10, 11). Although it is clear that an AP site on the template strand is a strong block for DNA pol δ-dependent synthesis on single-stranded DNA, the functional consequences of such a lesion on the ability of DNA pol δ to carry on strand displacement synthesis have never been investigated so far. Given the high frequency of spontaneous hydrolysis and/or cytidine deamination events, any detrimental effect of an AP site on the strand displacement activity of DNA pol δ might have important consequences both for lagging strand DNA synthesis and for long patch BER. In this work, we addressed this issue by constructing a series of synthetic gapped DNA templates with a single AP site at different positions with respect to the downstream primer to be displaced by DNA pol δ (see Fig. 1A). We show that an AP site immediately upstream of a single- to double-strand DNA junction constitutes a strong block to the strand displacement activity of DNA pol δ, even in the presence of RP-A and PCNA. Such a block could be resolved only through a “polymerase switch” involving the concerted physical and functional interaction of DNA pol β and Fen-1. The closely related DNA pol λ could only partially substitute for DNA pol β. Based on our data, we propose that stalling of a replication fork by an AP site not only is a consequence of its ability to inhibit nucleotide incorporation by the replicative DNA pols but can also stem from its effects on strand displacement during Okazaki fragment maturation. In summary, our data suggest the existence of a novel repair pathway that might be important in preventing replication fork stalling and identify a previously unnoticed deleterious effect of the AP site lesion on normal cell metabolism.Open in a separate windowFIGURE 1.An abasic site immediately upstream of a double-stranded DNA region inhibits the strand displacement activity of DNA polymerase δ. The reactions were performed as described under “Experimental Procedures.” A, schematic representation of the various DNA templates used. The size of the resulting gaps is indicated in nt. The position of the AP site on the 100-mer template strand is indicated relative to the 3′ end. Base pairs in the vicinity of the lesion are indicated by dashes. The size of the gaps (35–38 nt) is consistent with the size of ssDNA covered by a single RP-A molecule, which has to be released during Okazaki fragment synthesis when the DNA pol is approaching the 5′-end of the downstream fragment. When the AP site is covered by the downstream terminator oligonucleotide (Gap-3 and Gap-1 templates) the nucleotide placed on the opposite strand is C to mimic the situation generated by spontaneous loss of a guanine or excision of an oxidized guanine, whereas when the AP site is covered by the primer (nicked AP template), the nucleotide placed on the opposite strand is A to mimic the most frequent incorporation event occurring opposite an AP site. B, human PCNA was titrated in the presence of 15 nm (lanes 2–4 and 10–12) or 30 nm (lanes 6–8 and 14–16) recombinant human four subunit DNA pol δ, on a linear control (lanes 1–8) or a 38-nt gap control (lanes 9–16) template. Lanes 1, 5, 9, and 13, control reactions in the absence of PCNA. C, human PCNA was titrated in the presence of 60 nm DNA pol δ, on a linear AP (lanes 2–4) or 38-nt gap AP (lanes 6–9) template. Lanes 1 and 5, control reactions in the absence of PCNA.     

Mismatch repair outside of replication

Comment on: Rodriguez GP, et al. Proc Natl Acad Sci USA 2012; 109:6153-8.DNA mismatch repair (MMR) is important for preventing mutations due to errors occurring during replication. Mismatches that are not removed by the proofreading function of the replicative DNA polymerases are recognized by a MutS protein in bacteria or usually in eukaryotes by a MutSα or MutSβ heterodimer.¹ After recognition, downstream events involving MutL in bacteria and usually MutLα in eukaryotes result in the removal of the newly synthesized DNA strand and resynthesis of the region, avoiding a change in DNA sequence.¹ However, there are many situations in which MMR is active outside of the context of genome replication, and an important question is what determines which strand of DNA is removed in those cases once a mismatch is detected. Using a reversion assay that detects point mutations in the TRP5 gene of yeast, we recently demonstrated that mispairs that escape detection at the replication fork can be recognized later by MMR and repaired in a manner independent of the replicating strand.² In that case, MMR was found to be acting in a mutagenic manner and could restore growth to cells that were in a nondividing state.When a mismatch is detected, how does MMR determine which strand to remove? A variety of experiments have demonstrated that eukaryotic MMR is directed to remove the DNA strand containing a nick, which could occur in the newly synthesized DNA strand through a variety of processes.^1,3 Evidence suggests that an important source of those nicks is activation of the latent endonuclease activity of MutLα; MutLα interacts with PCNA in a manner that could discriminate the template and primer stands of replication and thus give proper strand discrimination to MMR.⁴ Strand discrimination also likely involves MutSα and MutSβ, as both complexes have been shown to have robust interactions with PCNA.¹ However, two MMR pathways involving MutSα have recently been observed: one with MutSα as an integral component of replication factories and the other in which MutSα presumably scans the genome for mismatches and is independent of PCNA interaction.⁵ This latter pathway of MutSα, untethered to normal replication, is of interest here.One major function of MMR outside of replication is its anti-recombination role, by which it prevents aberrant recombination between non-identical sequences.⁶ MMR also functions in meiotic recombination and is responsible for the formation of gene conversion gradients, which can best be understood as arising from directed MMR near the site of initiating double-strand breaks, and randomly directed MMR further from the site of such strand discrimination signals.⁷ Although in meiotic recombination it appears that MMR acts without strand discrimination in some cases, the result is not an increase in mutation, as the end result is a choice between two pre-existing alleles.There are at least three situations in which MMR acting outside of replication appears to play a pro-mutagenic role. One is in the expansion of triplet repeat sequences, which play a role in a number of neurodegenerative diseases.^3,4 Although MMR would be expected to prevent repeat slippage during replication, there is biochemical evidence that slippage loops formed in triplet repeat sequences in a non-replicating state could load a PCNA-MutLα complex that would nick DNA in a random manner, leading to large expansions.⁴ There is also evidence from various mouse model systems that suggest a role for MMR in promoting repeat expansions.^3,8The second situation is somatic hypermutation. It had been found in 1998 that somatic hypermutation was, contrary to expectations, dependent on the presence of active MMR,⁹ but there was no mechanistic way to explain that dependence. A combination of patch replication by a relatively inaccurate translesion DNA polymerase and MMR acting randomly in terms of DNA strand, offers the best rationale for this process.^2,3The third situation, which also has the broadest sweep, is that of cancer. If several different pathways must be altered by mutation in order for a tumor to form, how could that happen in cells that either divide very slowly, or are in a nondividing state? Two years after the initial discovery of the linkage between MMR defects and cancer, MacPhee hypothesized that MMR acting in a “randomly templated” mode could be responsible for the formation of mutations in nondividing cells, which could reenter a growth phase as a result of those mutations;¹⁰ that corresponds to the observation we recently made in yeast, illustrated in Figure 1.² This is precisely the type of activity envisioned by MacPhee. What is not yet known is the signal giving strand discrimination to MMR; it could possibly be a random loading of MutLα,⁴ or it could be the presence of a random nick in the DNA strand close enough to the mismatch to be used for strand discrimination.Open in a separate windowFigure 1. Mutagenic MMR in nondividing cells. Cells were electroporated with 8-oxoGTP and then plated on Trp medium. The 8-oxoGTP was incorporated throughout the genome, including in some cells at the position indicated above which must revert via a TA→GC transversion in order for the cell to become Trp+. Once plated, the Trp- cells were unable to undergo even one round of replication, although they remained viable for over two weeks. In the absence of MMR, very few revertants appeared. However, in the presence of MMR, many revertants arose within the 3 d expected for cells that would have begun growth immediately after plating, but an even larger number of revertant colonies arose later on the plates up to a period of a week later. Thus only cells with active MMR were able to regain growth from a nondividing state due to the mutation created by MMR activity.The importance of MMR in preventing mutations during replication is unquestioned. However, the multiple activities of MMR outside of replication tend to be less appreciated. When MMR recognizes mismatches in DNA outside of the context of replication, the issue of what gives strand discrimination to MMR becomes critical. If the signal is randomly generated, MMR activity can be mutagenic, as we have found.²     

Lambda Red Recombineering in Escherichia coli Occurs Through a Fully Single-Stranded Intermediate

