A novel, topologically constrained DNA molecule containing a double Holliday junction: design, synthesis, and initial biochemical characterization

The double Holliday junction (dHJ) is a central intermediate to homologous recombination, but biochemical analysis of the metabolism of this structure has been hindered by the lack of a substrate that adequately replicates the endogenous structure. We have synthesized a novel dHJ substrate that consists of two small, double stranded DNA circles conjoined by two Holliday junctions (HJs). Its biochemical synthesis is based on the production of two pairs of single stranded circles from phagemids, followed by their sequential annealing with reverse gyrase. The sequence between the two HJs is identical on both strands, allowing the HJs to migrate without the generation of unpaired regions of DNA, whereas the distance between the HJs is on the order of gene conversion tracts thus far measured in Drosophila and mouse model systems. The structure of this substrate also provides similar topological constraint as would occur in an endogenous dHJ. Digestion of the dHJ substrate by T7 endonuclease I resolves the substrate into crossover and non-crossover products, as predicted by the Szostak model of double strand break repair. This substrate will greatly facilitate the examination of the mechanism of resolution of double Holliday junctions.     

GEN1 promotes Holliday junction resolution by a coordinated nick and counter-nick mechanism

Holliday junctions (HJs) that physically link sister chromatids or homologous chromosomes are formed as intermediates during DNA repair by homologous recombination. Persistent recombination intermediates are acted upon by structure-selective endonucleases that are required for proper chromosome segregation at mitosis. Here, we have purified full-length human GEN1 protein and show that it promotes Holliday junction resolution by a mechanism that is analogous to that exhibited by the prototypic HJ resolvase E. coli RuvC. We find that GEN1 cleaves HJs by a nick and counter-nick mechanism involving dual co-ordinated incisions that lead to the formation of ligatable nicked duplex products. As observed with RuvC, cleavage of the first strand is rate limiting, while second strand cleavage is rapid. In contrast to RuvC, however, GEN1 is largely monomeric in solution, but dimerizes on the HJ. Using HJs containing non-cleavable phosphorothioate-containing linkages in one strand, we show that the two incisions can be uncoupled and that the first nick occurs upon GEN1 dimerization at the junction. These results indicate that the mechanism of HJ resolution is largely conserved from bacteria to man, despite a lack of sequence homology between the resolvases.     

DNA strand displacement, strand annealing and strand swapping by the Drosophila Bloom's syndrome helicase

Genetic analysis of the Drosophila Bloom's syndrome helicase homolog (mus309/DmBLM) indicates that DmBLM is required for the synthesis-dependent strand annealing (SDSA) pathway of homologous recombination. Here we report the first biochemical study of DmBLM. Recombinant, epitope-tagged DmBLM was expressed in Drosophila cell culture and highly purified protein was prepared from nuclear extracts. Purified DmBLM exists exclusively as a high molecular weight (~1.17 MDa) species, is a DNA-dependent ATPase, has 3′→5′ DNA helicase activity, prefers forked substrate DNAs and anneals complementary DNAs. High-affinity DNA binding is ATP-dependent and low-affinity ATP-independent interactions contribute to forked substrate DNA binding and drive strand annealing. DmBLM combines DNA strand displacement with DNA strand annealing to catalyze the displacement of one DNA strand while annealing a second complementary DNA strand.     

Mlh1-Mlh3, a Meiotic Crossover and DNA Mismatch Repair Factor,Is a Msh2-Msh3-stimulated Endonuclease

