The enzymatic basis of processivity in lambda exonuclease

λ Exonuclease is a highly processive 5′→3′ exonuclease that degrades double-stranded (ds)DNA. The single-stranded DNA produced by λ exonuclease is utilized by homologous pairing proteins to carry out homologous recombination. The extensive studies of λ biology, λ exonuclease enzymology and the availability of the X-ray crystallographic structure of λ exonuclease make it a suitable model to dissect the mechanisms of processivity. λ Exonuclease is a toroidal homotrimeric molecule and this quaternary structure is a recurring theme in proteins engaged in processive reactions in nucleic acid metabolism. We have identified residues in λ exonuclease involved in recognizing the 5′-phosphate at the ends of broken dsDNA. The preference of λ exonuclease for a phosphate moiety at 5′ dsDNA ends has been established in previous studies; our results indicate that the low activity in the absence of the 5′-phosphate is due to the formation of inert enzyme–substrate complexes. By examining a λ exonuclease mutant impaired in 5′-phosphate recognition, the significance of catalytic efficiency in modulating the processivity of λ exonuclease has been elucidated. We propose a model in which processivity of λ exonuclease is expressed as the net result of competition between pathways that either induce forward translocation or promote reverse translocation and dissociation.     

A Bridge Crosses the Active-Site Canyon of the Epstein-Barr Virus Nuclease with DNase and RNase Activities

Epstein-Barr virus, a double-stranded DNA (dsDNA) virus, is a major human pathogen from the herpesvirus family. The nuclease is one of the lytic cycle proteins required for successful viral replication. In addition to the previously described endonuclease and exonuclease activities on single-stranded DNA and dsDNA substrates, we observed an RNase activity for Epstein-Barr virus nuclease in the presence of Mn²⁺, giving a possible explanation for its role in host mRNA degradation. Its crystal structure shows a catalytic core of the D-(D/E)XK nuclease superfamily closely related to the exonuclease from bacteriophage lambda with a bridge across the active-site canyon. This bridge may reduce endonuclease activity, ensure processivity or play a role in strand separation of dsDNA substrates. As the DNA strand that is subject to cleavage is likely to make a sharp turn in front of the bridge, endonuclease activity on single-stranded DNA stretches appears to be possible, explaining the cleavage of circular substrates.     

The specificity of lambda exonuclease. Interactions with single-stranded DNA.

The lambda exonuclease, an enzyme that has been implicated in genetic recombination, rapidly and processively degrades native DNA, starting at the 5' terminus. The enzyme will also degrade the 5'-terminated strand at a single-stranded branch. The experiments reported here reveal various interactions of the enzyme with single-stranded DNA. The rate of digestion is related inversely to the length of single strands. Chains of 100 nucleotides are digested at about 10% the rate of digestion of native DNA. Digestion of the single-stranded ends of lambda DNA does not appear to occur processively. The enzyme binds to circular as well as linear single strands and the affinity for single strands is also related inversely to the chain length. In an equimolar mixture of single- and double-stranded DNA the action of lambda exonuclease on the latteris about half-inhibited. At 20 degrees the initiation of digestion at the 5' terminus of duplex DNA is blocked sterically when such DNA has 3'-terminal single strands that are longer than 100 nucleotides. Information about these properties is important for the practical use of lambda exonuclease as well as for reflections on the role of the enzyme in genetic recombination.     

Characterization of human herpesvirus 6A/B U94 as ATPase,helicase, exonuclease and DNA-binding proteins

Human herpesvirus-6A (HHV-6A) and HHV-6B integrate their genomes into the telomeres of human chromosomes, however, the mechanisms leading to integration remain unknown. HHV-6A/B encode a protein that has been proposed to be involved in integration termed U94, an ortholog of adeno-associated virus type 2 (AAV-2) Rep68 integrase. In this report, we addressed whether purified recombinant maltose-binding protein (MBP)-U94 fusion proteins of HHV-6A/B possess biological functions compatible with viral integration. We could demonstrate that MBP-U94 efficiently binds both dsDNA and ssDNA containing telomeric repeats using gel shift assay and surface plasmon resonance. MBP-U94 is also able to hydrolyze adenosine triphosphate (ATP) to ADP, providing the energy for further catalytic activities. In addition, U94 displays a 3′ to 5′ exonuclease activity on dsDNA with a preference for 3′-recessed ends. Once the DNA strand reaches 8–10 nt in length, the enzyme dissociates it from the complementary strand. Lastly, MBP-U94 compromises the integrity of a synthetic telomeric D-loop through exonuclease attack at the 3′ end of the invading strand. The preferential DNA binding of MBP-U94 to telomeric sequences, its ability to hydrolyze ATP and its exonuclease/helicase activities suggest that U94 possesses all functions required for HHV-6A/B chromosomal integration.     

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.     