The phage lambda-derived Red recombination system is a powerful tool for making targeted genetic changes in Escherichia coli, providing a simple and versatile method for generating insertion, deletion, and point mutations on chromosomal, plasmid, or BAC targets. However, despite the common use of this system, the detailed mechanism by which lambda Red mediates double-stranded DNA recombination remains uncertain. Current mechanisms posit a recombination intermediate in which both 5′ ends of double-stranded DNA are recessed by λ exonuclease, leaving behind 3′ overhangs. Here, we propose an alternative in which lambda exonuclease entirely degrades one strand, while leaving the other strand intact as single-stranded DNA. This single-stranded intermediate then recombines via beta recombinase-catalyzed annealing at the replication fork. We support this by showing that single-stranded gene insertion cassettes are recombinogenic and that these cassettes preferentially target the lagging strand during DNA replication. Furthermore, a double-stranded DNA cassette containing multiple internal mismatches shows strand-specific mutations cosegregating roughly 80% of the time. These observations are more consistent with our model than with previously proposed models. Finally, by using phosphorothioate linkages to protect the lagging-targeting strand of a double-stranded DNA cassette, we illustrate how our new mechanistic knowledge can be used to enhance lambda Red recombination frequency. The mechanistic insights revealed by this work may facilitate further improvements to the versatility of lambda Red recombination.OVER the past decade, lambda Red recombination (“recombineering”) has been used as a powerful technique for making precisely defined insertions, deletions, and point mutations in Escherichia coli, requiring as few as 35 bp of homology on each side of the desired alteration (Thomason et al. 2007a; Sharan et al. 2009). With this system, single-stranded DNA (ssDNA) oligonucleotides have been used to efficiently modify E. coli chromosomal targets (Ellis et al. 2001; Costantino and Court 2003), BACs (Swaminathan et al. 2001), and plasmids (Thomason et al. 2007b), as well as to rapidly optimize a metabolic pathway coding for the production of lycopene (Wang et al. 2009). Furthermore, linear double-stranded DNA (dsDNA) recombineering has been used to replace chromosomal genes (Murphy 1998; Murphy et al. 2000), to disrupt gene function (Datsenko and Wanner 2000), and to develop novel cloning methods (Lee et al. 2001; Li and Elledge 2005). Large-scale dsDNA recombineering projects include creating a library of single-gene knockout E. coli strains (Baba et al. 2006) and removing 15% of the genomic material from a single E. coli strain (Posfai et al. 2006). Linear dsDNA recombineering has also been used to insert heterologous genes and entire pathways into the E. coli chromosome (Zhang et al. 1998; Wang and Pfeifer 2008) and BACs (Lee et al. 2001; Warming et al. 2005), including those used for downstream applications in eukaryotes (Chaveroche et al. 2000; Bouvier and Cheng 2009). However, despite the broad use of this method, the mechanism of lambda Red recombination has not achieved scientific consensus, particularly in the case of dsDNA recombination. A clearer understanding of the mechanism underlying this process could suggest ways to improve the functionality, ease, and versatility of lambda Red recombination.Three phage-derived lambda Red proteins are necessary for carrying out dsDNA recombination: Gam, Exo, and Beta. Gam prevents the degradation of linear dsDNA by the E. coli RecBCD and SbcCD nucleases; lambda exonuclease (Exo) degrades dsDNA in a 5′ to 3′ manner, leaving single-stranded DNA in the recessed regions; and Beta binds to the single-stranded regions produced by Exo and facilitates recombination by promoting annealing to the homologous genomic target site (Sawitzke et al. 2007). Current mechanisms claim that Exo binds to both 5′ ends of the dsDNA and degrades in both directions simultaneously to produce a double-stranded region flanked on both sides by 3′ overhangs (Sharan et al. 2009; Szczepanska 2009). However, a comprehensive explanation of how this construct ultimately recombines with the chromosome has not yet been advanced.Initially, it was proposed that this recombination occurs via strand invasion (Thaler et al. 1987). However, it has more recently been shown that strand invasion is unlikely to be the dominant mechanism in the absence of long regions of homology, as recombination remains highly proficient in a recA^- background (Yu et al. 2000). Furthermore, a detailed analysis of lambda Red recombination products showed characteristics consistent with strand annealing rather than a strand invasion model (Stahl et al. 1997). Finally, lambda Red dsDNA recombination has been shown to preferentially target the lagging strand during DNA replication, which suggests strand annealing rather than strand invasion (Lim et al. 2008; Poteete 2008).To explain these results, Court et al. (2002) proposed a strand-annealing model for insertional dsDNA recombination (Figure 1A), in which one single-stranded 3′ end anneals to its homologous target at the replication fork. The replication fork then stalls, due to the presence of a large dsDNA nonhomology (i.e., the insertion cassette). The stalled replication fork is ultimately rescued by the other replication fork traveling in the opposite direction around the circular bacterial chromosome. The other 3′ end of the recombinogenic DNA anneals to the homology region exposed by the second replication fork, forming a crossover structure, which is then resolved by unspecified E. coli enzymes (Court et al. 2002).Open in a separate windowFigure 1.—Previously proposed lambda Red-mediated dsDNA recombination mechanisms. Heterologous dsDNA is shown in green; Exo is an orange oval, and Beta is a yellow oval. In both mechanisms the recombination intermediate is proposed to be a dsDNA core flanked on either side by 3′ ssDNA overhangs. (A) The Court mechanism posits that (1) Beta facilitates annealing of one 3′ overhang to the lagging strand of the replication fork. (2) This replication fork then stalls and backtracks so that the leading strand can template switch onto the synthetic dsDNA. The heterologous dsDNA blocks further replication from this fork. (3) Once the second replication fork reaches the stalled fork, the other 3′ end of the integration cassette is annealed to the lagging strand in the same manner as prior. Finally, the crossover junctions must be resolved by unspecified E. coli enzymes (Court et al. 2002). (B) The Poteete mechanism suggests that (1) Beta facilitates 3′ overhang annealing to the lagging strand of the replication fork and (2) positions the invading strand to serve as the new template for leading-strand synthesis. This structure is resolved by an unspecified host endonuclease (red triangle), and (3) the synthetic dsDNA becomes template for both lagging and leading-strand synthesis. A second template switch must then occur at the other end of the synthetic dsDNA (Poteete 2008). The figure was adapted from the references cited.The Court mechanism was challenged by Poteete (2008), who showed that the dsDNA recombination of a linear lambda phage chromosome occurs readily onto a unidirectionally replicating plasmid, which does not have the second replication fork required by the Court mechanism (Court et al. 2002). Thus, Poteete proposed an alternate mechanism (Poteete 2008), termed “replisome invasion” (Figure 1B), in which a 3′ overhang of the Exo-processed dsDNA first anneals to its complementary sequence on the lagging strand of the recombination target. Subsequently, this overhang displaces the leading strand, thereby serving as the new template for leading-strand synthesis. The resulting structure is resolved by an unspecified endonuclease, after which the recombinogenic DNA becomes the template for the synthesis of both new strands. In the context of recombineering using a linear dsDNA cassette, the author indicates that a second strand-switching event must occur at the other end of the incoming dsDNA.While Poteete''s mechanism addresses some of the weaknesses of the Court mechanism, it remains largely speculative. This mechanism does not identify the endonuclease responsible for resolving the structure after the first template switching event, nor does it explain how the recombinogenic DNA and replication machinery form a new replication fork. Additionally, this template-switching mechanism would have to operate two times in a well-controlled manner, which may not be consistent with the high-recombination frequencies often observed (Murphy et al. 2000) for lambda Red-mediated dsDNA insertion. Finally, little experimental evidence has been advanced to directly support this hypothesis.To address the deficiencies in these mechanisms, we propose that lambda Red dsDNA recombination proceeds via a ssDNA intermediate rather than a dsDNA core flanked by 3′ overhangs (Figure 2). In this mechanism, Exo binds to one of the two dsDNA strands and degrades that strand completely, leaving behind full-length ssDNA. This ssDNA then anneals to its homology target at the lagging strand of the replication fork and is incorporated as part of the newly synthesized strand as if it were an Okazaki fragment. This process is analogous to the accepted mechanism for the lambda Red-mediated recombination of ssDNA oligonucleotides (Court et al. 2002) and, therefore, unifies the mechanisms for ssDNA and dsDNA recombination. Notably, our mechanism uses one replication fork for the incorporation of a full-length heterologous cassette, thereby addressing Poteete''s criticism of the Court mechanism.Open in a separate windowFigure 2.—Lambda Red mediated dsDNA recombination proceeds via a ssDNA intermediate. Instead of a recombination intermediate involving dsDNA flanked by 3′-ssDNA overhangs, we propose that one strand of linear dsDNA is entirely degraded by Exo (orange oval). Beta (yellow oval) then facilitates annealing to the lagging strand of the replication fork in place of an Okazaki fragment. The heterologous region does not anneal to the genomic sequence. This mechanism could account for gene replacement (as shown) or for insertions in which no genomic DNA is removed.The degradation of an entire strand by lambda Exo is feasible, given the highly processive nature of the enzyme (Subramanian et al. 2003). Whereas previously proposed mechanisms assume that both dsDNA ends are degraded approximately simultaneously, our hypothesis implies that some dsDNA molecules will be entirely degraded to ssDNA before a second Exo can bind to the other end. In this article, we demonstrate that single-stranded DNA is a viable recombinogenic intermediate with lagging-strand bias. Furthermore, we show that genetic information from one strand of a recombinogenic dsDNA cassette cosegregates during lambda Red-mediated recombination. These results provide strong support of our proposed mechanism.     