Crossing over between homologous chromosomes is initiated in meiotic prophase in most sexually reproducing organisms by the appearance of programmed double strand breaks throughout the genome. In Saccharomyces cerevisiae the double-strand breaks are resected to form three prime single-strand tails that primarily invade complementary sequences in unbroken homologs. These invasion intermediates are converted into double Holliday junctions and then resolved into crossovers that facilitate homolog segregation during Meiosis I. Work in yeast suggests that Msh4-Msh5 stabilizes invasion intermediates and double Holliday junctions, which are resolved into crossovers in steps requiring Sgs1 helicase, Exo1, and a putative endonuclease activity encoded by the DNA mismatch repair factor Mlh1-Mlh3. We purified Mlh1-Mlh3 and showed that it is a metal-dependent and Msh2-Msh3-stimulated endonuclease that makes single-strand breaks in supercoiled DNA. These observations support a direct role for an Mlh1-Mlh3 endonuclease activity in resolving recombination intermediates and in DNA mismatch repair.     

ruvA Mutants that resolve Holliday junctions but do not reverse replication forks

RuvAB and RuvABC complexes catalyze branch migration and resolution of Holliday junctions (HJs) respectively. In addition to their action in the last steps of homologous recombination, they process HJs made by replication fork reversal, a reaction which occurs at inactivated replication forks by the annealing of blocked leading and lagging strand ends. RuvAB was recently proposed to bind replication forks and directly catalyze their conversion into HJs. We report here the isolation and characterization of two separation-of-function ruvA mutants that resolve HJs, based on their capacity to promote conjugational recombination and recombinational repair of UV and mitomycin C lesions, but have lost the capacity to reverse forks. In vivo and in vitro evidence indicate that the ruvA mutations affect DNA binding and the stimulation of RuvB helicase activity. This work shows that RuvA's actions at forks and at HJs can be genetically separated, and that RuvA mutants compromised for fork reversal remain fully capable of homologous recombination.     

The Bloom's syndrome helicase promotes the annealing of complementary single-stranded DNA

The product of the gene mutated in Bloom's syndrome, BLM, is a 3′–5′ DNA helicase belonging to the highly conserved RecQ family. In addition to a conventional DNA strand separation activity, BLM catalyzes both the disruption of non-B-form DNA, such as G-quadruplexes, and the branch migration of Holliday junctions. Here, we have characterized a new activity for BLM: the promotion of single-stranded DNA (ssDNA) annealing. This activity does not require Mg²⁺, is inhibited by ssDNA binding proteins and ATP, and is dependent on DNA length. Through analysis of various truncation mutants of BLM, we show that the C-terminal domain is essential for strand annealing and identify a 60 amino acid stretch of this domain as being important for both ssDNA binding and strand annealing. We present a model in which the ssDNA annealing activity of BLM facilitates its role in the processing of DNA intermediates that arise during repair of damaged replication forks.     

A tale of tails: insights into the coordination of 3′ end processing during homologous recombination

Eukaryotic genomes harbor a large number of homologous repeat sequences that are capable of recombining. Their potential to disrupt genome stability highlights the need to understand how homologous recombination processes are coordinated. The Saccharomyces cerevisiae Rad1–Rad10 endonuclease performs an essential role in recombination between repeated sequences, by processing 3′ single‐stranded intermediates formed during single‐strand annealing and gene conversion events. Several recent studies have focused on factors involved in Rad1–Rad10‐dependent removal of 3′ nonhomologous tails during homologous recombination, including Msh2–Msh3, Slx4, and the newly identified Saw1 protein. Together, this new work provides a model for how Rad1–Rad10‐dependent end processing is coordinated: Msh2–Msh3 stabilizes and prepares double‐strand/single‐strand junctions for Rad1–Rad10 cleavage, Saw1 recruits Rad1–Rad10 to 3′ tails, and Slx4 mediates crosstalk between the DNA damage checkpoint machinery and Rad1–Rad10.     