Bipolar DNA translocation contributes to highly processive DNA unwinding by RecBCD enzyme

We recently demonstrated that the RecBCD enzyme is a bipolar DNA helicase that employs two single-stranded DNA motors of opposite polarity to drive translocation and unwinding of duplex DNA. We hypothesized that this organization may explain the exceptionally high rate and processivity of DNA unwinding catalyzed by the RecBCD enzyme. Using a stopped-flow dye displacement assay for unwinding activity, we test this idea by analyzing mutant RecBCD enzymes in which either of the two helicase motors is inactivated by mutagenesis. Like the wild-type RecBCD enzyme, the two mutant proteins maintain the ability to bind tightly to blunt duplex DNA ends in the absence of ATP. However, the rate of forward translocation for the RecB motor-defective enzyme is only approximately 30% of the wild-type rate, whereas for the RecD motor-defective enzyme, it is approximately 50%. More significantly, the processivity of translocation is substantially reduced by approximately 25- and 6-fold for each mutant enzyme, respectively. Despite retaining the capacity to bind blunt dsDNA, the RecB-mutant enzyme has lost the ability to unwind DNA unless the substrate contains a short 5'-terminated single-stranded DNA overhang. The consequences of this observation for the architecture of the single-stranded DNA motors in the initiation complex are discussed.     

Electron microscopic analysis of DNA forks generated by Escherichia coli DNA helicase II

T7 phage DNA eroded with lambda exonuclease (to create 3'-protruding strands) or exonuclease III (to create 5'-protruding strands) was treated under unwinding assay conditions with DNA helicase II. Single-stranded DNA-binding protein (of Escherichia coli or phage T4) was added to disentangle the denatured DNA and the complexes were examined in the electron microscope. DNA helicase II complexes filtered through a gel column before assay retain the ability to generate forks suggesting that DNA helicase II unwinds in a preformed complex by translocating along the bound DNA strand. The enzyme initiates preferentially at the ends of the lambda-exonuclease-treated duplexes and is found at a fork on the initially protruding strand. It also initiates at the ends of the exonuclease-III-treated duplexes where, as with approximately 5% of the forks traceable back to a single-stranded gap, it is found on the initially recessed strand. The results are consistent with the view that DNA helicase II unwinds in the 3'-5' direction relative to the bound strand. They also confirm that the enzyme can initiate at the end of a fully base-paired strand. At a fork, DNA helicase II is bound as a tract of molecules of approximately 110 nm in length. Tracts of enzyme assemble from non-cooperatively bound molecules in the presence of ATP. During unwinding, DNA helicase II apparently can translocate to the displaced strand which conceivably can deplete the leading strand of the enzyme. Continued adsorption of enzyme to DNA might replenish forks arrested by strand switch of the unwinding enzyme.     

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.     

ATP hydrolysis and the displaced strand are two factors that determine the polarity of RecA-promoted DNA strand exchange.

When the recA protein (RecA) of Escherichia coli promotes strand exchange between single-stranded DNA (ssDNA) circles and linear double-stranded DNAs (dsDNA) with complementary 5' or 3' ends a polarity is observed. This property of RecA depends on ATP hydrolysis and the ssDNA that is displaced in the reaction since no polarity is observed in the presence of the non-hydrolyzable ATP analog, ATP gamma S, or in the presence of single-strand specific exonucleases. Based on these results a model is presented in which both the 5' and 3' complementary ends of the linear dsDNA initiate pairing with the ssDNA circle but only one end remains stably paired. According to this model, the association/dissociation of RecA in the 5' to 3' direction on the displaced strand determines the polarity of strand exchange by favoring or blocking its reinvasion into the newly formed dsDNA. Reinvasion is favored when the displaced strand is coated with RecA whereas it is blocked when it lacks RecA, remains covered by single-stranded DNA binding protein or is removed by a single-strand specific exonuclease. The requirement for ATP hydrolysis is explained if the binding of RecA to the displaced strand occurs via the dissociation and/or transfer of RecA, two functions that depend on ATP hydrolysis. The energy for strand exchange derives from the higher binding constant of RecA for the newly formed dsDNA as compared with that for ssDNA and not from ATP hydrolysis.     

DNA Replication Catalyzed by Herpes Simplex Virus Type 1 Proteins Reveals Trombone Loops at the Fork

Using purified replication factors encoded by herpes simplex virus type 1 and a 70-base minicircle template, we obtained robust DNA synthesis with leading strand products of >20,000 nucleotides and lagging strand fragments from 600 to 9,000 nucleotides as seen by alkaline gel electrophoresis. ICP8 was crucial for the synthesis on both strands. Visualization of the deproteinized products using electron microscopy revealed long, linear dsDNAs, and in 87%, one end, presumably the end with the 70-base circle, was single-stranded. The remaining 13% had multiple single-stranded segments separated by dsDNA segments 500 to 1,000 nucleotides in length located at one end. These features are diagnostic of the trombone mechanism of replication. Indeed, when the products were examined with the replication proteins bound, a dsDNA loop was frequently associated with the replication complex located at one end of the replicated DNA. Furthermore, the frequency of loops correlated with the fraction of DNA undergoing Okazaki fragment synthesis.     