Establishing the human rolling circle reaction

Comment on: Kang YH, et al. Proc Natl Acad Sci USA 2012; 109:6042-7.In eukaryotes, the complex comprised of Mcm2–7, Cdc45 and GINS (CMG) is essential for DNA replication. Several lines of evidence indicate that the Mcm2–7 complex is the motor of the replicative helicase (reviewed in ref. ¹), which is activated by its association with Cdc45 and GINS.² Recently, we described the isolation and characterization of the human (h) CMG complex.³ In HeLa cells, this complex was formed only on chromatin and, following its isolation from cells, exhibited DNA helicase activity. Purified from Sf9 cells, hCMG possesses 3′→5′ DNA helicase activity, indicating that it moves ahead of the leading-strand DNA polymerase (pol). In contrast, the prokaryotic helicase DnaB, which unwinds DNA in the 5′→3′ direction, moves on the lagging strand. Detailed information about the progression of the prokaryotic replication fork was obtained using the rolling-circle method (ref. ⁴ and references therein). These studies permitted a detailed characterization of the joint action of the replicative pol and replicative helicase. In the rolling-circle reaction, the pol extends the 3′ end of a primer annealed to a minicircle that is then unwound simultaneously by the helicase (for a possible arrangement of proteins at the replication fork, see Fig. 1). The emerging single-stranded 5′-tail provides the template for lagging-strand synthesis. In most experiments, minicircles were engineered to contain only three nucleotides, allowing the distinction between leading- and lagging-strand nucleotide incorporation.Open in a separate windowFigure 1. Model of the human replication fork. The CMG complex unwinds DNA in the 3′→5′ direction. Polα/primase synthesizes primers to initiate leading- and lagging-strand synthesis. Polε and polδ are assigned as leading- and lagging-strand polymerases based on evidence in yeast.^5,6 Both pols require the processivity factor PCNA. RPA binds to single-stranded DNA. Additional proteins are required for DNA replication, of which only Ctf4 and Mcm10 are shown for simplicity.We initiated experiments to develop a eukaryotic replication fork in order to investigate whether the hCMG helicase activity could be coupled with the replicative pols.³ We set up rolling circle reactions using a 200-nt minicircle, the putative leading strand pol ε⁵ and hCMG and showed that DNA chains longer than 10 kb were produced (representing > 50 turns of the circle). The putative lagging strand pol δ,⁶ however, did not replace hpol ε in this reaction, though both pols extended primers on single-stranded M13 to full-length products (about 7 kb). It is tempting to speculate that an interaction between hCMG and hpol ε, but not hpol δ, contributes to their different activities. Specific interaction between GINS, a component of the CMG complex, and hpol ε has been demonstrated.⁷ However, it is presently unclear whether this contributes to the observed preferential role of pol ε and thus requires further examination.The processivity of the CMG complex alone was about 500 bp, which was stimulated to about 1 kbp by the addition of a single-strand DNA binding protein, either E. coli SSB or hRPA. The rolling circle reaction is also dependent on E. coli SSB, presumably to sequester the emerging single-stranded 5′ tail. Surprisingly, hRPA did not replace E. coli SSB in the rolling circle reaction. This was attributed to its inhibitory effects on pol ε activity in vitro. The influence of hRPA on eukaryotic fork progression is presently unclear. In the in vitro SV40 viral DNA replication system, hRPA is essential for DNA synthesis and cannot be replaced by E. coli SSB (reviewed in ref. ⁸). In this system, the SV40 large T-antigen acts as the replicative helicase, and hRPA is essential for its interaction with the hpolα/primase complex, which positions primase to initiate RNA chains. In the SV40 replication reaction, hpol δ synthesizes both leading and lagging strands. Surprisingly, while prokaryotic pols (and their processivity factors) can replace hpol δ and its auxiliary proteins in the in vitro SV40 elongation reaction, hpol ε does not play a role,⁸ suggesting that, in this system, the action of hpol ε is preferentially excluded. Importantly, no rolling circle synthesis was detected when hpol δ was used in lieu of hpol ε.³ Whether a similar mechanism leading to the exclusion of hpol δ from leading-strand synthesis is operational with the CMG helicase remains to be investigated. Using an archaeal system consisting of Pol B, RFC, PCNA, the 3′→5′ DNA helicase Mcm and the DNA primase, we have performed both leading- and lagging-strand synthesis on a rolling circle substrate.⁹ Currently, our efforts are focused on the synthesis of the lagging-strand with human proteins.In cells, the replication machinery duplicates chromatinized DNA. Thus, it is likely that chromatin remodeling factors and nucleosome chaperones play roles in the progression of the replication fork. In support of this notion, FACT was identified as a component of the yeast replisome progression complex.¹⁰ Various other proteins associate with the replication fork, such as Mcm10, Ctf4, Tim-Tipin and Claspin. The effects of these proteins on the in vitro replication reaction in eukaryotes remain to be examined.     

Setting the stage for cohesion establishment by the replication fork

Comment on: Rudra S, et al. Cell Cycle 2012; 2114-21The complex process of semi-conservative DNA replication involves a mechanism whereby the leading and lagging strands with opposite polarity serve as templates for concerted synthesis of complementary base pairs.¹ Lagging-strand synthesis creates discontinuous Okazaki fragments that require timely processing of the 5′ flaps, so that adjacent nascent DNA strands are ligated together to insure genomic stability. While the genetic and molecular requirements of Okazaki fragment maturation have been studied in much detail, the precise temporal and spatial relationship of lagging-strand processing to sister chromatid cohesion remains unclear.² The newly replicated daughter duplex DNA molecules (i.e., the sister chromatids) become tethered during DNA replication and remain paired in order to permit proper segregation of the chromosomes to respective poles during mitosis and nuclear division. Elegant genetic studies in yeast have implicated posttranslational modification of cohesins (specialized protein complexes responsible for tethering sister pairs) by Ctf7/Eco1 acetylase as a key regulatory step in the process, enabling cohesins to perform their function in capturing the newly synthesized sister chromatids. Previous work suggested that genetic and physical interactions among the yeast acetyltransferase Ctf7/Eco1, helicase Chl1, Flap Endonuclease (Fen1) and accessory replication factors [e.g., RFC (clamp loader) and PCNA (clamp)] play an integral role in cohesion establishment. Based on these pieces of evidence, several models to explain the relationship between replication fork dynamics and sister chromatid cohesion have been proposed; however, our understanding of the precise timing of cohesin acetylation and the passage of the replication fork machinery has remained murky at best. Given the importance of proper chromosome segregation for chromosomal stability and the suppression of developmental disorders and tumorigenesis, a comprehensive understanding of the molecular acrobatics involved in sister chromatid cohesion is highly important.In a recent study, the temporal relationship between sister chromatid establishment and lagging-strand synthesis was illuminated.³ The authors have elucidated the link between the catalytic functions of DNA unwinding, flap processing and acetylation, which supports a model of cohesion deposition and establishment that occurs after the passage of the replication fork, similar to how genomic DNA becomes chromatinized. This is a significant advance from an earlier and very popular model of sister chromatid cohesion predicted that Ctf7/Eco1 acetylated cohesin proteins before the encounter by the DNA replication fork, which was thought to permit fork progression and the proper cohesion state for sister chromatid tethering (for review, see ref. ²). Instead, the genetic evidence presented by the Skibbens lab supports a model whereby cohesion establishment is temporally coupled to lagging-strand processing.³ In support of the genetic proof, Rudra and Skibbens went on to show that both Ctf7/Eco1 and Chl1 are associated with the lagging-strand processing nuclease Fen1. Altogether, the experimental results implicate a post-fork establishment model that is analogous to how histone protein complexes are deposited onto newly synthesized sister chromatids and become posttranslationally modified to confer epigenetic status.The discovery from the Skibbens lab that cohesion establishment is closely orchestrated with Okazaki fragment processing prompts a new line of inquiry about the control of flap processing by acetylation and its dual purpose for proper sister chromatid cohesion and replication fidelity in eukaryotes (Fig. 1). The catalytic activity of human FEN-1^4,5 and a functionally related endonuclease known as Dna2⁴ have been shown to be modulated by p300 acetylation, which suggested a model for creating long flap intermediates to promote genomic stability and suppress mutagenesis. Given evidence that ChlR1 is implicated in the genetic disorder Warsaw Breakage syndrome and that the human homolog of yeast Chl1⁶ interacts with the RFC complex and Fen1,⁷ it will be informative to determine if acetyltransferases such as the human orthologs Esco1 and Esco2, the latter mutated in the cohesinopathy Roberts syndrome,⁸ and perhaps other acetyltransferases (e.g., p300) are master regulators of lagging-strand synthesis that not only affect replication fidelity and genomic stability, but also sister chromatid cohesion. Coordination of sister chromatid cohesion establishment with lagging strand synthesis may also involve replication fork stabilization by the Timeless-Tipin protein complex implicated in replication checkpoint.⁹ Defects in the efficient coupling of lagging-strand synthesis to sister chromatid cohesion may contribute to the chromosomal instability characteristic of age-related diseases and cancer.Open in a separate windowFigure 1. Interplay between acetylation, replication fork dynamics and cohesion establishment important for chromosomal integrity.     

Biphasic ethylene production during the hypersensitive response in Arabidopsis: A window into defense priming mechanisms?