Repairing a double-strand chromosome break by homologous recombination: revisiting Robin Holliday's model

Since the pioneering model for homologous recombination proposed by Robin Holliday in 1964, there has been great progress in understanding how recombination occurs at a molecular level. In the budding yeast Saccharomyces cerevisiae, one can follow recombination by physically monitoring DNA after the synchronous induction of a double-strand break (DSB) in both wild-type and mutant cells. A particularly well-studied system has been the switching of yeast mating-type (MAT) genes, where a DSB can be induced synchronously by expression of the site-specific HO endonuclease. Similar studies can be performed in meiotic cells, where DSBs are created by the Spo11 nuclease. There appear to be at least two competing mechanisms of homologous recombination: a synthesis-dependent strand annealing pathway leading to noncrossovers and a two-end strand invasion mechanism leading to formation and resolution of Holliday junctions (HJs), leading to crossovers. The establishment of a modified replication fork during DSB repair links gene conversion to another important repair process, break-induced replication. Despite recent revelations, almost 40 years after Holliday's model was published, the essential ideas he proposed of strand invasion and heteroduplex DNA formation, the formation and resolution of HJs, and mismatch repair, remain the basis of our thinking.     

Mobile D-loops are a preferred substrate for the Bloom's syndrome helicase

The Bloom's syndrome helicase, BLM, is a member of the highly conserved RecQ family, and possesses both DNA unwinding and DNA strand annealing activities. BLM also promotes branch migration of Holliday junctions. One role for BLM is to act in conjunction with topoisomerase IIIα to process homologous recombination (HR) intermediates containing a double Holliday junction by a process termed dissolution. However, several lines of evidence suggest that BLM may also act early in one or more of the recombination pathways to eliminate illegitimate or aberrantly paired DNA joint molecules. We have investigated whether BLM can disrupt DNA displacement loops (D-loops), which represent the initial strand invasion step of HR. We show that mobile D-loops created by the RecA recombinase are a highly preferred substrate for BLM with the invading strand being displaced from the duplex. We have identified structural features of the D-loop that determine the efficiency with which BLM promotes D-loop dissociation. We discuss these results in the context of models for the role of BLM as an ‘anti-recombinase’.     

Single strand DNA binding and annealing activities in the yeast recombination factor Rad59

Saccharomyces cerevisiae RAD59 gene is required for homologous recombination processes and normal level of resistance to ionizing radiation. To study the biochemical functions of Rad59, it was overproduced in yeast and purified to near homogeneity. Rad59 binds DNA, showing much higher affinity for ssDNA than dsDNA. Rad59 also anneals complementary DNA strands, and order of addition experiments indicate that maximal annealing efficiency is achieved when both complementary DNA strands are present upon addition of Rad59. Thus, Rad59 resembles its homolog Rad52 in being able to bind ssDNA and anneal complementary DNA strands. However, unlike Rad52, DNA annealing by Rad59 is not accelerated by the ssDNA binding factor RPA. DNA binding and strand annealing are likely to be important for the biological functions of Rad59 in general recombination and in the single-strand annealing pathway of recombination.     

The roles of the Saccharomyces cerevisiae RecQ helicase SGS1 in meiotic genome surveillance

Background

The Saccharomyces cerevisiae RecQ helicase Sgs1 is essential for mitotic and meiotic genome stability. The stage at which Sgs1 acts during meiosis is subject to debate. Cytological experiments showed that a deletion of SGS1 leads to an increase in synapsis initiation complexes and axial associations leading to the proposal that it has an early role in unwinding surplus strand invasion events. Physical studies of recombination intermediates implicate it in the dissolution of double Holliday junctions between sister chromatids.

Methodology/Principal Findings

In this work, we observed an increase in meiotic recombination between diverged sequences (homeologous recombination) and an increase in unequal sister chromatid events when SGS1 is deleted. The first of these observations is most consistent with an early role of Sgs1 in unwinding inappropriate strand invasion events while the second is consistent with unwinding or dissolution of recombination intermediates in an Mlh1- and Top3-dependent manner. We also provide data that suggest that Sgs1 is involved in the rejection of ‘second strand capture’ when sequence divergence is present. Finally, we have identified a novel class of tetrads where non-sister spores (pairs of spores where each contains a centromere marker from a different parent) are inviable. We propose a model for this unusual pattern of viability based on the inability of sgs1 mutants to untangle intertwined chromosomes. Our data suggest that this role of Sgs1 is not dependent on its interaction with Top3. We propose that in the absence of SGS1 chromosomes may sometimes remain entangled at the end of pre-meiotic replication. This, combined with reciprocal crossing over, could lead to physical destruction of the recombined and entangled chromosomes. We hypothesise that Sgs1, acting in concert with the topoisomerase Top2, resolves these structures.