The hypersensitive response (HR) is a cell death phenomenon associated with localized resistance to pathogens. Biphasic patterns in the generation of H₂O₂, salicylic acid and ethylene have been observed in tobacco during the early stages of the HR. These biphasic models reflect an initial elicitation by pathogen-associated molecular patterns followed by a second phase, induced by pathogen-encoded avirulence gene products. The first phase has been proposed to potentiate the second, to increase the efficacy of plant resistance to disease. This potentiation is comparable to the “priming” of plant defenses which is seen when plants display systemic resistance to disease. The events regulating the generation of the biphasic wave, or priming, remains obscure, however recently we demonstrated a key role for nitric oxide in this process in a HR occurring in tobacco. Here we use laser photoacoustic detection to demonstrate that biphasic ethylene production also occurs during a HR occurring in Arabidopsis. We suggest that ethylene emanation during the HR represents a ready means of visualising biphasic events during the HR and that exploiting the genomic resources offered by this model species will facilitate the development of a mechanistic understanding of potentiating/priming processes.Key words: hypersensitive response, biphasic patterns, potentiation, defense priming, ethylene, ArabidopsisThe Hypersensitive Response (HR) is a cell death process which occurs at the site of attempted pathogen attack and which has been associated with host resistance.¹ Much work on the regulation of the HR has indicated the importance of H₂O₂,² and NO.³ A feature of H₂O₂ generation during the HR is its biphasic pattern (Fig. 1A). The first rise reflects elicitation by pathogen-associated molecular patterns (PAMPs)⁴ and the second reflects the interaction between a pathogen-encoded avirulence (avr) gene product with a plant resistance (R) gene. A key aspect of the first rise is the initiation of salicylic acid (SA) synthesis which potentiates the second rise and hence the potency of plant defense and the HR.⁵Open in a separate windowFigure 1Patterns of defense signal generation during the Pseudomonas syringae pv. phaseolicola elicited-hypersensitive response in tobacco (Nicotiana tabacum). Generation of (A) H₂O₂ (●, Mur¹⁸); (B) nitric oxide (◇; Mur¹² (C) salicylic acid (SA, ■¹⁹) and (D) ethylene (○ Mur⁹) during a HR elicited by Pseudomonas syringae pv. phaseolicola (Psph) in tobacco cv. Samsun NN. In (A) a phase where SA acts to augment the second rise in H₂O₂—the potentiation phase—is highlighted. The potentiation phase is likely to be similar to defense “priming”.⁶ Methodological details are contained within the appropriate references. (E) A possible model for biphasic defense signal regulation during the Psph-elicited HR in tobacco. During an initial phase NO and H₂O₂ act to initiate SA biosynthesis, where SA and NO act to initiate a “H₂O₂ biphasic switch”. This could initially suppress both SA and the H₂O₂ generation but subsequently acts to potentiate a second phase of H₂O₂ generation. This in turn increases SA biosynthesis which could act with NO to initiate the “C₂H₄ biphasic switch” to potentiate ethylene production. These (and other) signals contribute to initiation of the HR and SAR.This potentiation mechanism appears to be similar to defense priming; when whole plants display systemic resistance to disease as opposed to a localized resistance against pathogens. Priming can be initiated (the “primary stimulus”) following attack with a necrotizing pathogen (leading to “systemic acquired resistance”, SAR) or non-pathogenic rhizosphere bacteria (to confer “induced systemic resistance”, ISR). In the primed state a plant stimulates a range of plant defense genes, produces anti-microbial phytoalexins and deposits cell wall strengthening molecules, but only on imposition of a “secondary stimulus”.⁶ Such secondary stimuli include SA³ or PAMPs⁷ and is likely to be mechanistically similar to the potentiation step in the biphasic pattern of H₂O₂ generation (shaded in Fig. 1A). Accordingly, the two phases in the biphasic wave represent primary and secondary stimuli in priming.Highlighting a similarity between local HR-based events and priming, adds further impetus to efforts aiming to describe the underlying mechanism(s), however both phenomena remain poorly understood. Besides SA, both jasmonates and abscisic acid (ABA) have been shown to prime defenses as have a range of non-plant chemicals, with β-aminobutyric acid (BABA) being perhaps most widely used.^6,8 Mutants which fail to exhibit BABA-mediated potentiation were defective in either a cyclin-dependent kinase-like protein, a polyphosphoinositide phosphatase or an ABA biosynthetic enzyme.⁸We have recently investigated biphasic ethylene production during the HR in tobacco elicited by the nonhost HR-eliciting bacterial pathogen Pseudomonas syringae pv. phaseolicola.⁹ As with H₂O₂ generation, this pattern reflected PAMP-and AVR-dependent elicitation events and included a SA-mediated potentiation stage. Crucially, we also showed that NO was a vital component in the SA-potentiation mechanism. When this finding is integrated with our other measurements of defense signal generation in the same host-pathogen system the complexity in the signaling network is revealed (Fig. 1). NO generation (Fig. 1B) appeared to be coincident with the first rise in H₂O₂ (Fig. 1A) which initiated SA biosynthesis^10,11 and together would contribute to the first small, but transient, rise in that hormone (Fig. 1C). In line with established models⁵ this momentary rise in SA coincides with the potentiation phase (shaded in Fig. 1A) required to augment the second rise in ROS. However, ethylene production seems to be correlated poorly with the patterns of NO, H₂O₂ and SA (Fig. 1D). Nevertheless, biphasic ethylene production was found to reflect PAMP and AVR-dependent recognition and included a SA-mediated potentiation step.⁹ Hence, ethylene production could be used as a post-hoc indicator of the potentiation mechanism. Therefore, our discovery that the second wave of ethylene production—a “biphasic switch”—is influenced by NO acting with SA could also be relevant to the H₂O₂ generation. Significantly, the second phases in both H₂O₂ and ethylene production occur exactly where SA and NO production coincides; in the case of H₂O₂ generation 2–4 h post challenge and with ethylene 6 h onwards (Fig. 1E).Thus, ethylene production represents a readily assayable marker to indicate perturbations in the underlying biphasic and possible priming mechanisms. As we have demonstrated, laser photoacoustic detection (LAPD) is a powerful on-line approach to determine in planta ethylene production in tobacco^9,12 but any mechanistic investigations would be greatly facilitated if the genetic resources offered by the model species Arabidopsis could be exploited.To address this, Arabidopsis Col-0 rosettes were vacuum infiltrated with either Pseudomonas syringae pv. tomato (Pst) avrRpm1 (HR-eliciting), the virulent Pst strain and the non-HR eliciting and non-virulent Pst hrpA strain. Ethylene production was monitored by LAPD (Fig. 2A). Significantly, Pst avrRpm1 initiated a biphasic pattern of ethylene production whose kinetics were very similar to that seen in tobacco (compare Figs. 2A with ​with1D).1D). Inoculations with Pst and Pst hrpA only displayed the first PAMP-dependent rise in ethylene production. Thus, these data establish that Arabidopsis can be used to investigate biphasic switch mechanism(s) in ethylene production during the HR and possibly defense priming. When considering such mechanisms, it is relevant to highlight the work of Foschi et al.¹³ who observed that biphasic activation of a monomeric G protein to cause phase-specific activation of different kinase cascades. Interestingly, ethylene has been noted to initiate biphasic activation of G proteins and kinases in Arabidopsis, although differing in kinetics to the phases seen during the HR.¹⁴ Further, plant defense priming has been associated with the increased accumulation of MAP kinase protein.⁶Open in a separate windowFigure 2Ethylene in the Pseudomonas syringae pv. tomato elicited-hypersensitive response in Arabidopsis thaliana. (A) Ethylene production from 5 week old short day (8 h light 100 µmol.m².sec⁻¹) grown Arabidopsis rosette leaves which were vacuum infiltrated with bacterial suspensions (2 × 10⁶ colony forming units.ml⁻¹) of Pseudomonas syringae pv. tomato (Pst) strains detected using laser photoacoustic detection (LAPD). Experimental details of the ethylene detection by LAPD are detailed in Mur et al.⁹ The intercellular spaces in leaves were infiltrated with the HR-eliciting strain Pst avrRpm1, (■), the virulent strain Pst (△) or the non-virulent and non-HR eliciting derivative, Pst hrpA (◇). (B) The appearance of Arabidopsis Col-0 and etr1-1 leaves at various h following injection with 2 × 10⁶ c.f.u.mL⁻¹ with of Pst avrRpm1. (C) Explants (1 cm diameter discs) from Arabidopsis leaf areas infiltrated with suspensions of Pst avrRpm1 were placed in a 1.5 cm diameter well, bathed in 1 mL de-ionized H₂O. Changes in the conductivity of the bathing solution, as an indicator of electrolyte leakage from either wild type Col-0 (◆), mutants which were compromised in ethylene signaling; etr1-1 (□), ein2-2 (▲) or which overproduced ethylene; eto2-1 (●) were measured using a conductivity meter. Methodological details are set out in Mur et al.⁹A further point requires consideration; the role of ethylene as a direct contributor to plant defense.¹⁵ The contribution of ethylene to the HR has been disputed,¹⁶ but in tobacco we have observed that altered ethylene production influenced the formation of a P. syringae pv. phaseolicola elicited HR.⁹ In Arabidopsis, cell death in the ethylene receptor mutant etr1-1 following inoculation with Pst avrRpm1 is delayed compared to wild type (Fig. 2B). When electrolyte leakage was used to quantify Pst avrRpm1 cell death, both etr1-1 and the ethylene insensitive signaling mutant ein2-1 exhibited slower death than wild-type but in the ethylene overproducing mutant eto2, cell death was augmented (Fig. 2C). These data indicate that ethylene influences the kinetics of the HR.Taking these data together we suggest that the complexity of signal interaction during the HR or in SAR/ISR could be further dissected by combining the genetic resources of Arabidopsis with measurements of ethylene production using such sensitive approaches as LAPD.     

UDP Facilitates Microglial Phagocytosis Through P2Y6 Receptors

Microglia engage in the clearance of dead cells or dangerous debris. When neighboring cells are injured, the cells release or leak ATP into extracellular space and microglia rapidly move toward or extend a process to the nucleotides as chemotaxis through P2Y12 receptors. In the meanwhile, microglia express the metabotropic P2Y6 receptors, the activation of which by uridine 5′-diphosphate (UDP) triggers microglial phagocytosis in a concentration-dependent fashion. UDP/UTP was leaked when hippocampal neurons were damaged by kainic acid in vivo and in vitro. Systemic administration of kainic acid in rats resulted in neuronal cell death in the hippocampal CA1 and CA3 regions, where increases in mRNA for P2Y6 receptors in activated microglia. Thus, the P2Y6 receptor is upregulated when neurons are damaged, and would function as a sensor for phagocytosis by sensing diffusible UDP signals.Key Words: microglia, phagocytosis, P2Y6 receptors, UDPAccumulating findings indicate that nucleotides play an important role in neuron to glia communication through P2 purinoceptors, even though ATP is recognized primarily to be a source of free energy and nucleotides are key molecules in cells. P2 purinoceptors are divided into two families, ionotropic receptors (P2X) and metabotropic receptors (P2Y) (Fig. 1). P2X receptors (seven types; P2X₁-P2X₇) contain intrinsic pores that open by binding with ATP. P2Y (eight types; P2Y_{1,2,4,6 and 11–14}) are activated by nucleotides and couple to intracellular second-messenger systems through heteromeric G-proteins.¹ Microglia express P2X₄, P2X₇, P2Y₂, P2Y₆ and P2Y₁₂¹ and are known as resident macrophages in CNS, accounting for 5–10% of the total population of glia.^2,3 When neurons are injured or dead, microglia are activated, resulting in their interaction with immune cells, active migration to the site of injury, release of pro-inflammatory substances and the phagocytosis of damaged cells or debris. For such activation of microglial motilities, extracellular nucleotides have a central role. Extracellular ATP functions as a chemoattractant. Microglial chemotaxis by ATP via P2Y₁₂ receptors was originally found by Honda et al.,⁴ and has recently been confirmed in vivo in P2Y₁₂ receptor knockout animals.⁵ Neuronal injury results in the release or leakage of ATP that appears to be a “find-me” signal from damaged neurons to microglia to cause chemotaxis. In addition to microglial migration by ATP, another nucleotide, UDP, an endogenous agonist of the P2Y₆ receptor, greatly activates the motility of microglia and orders microglia to engulf damaged neurons.⁶Open in a separate windowFigure 1P2 purinergic receptors (ATP receptors).Phagocytosis is a specialized form of endocytosis taking relatively large particles (> 1.0 µm) into vacuoles and has a central role in tissue remodeling, inflammation and the defense against infectious agents.⁷ Phagocytosis is initiated by the activation of cell-surface phagocytosis receptors, including Fc receptors, complement receptors, integrins, endotoxin receptors (CD18, CD14), mannose receptors and scavenger receptors⁸ which are activated by corresponding extracellular ligands called as “eat-me” signals. Since recognition is the most important step for phagocytosis, extensive studies on phagocytosis receptors have been reported. With regard to apoptotic cells, it is well known that dying cells express so called “eat-me” signals such as phosphatidylserine (PS) on their surface membrane,⁸ by which microglia recognize the apoptotic cells in order to catch and remove them.⁸ As for amyloid β protein (Aβ), a key molecule that mediates Alzheimer''s disease, microglia remove Aβ presumably via Fc receptor-dependent phagocytosis.^9,10 It, however, is unclear how phagocytotic cells come to the target cells or debris. Our findings suggest that nucleotides might be the molecules to guide phagocytotic cells to the targets.We found that exogenously applied UDP caused microglial phagocytosis through P2Y₆ in a concentration-dependent manner, and that neuronal injury caused by kainic acid (KA) upregulated P2Y₆ receptors in microglia, the KA evoked neuronal injury resulted in an increase in extracellular UTP, which was immediately metabolized into UDP in vivo and in vitro. We also found that UDP leaked from injured neurons caused P2Y₆ receptor-dependent phagocytosis in vivo and in vitro. Thus, UDP could be a diffusible molecule that signals the crisis of damaged neurons to microglia, triggering phagocytosis. Nucleotides seem to have the ability to act as “eat-us” signals for necrotic cells suffering traumatic or ischemic injury because such necrotic cells cause swelling, followed by shrinkage, leading to the leakage of cytoplasmic molecules including a large amount of ATP and UTP and extracellular nucleotides are immediately degraded by ecto-nucleotideases, suggesting that leaked nucleotides could be transient and localized signals that alert to the crisis created by the presence of the necrotic cells. These findings suggest that microglia might be attracted by ATP/ADP^4,5,11,12 and subsequently recognize UDP, starting to recognize “eat-me” signals attached to the targets and engulf them (Fig. 2). It is interesting that ATP/ADP is not able to efficiently activate P2Y₆ receptors, nor can UDP act on P2Y₁₂ receptors. Thus, adenine and uridine nucleotides would regulate microglial motilities, i.e. chemotaxis and phagocytosis, in a coordinated fashion.Open in a separate windowFigure 2Illustration of nucleotide-activated microglial chemotaxix and phagocytosis. Activated microglia might be attracted by ATP/ADP is not able to efficiently activate P2Y6 receptors, nor ca UDP act on P2Y12 receptors.     

The Dissolution of Double Holliday Junctions

Double Holliday junctions (dHJS) are important intermediates of homologous recombination. The separate junctions can each be cleaved by DNA structure-selective endonucleases known as Holliday junction resolvases. Alternatively, double Holliday junctions can be processed by a reaction known as “double Holliday junction dissolution.” This reaction requires the cooperative action of a so-called “dissolvasome” comprising a Holliday junction branch migration enzyme (Sgs1/BLM RecQ helicase) and a type IA topoisomerase (Top3/TopoIIIα) in complex with its OB (oligonucleotide/oligosaccharide binding) fold containing accessory factor (Rmi1). This review details our current knowledge of the dissolution process and the players involved in catalyzing this mechanistically complex means of completing homologous recombination reactions.For decades, homologous recombination (HR) was defined as a mechanism for the production of new allelic combinations during meiosis because it can generate so-called crossing-over (see Mehta and Haber 2014). Crossovers are likely generated by the asymmetric cleavage of a key intermediate in HR, the dHJ, by the action of structure-selective endonucleases called “resolvases” (Fig. 1A) (see Wyatt and West 2014). In addition to its essential function during meiosis, HR has proven to be a crucial DNA repair pathway in mitotic cells. Precisely because it has the potential to generate crossing-over, the resolution of dHJ by resolvases affords a high risk of genomic instability in these circumstances. Indeed, when HR is engaged between two homologous chromosomes or two homeologous sequences, dHJ resolution could lead, respectively, to loss of heterozygosity or gross chromosomal rearrangements. Thus, an alternative mechanism allowing dHJ processing without crossing-over would appear essential when HR is used for DNA repair. Such a mechanism, termed dHJ dissolution, is thought to be a major route for dissipation of dHJs arising from HR repair (LaRocque et al. 2011; Krejci et al. 2012). During dHJ dissolution, the two HJs are branch migrated toward one another until they form a hemicatenated intermediate that can be decatenated by a topoisomerase (Fig. 1B). This sophisticated reaction is performed by the so-called “dissolvasome” complex composed of a specific RecQ helicase (BLM in humans/Sgs1 in budding yeast) and a type IA topoisomerase known as topoisomerase III (Fig. 2; for general reviews about RecQ helicases and topoisomerases, see Champoux 2001; Wang 2002; Bachrati and Hickson 2003; Viard and de la Tour 2007; Chu and Hickson 2009; Vindigni and Hickson 2009.Open in a separate windowFigure 1.Double Holliday junction processing pathways. (A) During HJ resolution, each HJ of a dHJ is cleaved by a structure-selective endonuclease (resolvase). Depending on the combination of cleavage orientations, which can be asymmetric or symmetric, this process can generate both crossover and noncrossover products. In contrast, during dissolution (B), each strand engaged in the dHJ is reassociated with its original complementary strand, preventing exchange of genetic material between the two homologous sequences (and hence generating exclusively noncrossover products). DHJ dissolution (B) is initiated by migration of the HJs toward one another. The fusion/collapse of the two HJs results in a hemicatenated intermediate. Decatenation of this intermediate regenerates the original DNA species present before the initiation of HR.Open in a separate windowFigure 2.Domain organization of RecQ helicases, topoisomerases IA, and RMI proteins. (A) Most of the RecQ helicase members share a superfamily 2 helicase domain (SF2), a RecQ conserved domain (RQC), and a helicase and RNase D carboxy-terminal domain (HRDC). Besides this “RecQ core” domain, some RecQ helicases contain amino-terminal and carboxy-terminal extensions that vary in size, sequence, and functionality (e.g., SLD2 homology domain in RECQ4, and a signature motif in the carboxy-terminal domain of RECQ5). The hatched boxes denote partially degenerate RQC domains. BLM/Sgs1 helicases share a common domain organization, including an amino-terminal extension that includes domains for interaction with both TopoIII/RMI1 (TR) and replication protein A (RPA), in addition to a region that has been proposed to be required for DNA strand exchange (SE) activity. (B) All type IA topoisomerases contain a conserved catalytic domain (topoisomerase IA). Some topoisomerase IA enzymes also exhibit a carboxy-terminal extension, frequently composed of zinc finger motifs (black boxes), which is believed to mediate protein–DNA and protein–protein interactions. The contribution of the carboxy-terminal extension to dissolution is unknown. The regions interacting with other components of the dissolvasome are unknown. (C) In RMI1 proteins, only the DUF1676 and the OB-fold domain 1 (OB1) are conserved from yeast to human. The OB1 associates with both BLM/Sgs1 and topoisomerase III (BT/ST). In addition, human RMI1 exhibits a carboxy-terminal extension, composed of a middle region, which mediates RPA binding, and a second OB fold (OB2), which is able to associate with RMI2. RMI2 is also an OB-fold protein (OB3) that stably associates with the dissolvasome in human cells. In total, therefore, the human RMI1/2 complex contains three OB folds.In this review, we first take a historical look at the experimental evidence that led some groups to formulate the proposal that a reaction akin to dissolution must exist, and which then led Wu and Hickson (2003) to confirm its existence by reconstitution of the dissolution reaction in vitro using purified proteins. Following that, we will review the individual and combined roles of the components of what we will term the dHJ dissolvasome. Although many mechanistic aspects of dHJ dissolution remain obscure, several biochemical studies have provided a general understanding of this conceptually simple, but mechanistically complex, reaction.     