Conclusions

This work provides evidence that Sgs1 interacts with various partner proteins to maintain genome stability throughout meiosis.     

Synergic and Opposing Activities of Thermophilic RecQ-like Helicase and Topoisomerase 3 Proteins in Holliday Junction Processing and Replication Fork Stabilization

RecQ family helicases and topoisomerase 3 enzymes form evolutionary conserved complexes that play essential functions in DNA replication, recombination, and repair, and in vitro, show coordinate activities on model recombination and replication intermediates. Malfunctioning of these complexes in humans is associated with genomic instability and cancer-prone syndromes. Although both RecQ-like and topoisomerase 3 enzymes are present in archaea, only a few of them have been studied, and no information about their functional interaction is available. We tested the combined activities of the RecQ-like helicase, Hel112, and the topoisomerase 3, SsTop3, from the thermophilic archaeon Sulfolobus solfataricus. Hel112 showed coordinate DNA unwinding and annealing activities, a feature shared by eukaryotic RecQ homologs, which resulted in processing of synthetic Holliday junctions and stabilization of model replication forks. SsTop3 catalyzed DNA relaxation and annealing. When assayed in combination, SsTop3 inhibited the Hel112 helicase activity on Holliday junctions and stimulated formation and stabilization of such structures. In contrast, Hel112 did not affect the SsTop3 DNA relaxation activity. RecQ-topoisomerase 3 complexes show structural similarity with the thermophile-specific enzyme reverse gyrase, which catalyzes positive supercoiling of DNA and was suggested to play a role in genome stability at high temperature. Despite such similarity and the high temperature of reaction, the SsTop3-Hel112 complex does not induce positive supercoiling and is thus likely to play different roles. We propose that the interplay between Hel112 and SsTop3 might regulate the equilibrium between recombination and anti-recombination activities at replication forks.     

Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast

Very few gene conversions in mitotic cells are associated with crossovers, suggesting that these events are regulated. This may be important for the maintenance of genetic stability. We have analyzed the relationship between homologous recombination and crossing-over in haploid budding yeast and identified factors involved in the regulation of crossover outcomes. Gene conversions unaccompanied by a crossover appear 30 min before conversions accompanied by exchange, indicating that there are two different repair mechanisms in mitotic cells. Crossovers are rare (5%), but deleting the BLM/WRN homolog, SGS1, or the SRS2 helicase increases crossovers 2- to 3-fold. Overexpressing SRS2 nearly eliminates crossovers, whereas overexpression of RAD51 in srs2Delta cells almost completely eliminates the noncrossover recombination pathway. We suggest Sgs1 and its associated topoisomerase Top3 remove double Holliday junction intermediates from a crossover-producing repair pathway, thereby reducing crossovers. Srs2 promotes the noncrossover synthesis-dependent strand-annealing (SDSA) pathway, apparently by regulating Rad51 binding during strand exchange.     

Enzymatic processing of replication and recombination intermediates by the vaccinia virus DNA polymerase