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized λ DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).Open in a separate windowClick here to view.^{(103M, flv)}     

Timeless tunes: Replicating happy endings

Comment on: Leman AR, et al. Cell Cycle 2012; 11:2337-47.DNA replication is at the heart of the inheritance of genetic material. A single replication fork can progress through hundreds of kilobases of DNA, melting parental double-stranded DNA and leaving newly synthesized strands in its wake. A beautiful illustration showing how the replication machinery accomplishes this complex task is one of the triumphs of molecular biology. However, it is known that DNA replication is not always as processive as the textbooks suggest. Specifically, the rate of fork progression varies depending on the regions being replicated, and the replication fork even stalls in some circumstances, during replication of heterochromatin or damaged DNA, for example. A stalled replication fork has two fates. It may restart DNA replication, or it may collapse after prolonged stalling. A collapsed replication fork is particularly dangerous for the genome, because the DNA intermediate left by the collapsed fork may form a double-stranded break, a highly mutagenic lesion that can undergo illegitimate recombination. To circumvent replication fork collapse, cells are equipped with specialized proteins that stabilize the stalled replication fork. Timeless and Tipin are highly conserved in eukaryotes. from yeast to humans, and form a complex to protect stalled replication forks.In a paper published in Cell Cycle, Noguchi and his group investigated how Timeless plays a role in telomere replication in human cells.¹ Telomeres consist of tandem arrays of short repetitive DNA (TTAGGG/CCCTAA in mammals) at the ends of chromosomes and numerous associated proteins. Telomeres are essential for the stable maintenance of genomic DNA, because they protect the DNA termini from undergoing accidental recombination and exonuclease attack. Dysfunctional telomeres lead to genetic instability that eventually results in senescence and cancer development. Because of the heterochromatic nature of telomeres, it has been recognized that telomere DNA is one of the genomic regions that impede replication fork progression. Indeed, in vitro DNA replication experiments using SV40 DNA, and cell extracts demonstrated that telomere DNA is replicated less efficiently and incurs more fork stalling than non-telomeric DNA.² Moreover, overexpression of telomere-DNA binding protein TRF1 in HeLa cells led to an accumulation of replicating telomeres, consistent with a slower replication rate of telomeres under those circumstance. Furthermore, experiments using TRF1-deleted murine cells showed that TRF1 is essential for efficient telomere DNA replication.³ Collectively, these results confirm that the telomere is a difficult-to-replicate region.There is an apparent contradiction between two earlier studies, however, with TRF1 described as an anti-replication protein in one report² and a pro-replication protein in the other.³ One potential explanation for the inconsistency might be that TRF1 requires other protein(s) to perform its pro-replication function, and the second factor was missing in the TRF1-overexpression experiments. Noguchi and his colleagues investigated this possibility by testing whether Timeless is required for proficient telomere DNA replication.¹ They found that Timeless-knockdown cells displayed telomere length shortening and an increased frequency of dysfunctional telomeres. In vitro replication assays of SV40 DNA revealed that Timeless-depleted extracts supported non-telomere replication proficiently, while telomere replication was inefficient. They then demonstrated that addition of recombinant TRF1 to the replication system slowed telomere replication. Importantly, Timeless depletion and TRF1 addition did not produce additive effects on telomere replication, suggesting that Timeless and TRF1 function in the same pathway. These results suggest a model as described in Figure 1. A replication fork frequently stalls at telomeres because of the molecularly crowded nature of telomeric chromatin. Timeless presumably encounters TRF1 at telomeres and protects the stalled fork from undergoing collapse. In the absence of Timeless, the stalled forks easily collapse, leading to an abrupt shortening of telomeres. Several questions remain to be answered. Given that Timeless moves along the genomic DNA as a component of the replication machinery,⁴ it will be particularly interesting to see how Timeless (or the replication machinery) interacts with telomeric chromatin. In such studies, a dynamic transaction between the regional chromatin at telomeres and the replication machinery may be revealed.Open in a separate windowFigure 1. Hard life at telomeres. (A) Mammalian telomeres consist of repetitive DNA that potentially forms higher-ordered structures [G-quartet(G4)-DNA] and numerous proteins, including telomere DNA-binding protein TRF1. (B) Replication fork is frequently stalled at telomeres. Overexpressed TRF1 slows down fork progression at the telomere, while endogenous TRF1 together with Timeless protein facilitates it. Timeless protects the stalled replication fork from collapse. (C) Telomeres are unique in that the most distal replication fork is not coupled with another fork progressing inversely. (D) Prolonged fork stalling may lead to the formation of a DNA double-strand break. Because of the lack of another fork compensating the telomere replication (C), the break immediately results in the abrupt single-step shortening of telomere DNAs.     

Destabilization of Yeast Micro- and Minisatellite DNA Sequences by Mutations Affecting a Nuclease Involved in Okazaki Fragment Processing (rad27) and DNA Polymerase δ (pol3-t)