Poxvirus DNA polymerases play a critical role in promoting virus recombination. To test if vaccinia polymerase (E9L) could mediate this effect by catalyzing the post-synaptic processing of recombinant joint molecules, we prepared substrates bearing a nick, a 3′-unpaired overhang, a 5′ overhang, or both 3′ and 5′ overhangs. The sequence of the 5′ overhang was also modified to permit or preclude branch migration across the joint site. These substrates were incubated with E9L, and the fate of the primer strand characterized under steady-state reaction conditions. E9L rapidly excises a mispaired 3′ strand from a DNA duplex, producing a meta-stable nicked molecule that is a substrate for ligase. The reaction was not greatly affected by adding an unpaired 5′ strand, but since such molecules cannot be processed into nicked intermediates, the 3′-ended strand continued to be subjected to exonucleolytic attack. Incorporating homology into the 5′ overhang prevented this and permitted some strand assimilation, but such substrates also promoted strand-displacement DNA synthesis of a type predicted by the 1981 Moyer and Graves model for poxvirus replication. Single-strand annealing reactions are used by poxviruses to produce recombinant viruses and these data show that virus DNA polymerases can process DNA in such a manner as to both generate single-stranded substrates for such reactions and to facilitate the final processing of the reaction products.     

hXRCC2 enhances ADP/ATP processing and strand exchange by hRAD51

The assembly of bacterial RecA, and its human homolog hRAD51, into an operational ADP/ATP-regulated DNA-protein (nucleoprotein) filament is essential for homologous recombination repair (HRR). Yet hRAD51 lacks the coordinated ADP/ATP processing exhibited by RecA and is less efficient in HRR reactions in vitro. In this study, we demonstrate that hXRCC2, one of five other poorly understood non-redundant human mitotic RecA homologs (hRAD51B, hRAD51C, hRAD51D, hXRCC2, and hXRCC3), stimulates hRAD51 ATP processing. hXRCC2 also increases hRAD51-mediated DNA unwinding and strand exchange activities that are integral for HRR. Although there does not seem to be a long-lived interaction between hXRCC2 and hRAD51, we detail a strong adenosine nucleotide-regulated interaction between the hXRCC2-hRAD51D heterodimer and hRAD51. These observations begin to elucidate the separate and specialized functions of the human mitotic RecA homologs that enable an efficient nucleoprotein filament required for HRR.     

Unwinding forward and sliding back: an intermittent unwinding mode of the BLM helicase

There are lines of evidence that the Bloom syndrome helicase, BLM, catalyzes regression of stalled replication forks and disrupts displacement loops (D-loops) formed during homologous recombination (HR). Here we constructed a forked DNA with a 3′ single-stranded gap and a 5′ double-stranded handle to partly mimic a stalled DNA fork and used magnetic tweezers to study BLM-catalyzed unwinding of the forked DNA. We have directly observed that the BLM helicase may slide on the opposite strand for some distance after duplex unwinding at different forces. For DNA construct with a long hairpin, progressive unwinding of the hairpin is frequently interrupted by strand switching and backward sliding of the enzyme. Quantitative study of the uninterrupted unwinding length (time) has revealed a two-state-transition mechanism for strand-switching during the unwinding process. Mutational studies revealed that the RQC domain plays an important role in stabilizing the helicase/DNA interaction during both DNA unwinding and backward sliding of BLM. Especially, Lys1125 in the RQC domain, a highly conserved amino acid among RecQ helicases, may be involved in the backward sliding activity. We have also directly observed the in vitro pathway that BLM disrupts the mimic stalled replication fork. These results may shed new light on the mechanisms for BLM in DNA repair and homologous recombination.     

Unwinding of forked DNA structures by UvrD

Many studies have demonstrated the need for processing of blocked replication forks to underpin genome duplication. UvrD helicase in Escherichia coli has been implicated in the processing of damaged replication forks, or the recombination intermediates formed from damaged forks. Here we show that UvrD can unwind forked DNA structures, in part due to the ability of UvrD to initiate unwinding from discontinuities within the phosphodiester backbone of DNA. UvrD does therefore have the capacity to target DNA intermediates of replication and recombination. Such an activity resulted in unwinding of what would be the parental duplex DNA ahead of either a stalled replication fork or a D-loop formed by recombination. However, UvrD had a substrate preference for fork structures having a nascent lagging strand at the branch point but no leading strand. Furthermore, at such structures the polarity of UvrD altered so that unwinding of the lagging strand predominated. This reaction is reminiscent of the PriC-Rep pathway of replication restart, suggesting that UvrD and Rep may have at least partially redundant functions.     

Homologs of MutS and MutL during mammalian meiosis

In eukaryotes, homologs of the Escherichia coli MutS and MutL proteins are crucial for both meiotic recombination and post-replicative DNA mismatch repair. Both pathways require the formation of a MutS homolog complex which interacts with a second heterodimer, composed of two MutL homologs. During mammalian meiosis, it is likely that chromosome synapsis requires the presence of a MSH4-MSH5 heterodimer. PMS2, a MutL homolog, seems to play an important role in this process. A MSH4-MSH5 heterodimer is also likely present later with other MutL homologs (MLH1 and MLH3) and is involved in the crossing-over process. The phenotype of msh4-/- mutant mice and MSH4 immunolocalization on meiotic chromosomes suggest that MSH4 has an early function in mammalian meiotic recombination. Both MSH4 and PMS2 directly interact with the RAD51 DNA strand exchange protein. In addition, MSH4 and RAD51 proteins co-localize on mouse meiotic chromosome cores. These results suggest that MSH4 and its partners could act, just after strand exchange promoted by RAD51, to check the homology of DNA heteroduplexes.     

The Archaeal Topoisomerase Reverse Gyrase Is a Helix-destabilizing Protein That Unwinds Four-way DNA Junctions

Four-way junctions are non-B DNA structures that originate as intermediates of recombination and repair (Holliday junctions) or from the intrastrand annealing of palindromic sequences (cruciforms). These structures have important functional roles but may also severely interfere with DNA replication and other genetic processes; therefore, they are targeted by regulatory and architectural proteins, and dedicated pathways exist for their removal. Although it is well known that resolution of Holliday junctions occurs either by recombinases or by specialized helicases, less is known on the mechanisms dealing with secondary structures in nucleic acids. Reverse gyrase is a DNA topoisomerase, specific to microorganisms living at high temperatures, which comprises a type IA topoisomerase fused to an SF2 helicase-like module and catalyzes ATP hydrolysis-dependent DNA positive supercoiling. Reverse gyrase is likely involved in regulation of DNA structure and stability and might also participate in the cell response to DNA damage. By applying FRET technology to multiplex fluorophore gel imaging, we show here that reverse gyrase induces unwinding of synthetic four-way junctions as well as forked DNA substrates, following a mechanism independent of both the ATPase and the strand-cutting activity of the enzyme. The reaction requires high temperature and saturating protein concentrations. Our results suggest that reverse gyrase works like an ATP-independent helix-destabilizing protein specific for branched DNA structures. The results are discussed in light of reverse gyrase function and their general relevance for protein-mediated unwinding of complex DNA structures.     

BLM helicase measures DNA unwound before switching strands and hRPA promotes unwinding reinitiation

Bloom syndrome (BS) is a rare genetic disorder characterized by genomic instability and a high predisposition to cancer. The gene defective in BS, BLM, encodes a member of the RecQ family of 3′–5′ DNA helicases, and is proposed to function in recombinational repair during DNA replication. Here, we have utilized single‐molecule fluorescence resonance energy transfer microscopy to examine the behaviour of BLM on forked DNA substrates. Strikingly, BLM unwound individual DNA molecules in a repetitive manner, unwinding a short length of duplex DNA followed by rapid reannealing and reinitiation of unwinding in several successions. Our results show that a monomeric BLM can ‘measure’ how many base pairs it has unwound, and once it has unwound a critical length, it reverses the unwinding reaction through strand switching and translocating on the opposing strand. Repetitive unwinding persisted even in the presence of hRPA, and interaction between wild‐type BLM and hRPA was necessary for unwinding reinitiation on hRPA‐coated DNA. The reported activities may facilitate BLM processing of stalled replication forks and illegitimately formed recombination intermediates.