We examined the effects of mutations in the Saccharomyces cerevisiae RAD27 (encoding a nuclease involved in the processing of Okazaki fragments) and POL3 (encoding DNA polymerase δ) genes on the stability of a minisatellite sequence (20-bp repeats) and microsatellites (1- to 8-bp repeat units). Both the rad27 and pol3-t mutations destabilized both classes of repeats, although the types of tract alterations observed in the two mutant strains were different. The tract alterations observed in rad27 strains were primarily additions, and those observed in pol3-t strains were primarily deletions. Measurements of the rates of repetitive tract alterations in strains with both rad27 and pol3-t indicated that the stimulation of microsatellite instability by rad27 was reduced by the effects of the pol3-t mutation. We also found that rad27 and pol3-01 (an allele carrying a mutation in the “proofreading” exonuclease domain of DNA polymerase δ) mutations were synthetically lethal.All eukaryotic genomes thus far examined contain many simple repetitive DNA sequences, tracts of DNA with one or a small number of bases repeated multiple times (48). These repetitive regions can be classified as microsatellites (small repeat units in tandem arrays 10 to 60 bp in length) and minisatellites (larger repeat units in tandem arrays several hundred base pairs to several kilobase pairs in length). In this paper, arrays with repeat units 14 bp or less will be considered microsatellites and arrays with longer repeat units will be considered minisatellites.Previous studies show that simple repetitive sequences are unstable relative to “normal” DNA sequences, frequently undergoing additions or deletions of repeat units, in Escherichia coli (24), Saccharomyces cerevisiae (12), and mammals (59). This mutability has two important consequences. First, it results in polymorphic loci that are useful in genetic mapping and forensic studies (15, 59). Second, although these repetitive tracts are usually located outside of coding sequences, alterations in the lengths of microsatellites or minisatellites located within coding sequences can produce frameshift mutations or novel protein variants (20, 22, 26).From studies of the effects of various mutations on microsatellite stability in yeast and E. coli (40) and the analysis of mutational changes caused by DNA polymerase in vitro (21), it is likely that most alterations reflect DNA polymerase slippage events (47). These events involve the transient dissociation of the primer and template strands during the replication of a microsatellite (Fig. ​(Fig.1).1). If the strands reassociate to yield an unpaired repeat on the primer strand, the net result is an addition of repeats (following a second round of DNA replication). Unpaired repeats on the template strand would result in a deletion by the same mechanism. Open in a separate windowFIG. 1“Classical” model for the generation of microsatellite alterations by DNA polymerase slippage. Two single strands of a replicating DNA molecule are shown, with each repeat unit indicated by a rectangle. Arrows indicate the 3′ ends of the strand, and the top and bottom strands represent the elongating primer strand and the template strand, respectively. Step 1, the primer and template strand dissociate; step 2, the primer and template strands reassociate in a misaligned configuration, resulting in an unpaired repeat on either the template strand (left side) or primer strand (right side); step 3, DNA synthesis is completed. If the unpaired repeats are not excised by the DNA mismatch repair system, after the next round of DNA synthesis one DNA molecule will be shortened by one repeat (left side) or lengthened by one repeat (right side).A number of mutations have been shown to elevate microsatellite instability. In E. coli (24, 46), yeast (44, 45), and mammalian cells (27), mutations in genes affecting DNA mismatch repair dramatically elevate the instability of a dinucleotide microsatellite. The most likely explanation of this result is that the DNA mismatches (unpaired repeats) resulting from DNA polymerase slippage events are efficiently removed from the newly synthesized strand by the DNA mismatch repair system. Thus, in the absence of mismatch repair, tract instability is elevated. From genetic studies, it has been found that mismatch repair in yeast efficiently corrects DNA mismatches involving 1- to 14-base loops (the size of the repeat units in microsatellites) but fails to correct mismatches involving loops larger than 16 bases (the size of the repeat units in minisatellites) (3, 41, 53). An inefficient mechanism, not involving the classical DNA mismatch repair system, is capable of correcting large DNA loops formed during meiotic recombination (19).In addition to mutations affecting DNA mismatch repair, some mutations affecting DNA replication in yeast destabilize microsatellites. Yeast strains bearing a null mutation in the RAD27 (RTH1) gene have high levels of instability of the dinucleotide poly(GT) and the trinucleotide CAG, specifically elevating single-repeat insertions (18, 39). RAD27 encodes the homolog of the mammalian FEN-1 protein, a 5′-to-3′ exonuclease (10, 11, 33). This nuclease activity is required for removing the terminal ribonucleotide residue from the 5′ end of the Okazaki fragment (9, 14, 35, 54, 55, 57); this step is necessary for the two adjoining fragments to be ligated together. FEN-1 appears to be active as either an exonuclease in the presence of a single-stranded gap upstream of the 5′ terminus or an endonuclease on a 5′ flap structure (13, 34). Since yeast strains that contain a null mutation in RAD27 grow poorly but are viable (38, 43), it is likely that less efficient nuclease activities that are also capable of 5′ Okazaki fragment processing are present in yeast. In addition to destabilizing dinucleotide microsatellites, rad27 strains have high levels of spontaneous mitotic recombination, elevated rates of forward mutation, and increased sensitivity to the alkylating agent methyl methanesulfonate (MMS) (18, 38, 43). In contrast to the mutations normally seen in mismatch repair mutants, i.e., point mutations or small frameshifts, the types of mutations observed in the absence of Rad27p are duplications of sequences flanked by short direct repeats (4 to 7 bp in length) (49). These duplications were not affected by the DNA mismatch repair system.The same class of sequences that are duplicated in the rad27 strains show an elevated rate (up to 1,000-fold) of deletion in strains containing a temperature-sensitive allele (pol3-t) of the yeast gene encoding DNA polymerase δ (52, 53). This mutant (initially named tex1) was isolated in a strain that exhibited an increased excision rate of a bacterial transposon with long terminal repeats inserted within a yeast gene (7). The pol3-t allele, which encodes a mutation (Gly₆₄₁ to Ala₆₄₁) (51) located near the putative nucleotide binding and active-site domains of the enzyme (58), is thought to diminish the rate of lagging-strand synthesis resulting in long stretches of single-stranded DNA on the lagging-strand template (8). This single-stranded DNA may have the potential to form intrastrand base-paired structures, creating interactions between short direct repeats. These interactions would result in an increased frequency of deletions caused by DNA polymerase slippage.Since rad27 and pol3-t mutations elevate the rates of duplications and deletions associated with short separated repeats in nonrepetitive DNA sequences, Kunkel et al. (22) suggested that these mutations could also destabilize minisatellites. In this paper, we examine the effects of rad27 and pol3-t mutations on the stability of simple repeats in which the repeat unit length varies between 1 and 20 bp. Our results show that both mutations destabilize both microsatellites and minisatellites, but that the mechanisms involved in the destabilization are different for the two mutations.     

Protein and DNA Effectors Control the TraI Conjugative Helicase of Plasmid R1

The mechanisms controlling progression of conjugative DNA processing from a preinitiation stage of specific plasmid strand cleavage at the transfer origin to a stage competent for unwinding the DNA strand destined for transfer remain obscure. Linear heteroduplex substrates containing double-stranded DNA binding sites for plasmid R1 relaxosome proteins and various regions of open duplex for TraI helicase loading were constructed to model putative intermediate structures in the initiation pathway. The activity of TraI was compared in steady-state multiple turnover experiments that measured the net production of unwound DNA as well as transesterase-catalyzed cleavage at nic. Helicase efficiency was enhanced by the relaxosome components TraM and integration host factor. The magnitude of stimulation depended on the proximity of the specific protein binding sites to the position of open DNA. The cytoplasmic domain of the R1 coupling protein, TraDΔN130, stimulated helicase efficiency on all substrates in a manner consistent with cooperative interaction and sequence-independent DNA binding. Variation in the position of duplex opening also revealed an unsuspected autoinhibition of the unwinding reaction catalyzed by full-length TraI. The activity reduction was sequence dependent and was not observed with a truncated helicase, TraIΔN308, lacking the site-specific DNA binding transesterase domain. Given that transesterase and helicase domains are physically tethered in the wild-type protein, this observation suggests that an intramolecular switch controls helicase activation. The data support a model where protein-protein and DNA ligand interactions at the coupling protein interface coordinate the transition initiating production and uptake of the nucleoprotein secretion substrate.Controlled duplex DNA unwinding is a crucial prerequisite for the expression and maintenance of genomes. Genome-manipulating and -regulating proteins are central to that biological function in recognizing appropriate DNA targets at initiation sequences and unwinding the complementary strands to provide single-stranded DNA (ssDNA) templates for nucleic acid synthesis and other processing reactions. The protein machineries involved include nucleic acid helicases. DNA helicases are powerful enzymes that convert the energy of nucleoside triphosphate hydrolysis to directional DNA strand translocation and separation of the double helix into its constituent single strands (for reviews, see references 13, 14, 16, 38, 55, and 64). By necessity, these enzymes interact with DNA strands via mechanisms independent of sequence recognition. At replication initiation helicases gain controlled access to the double-stranded genome at positions determined by the DNA binding properties of initiator proteins that comprise an origin recognition complex (1, 9, 17, 31, 45, 66). The mechanisms supporting localized unwinding within the complex include initiator-induced DNA looping, wrapping, and bending and feature regions of low thermodynamic stability. The exposed ssDNA mediates helicase binding followed by directional translocation along that strand until the enzyme engages the duplex for unwinding.In the MOB_F family of conjugation systems, the plasmid DNA strand destined for transfer (T strand) is unwound from its complement by a dedicated conjugative helicase, TraI of F-like plasmids or TrwC of the IncW paradigm. These enzymes are remarkable in that the same polypeptides additionally harbor in a distinct domain a DNA transesterase activity. That function is required to recognize and cleave the precise phosphodiester bond, nic, in the T strand where unwinding of the secretion substrate begins. In current models the conjugative helicases are thus targeted to the transfer origin (oriT) of their cognate plasmid by the high-affinity DNA sequence interactions of their N-terminal DNA transesterase domains. In the bacterial cell, recruitment and activation of the conjugative helicase occur not on naked DNA but within an initiator complex called the relaxosome (67). For the F-like plasmid R1, sequence-specific DNA binding properties of the plasmid proteins TraI, TraY, TraM, and the host integration factor (IHF) direct assembly of the relaxosome at oriT (10, 12, 29, 33, 51, 52). Integration of protein TraM confers recognition features to the relaxosome, which permit its selective docking to TraD, the coupling protein associated with the conjugative type IV secretion system (T4CP) (2, 15, 49). In current models, the T4CP forms a hexameric translocation pore at the cytoplasmic membrane that not only governs substrate entry to the envelope spanning type IV secretion machinery but also provides energy for macromolecular transport via ATP hydrolysis (36, 50). These models propose that T4CPs provide not only a physical bridge between the plasmid and the type IV transporter but also a unique control function in distinguishing one plasmid (relaxosome) from another (7, 8). Before the current study (see accompanying report [41]), evidence indicating that regulation of the initiation of conjugative DNA processing also takes place at this interface had not been reported.F plasmid TraI protein, originally named Escherichia coli DNA helicase I, was initially characterized in the Hoffman-Berling laboratory (19). The purified enzyme exhibits properties in vitro consistent with its function in conjugative DNA strand transfer including a very high 1,100-bp/s rate of duplex unwinding, high processivity, and a 5′-to-3′ directional bias (relative to the strand to which it is bound) (34, 54). Together these features should readily support the observed rate of conjugative DNA translocation as well as concomitant replacement synthesis of the mobilized T strand from the 3′ OH product of nic cleavage.Comparatively little is known about the mechanisms of initiating TraI helicase activity. The enzyme requires ssDNA 5′ to the duplex junction (32), and a minimum length of 30 nucleotides (nt) is necessary to promote efficient duplex unwinding on substrates lacking oriT (11, 54). To our knowledge, oriT is the only sequence where the helicase activity is naturally initiated, however. Moreover, the unique fusion of a helicase to the site- and strand-specific DNA transesterase domains within MOB_F enzymes is expected to pose intriguing regulatory challenges during initiation. The combination within a single polypeptide of a site-specific DNA binding capacity with a helical motor activity would seem counterproductive. The extraordinary efficiency of these proteins in intercellular DNA strand transfer belies this prediction and instead hints strongly at a coordinated progression of the initiation pathway. Since relaxosome assembly is thus far insufficient to initiate helicase activity on supercoiled oriT substrates in vitro, we have developed a series of heteroduplex DNA substrates which support the unwinding reaction and model possible intermediate structures of R1 plasmid strand transfer initiation (10). In this system linear double-stranded DNA (dsDNA) substrates with a central region of sequence heterogeneity trap defined lengths of R1 oriT sequence in unwound conformation. Unexpectedly, efficient helicase activity initiated from a melted oriT duplex required ssDNA twice as long (60 nt) as that previously observed on substrates lacking this sequence (11).In the current report, we describe an application of these models where variation in the position of duplex opening in the vicinity of nic, as well as the additional presence of auxiliary relaxosome proteins, has revealed novel insights into control of a conjugative helicase involving both DNA and protein interactions. Moreover, we observe a sequence-independent stimulation of the unwinding reaction in the presence of T4CP TraD. These results support a model where docking of the preinitiation relaxosome assembly to the T4CP alters the composition and architecture of the complex in a manner essential to the subsequent initiation of T-strand unwinding.     

Base Flipping in V(D)J Recombination: Insights into the Mechanism of Hairpin Formation,the 12/23 Rule,and the Coordination of Double-Strand Breaks

Tn5 transposase cleaves the transposon end using a hairpin intermediate on the transposon end. This involves a flipped base that is stacked against a tryptophan residue in the protein. However, many other members of the cut-and-paste transposase family, including the RAG1 protein, produce a hairpin on the flanking DNA. We have investigated the reversed polarity of the reaction for RAG recombination. Although the RAG proteins appear to employ a base-flipping mechanism using aromatic residues, the putatively flipped base is not at the expected location and does not appear to stack against any of the said aromatic residues. We propose an alternative model in which a flipped base is accommodated in a nonspecific pocket or cleft within the recombinase. This is consistent with the location of the flipped base at position −1 in the coding flank, which can be occupied by purine or pyrimidine bases that would be difficult to stabilize using a single, highly specific, interaction. Finally, during this work we noticed that the putative base-flipping events on either side of the 12/23 recombination signal sequence paired complex are coupled to the nicking steps and serve to coordinate the double-strand breaks on either side of the complex.Antibody and T-cell receptor (TCR) diversity is generated by V(D)J recombination initiated by the RAG proteins, RAG1 and RAG2. The recombination signal sequences (RSSs), where recombination takes place, have a distinctive arrangement resembling transposon ends. The relationship between V(D)J recombination and transposition was established beyond doubt by the discovery of RAG-mediated transposition and by the identification of a triad of conserved active-site residues. This evidence placed RAG1 firmly within the family of transposases and retroviral integrases that have a characteristic DDE triad of amino acid residues that coordinate catalytic metal ions in the active site (1, 26, 30, 35, 39, 46). Later, the Transib family of transposons was identified as the likely ancestral group of RAG1 (33).In V(D)J recombination, the RAG proteins excise the DNA between a pair of RSSs. This fragment is the equivalent of an excised transposon, and it takes no further part in the canonical V(D)J recombination reaction. Instead, the variable regions of the genes encoding antibodies and TCR are created by the imprecise rejoining of the flanking DNA, referred to as the “coding flank.” A key feature of the cleaved coding flanks is that they have covalently closed hairpin ends. The asymmetric resolution of these hairpins contributes to the diversification of the coding sequences during rejoining. The hairpins themselves arise as a consequence of the molecular mechanism RAG-mediated RSS cleavage.The crystal structure for the catalytic core of the human immunodeficiency virus type 1 integrase protein revealed a structural fold shared in common with RNase H and the Holliday junction resolving enzyme RuvC (22). RNase H and RuvC monomers each perform a simple nicking reaction that requires a single phosphoryl transfer event. Cut-and-paste transposition, which requires at least three phosphoryl transfer steps at each transposon end, therefore presents a mechanistic challenge. One solution to this challenge was revealed by the discovery of the DNA-hairpin cleavage-intermediate in V(D)J recombination and Tn10 transposition (Fig. ​(Fig.1)1) (34, 57). However, it is interesting to note that the existence of this intermediate was first suggested by Coen and colleagues on the basis of the genomic scars produced by excision of the hAT family transposon Tam3 in Antirrhinum majus (14).Open in a separate windowFIG. 1.Hairpin-processing reactions of opposite polarity. Most prokaryotic and eukaryotic members of the DDE family have hairpin intermediates of opposite polarity. In this paper, we refer to the two strands of DNA as “first strand” or “second strand” depending on the order of cleavage. The first strand therefore corresponds to the transferred and nontransferred strands of the prokaryotic and eukaryotic elements, respectively. Scissile phosphates are in red. The transposon end and RSS are shown as gray triangles. (Left panel) In Tn5 and Tn10, the first step of the reaction is a nick on the bottom (first) strand that exposes the 3′-OH at the end of the transposon. The second strand is cleaved by a direct transesterification reaction, which generates a “proximal-hairpin” intermediate on the transposon end (5, 34). Resolution by a nick at the tip of the hairpin yields a blunt transposon end. Distortion of the DNA helix can be detected by permanganate sensitivity of the T−1 and T+2 residues on the second strand. The insert shows the crystal structure of the Tn5 transposon end, highlighting the flipped base at position +2 (19). Two tryptophan residues are also shown. One acts as a “wedge” or “probe” residue inserted into the DNA helix, while the other provides stacking interactions that stabilize the flipped base. The W323 probe residue resides within the catalytic core close to the DDE residue E326, whereas the W298 stacking residue is in the inserted subdomain (see text for further details). Base flipping takes place after the first nick and is probably maintained for all subsequent steps, including integration (3, 7). (Right panel) In V(D)J recombination and the hAT family of transposons, the polarity of the reaction is reversed. The first nick is on the top strand providing a 3′-OH group on the flanking DNA (53, 71, 77). Transesterification yields a “distal hairpin” intermediate on the flanking DNA that is processed by the host. The positions of relevant thymidine residues in our substrates are indicated.All DDE family transposases, including RAG1, cut the DNA to expose the 3′-OH at the end of the element (or RSS). However, the fate of the opposite strand and the order of strand cleavage events vary within the group (reviewed in references13, 18, and 55). Some enzymes, such as the retroviral integrases and the bacteriophage Mu transposase, nick and integrate the 3′-OH directly without second-strand cleavage. The cut-and-paste transposons, which cleave both strands of DNA, can be divided into two groups. With some notable exceptions such as the piggyBac element, most prokaryotic family members cleave the bottom strand of the recombination site first, whereas most eukaryotic members cleave the top strand first (8, 10, 20, 41, 47, 48, 77). For those family members with a hairpin mechanism, the inverted polarity of the first step dictates the reversal of all subsequent steps (Fig. ​(Fig.1).1). In consequence, most eukaryotic members of the family can achieve transposition with one less phosphoryl transfer reaction than the prokaryotic members, which are obliged to resolve the hairpin intermediate. The eukaryotic members can simply release the hairpin ends or, as in the case of RAG, hand them on to host factors for further processing (40).Insight into the hairpin mechanism was provided by a crystal structure for the Tn5 transpososome, in which the penultimate base on the second, nontransferred, strand was flipped from the helix and stacked against a tryptophan side chain in the protein (Fig. ​(Fig.1)1) (19). The flipped base seemed to provide the steric freedom that is presumed to be required for making and resolving the hairpin intermediate. Two groups searched for a residue in RAG1 that performs a function equivalent to the stacking tryptophan in the Tn5 transposase (27, 45). This work identified several candidate residues on the basis of their respective mechanistic defects and their rescue by modified DNA substrates.Here we have further assessed the candidate stacking residues using biochemical techniques previously used to study the dynamics of base flipping in Tn5 and Tn10 transposition (6, 7). We have identified a distortion at position −1 of the V(D)J coding flank DNA. It is introduced after the first nick at the RSS and is therefore reminiscent of the flipped base at the end of Tn5. The distortion is perfectly correlated with the ability of wild-type and mutant RAG-RSS complexes to perform the hairpin step of the reaction. We conclude that this base is probably equivalent to the flipped base in Tn5. However, none of the candidate aromatic residues seems to fulfill the function of the putative stacking tryptophan residue. We therefore propose a model in which base flipping in RAG recombination is significantly different from that in Tn5 transposition.Canonical V(D)J recombination occurs within a 12/23 RSS paired complex (24, 36, 60, 72, 73). This restriction is known as the 12/23 rule. More recently a further restriction, the so-called “beyond 12/23” (B12/23) rule has been proposed to explain the exclusion of direct Vβ-to-Jβ joining in the TCR β region, despite the presence of appropriately oriented pairs of 12 and 23 RSSs (4, 21, 31, 32).Little is known of the mechanisms that enforce the 12/23 rule or coordinate cleavage on either side of the complex. However, during this work, we observed that the coding flank distortion was coupled on either side of a 12/23 RSS paired complex: the distortion of a nicked coding flank is suppressed by an unnicked partner. We present a model and discuss the biological significance of this conformational coupling and its relevance to the B12/23 rule.