DNA end resection: Many nucleases make light work

Double-strand breaks (DSBs) are deleterious DNA lesions and if left unrepaired result in severe genomic instability. Cells use two main pathways to repair DSBs: homologous recombination (HR) or non-homologous end joining (NHEJ) depending on the phase of the cell cycle and the nature of the DSB ends. A key step where pathway choice is exerted is in the ‘licensing’ of 5′–3′ resection of the ends to produce recombinogenic 3′ single-stranded tails. These tails are substrate for binding by Rad51 to initiate pairing and strand invasion with homologous duplex DNA. Moreover, the single-stranded DNA generated after end processing is important to activate the DNA damage response. The mechanism of end processing is the focus of this review and we will describe recent findings that shed light on this important initiating step for HR. The conserved MRX/MRN complex appears to be a major regulator of DNA end processing. Sae2/CtIP functions with the MRX complex, either to activate the Mre11 nuclease or via the intrinsic endonuclease, in an initial step to trim the DSB ends. In a second step, redundant systems remove long tracts of DNA to reveal extensive 3′ single-stranded tails. One system is dependent on the helicase Sgs1 and the nuclease Dna2, and the other on the 5′–3′ exonuclease Exo1.     

Regulation of yeast DNA polymerase δ-mediated strand displacement synthesis by 5′-flaps

The strand displacement activity of DNA polymerase δ is strongly stimulated by its interaction with proliferating cell nuclear antigen (PCNA). However, inactivation of the 3′–5′ exonuclease activity is sufficient to allow the polymerase to carry out strand displacement even in the absence of PCNA. We have examined in vitro the basic biochemical properties that allow Pol δ-exo⁻ to carry out strand displacement synthesis and discovered that it is regulated by the 5′-flaps in the DNA strand to be displaced. Under conditions where Pol δ carries out strand displacement synthesis, the presence of long 5′-flaps or addition in trans of ssDNA suppress this activity. This suggests the presence of a secondary DNA binding site on the enzyme that is responsible for modulation of strand displacement activity. The inhibitory effect of a long 5′-flap can be suppressed by its interaction with single-stranded DNA binding proteins. However, this relief of flap-inhibition does not simply originate from binding of Replication Protein A to the flap and sequestering it. Interaction of Pol δ with PCNA eliminates flap-mediated inhibition of strand displacement synthesis by masking the secondary DNA site on the polymerase. These data suggest that in addition to enhancing the processivity of the polymerase PCNA is an allosteric modulator of other Pol δ activities.     

Coordinate 5' and 3' endonucleolytic trimming of terminally blocked blunt DNA double-strand break ends by Artemis nuclease and DNA-dependent protein kinase

Previous work showed that, in the presence of DNA-dependent protein kinase (DNA-PK), Artemis slowly trims 3′-phosphoglycolate-terminated blunt ends. To examine the trimming reaction in more detail, long internally labeled DNA substrates were treated with Artemis. In the absence of DNA-PK, Artemis catalyzed extensive 5′→3′ exonucleolytic resection of double-stranded DNA. This resection required a 5′-phosphate, but did not require ATP, and was accompanied by endonucleolytic cleavage of the resulting 3′ overhang. In the presence of DNA-PK, Artemis-mediated trimming was more limited, was ATP-dependent and did not require a 5′-phosphate. For a blunt end with either a 3′-phosphoglycolate or 3′-hydroxyl terminus, endonucleolytic trimming of 2–4 nucleotides from the 3′-terminal strand was accompanied by trimming of 6 nt from the 5′-terminal strand. The results suggest that autophosphorylated DNA-PK suppresses the exonuclease activity of Artemis toward blunt-ended DNA, and promotes slow and limited endonucleolytic trimming of the 5′-terminal strand, resulting in short 3′ overhangs that are trimmed endonucleolytically. Thus, Artemis and DNA-PK can convert terminally blocked DNA ends of diverse geometry and chemical structure to a form suitable for polymerase-mediated patching and ligation, with minimal loss of terminal sequence. Such processing could account for the very small deletions often found at DNA double-strand break repair sites.     

Structure-specific DNA binding by bacteriophage T5 5'-->3' exonuclease.

Phage T5 exonuclease is a 5'-->3'exodeoxyribonuclease that also exhibits endonucleolytic activity on flap structures (branched duplex DNA containing a free single-stranded 5'-end). Oligonucleotides were used to construct duplexes with either blunt ends, 5'-overhangs, 3'-overhangs, a flap or a forked end (pseudo-Y). The binding of T5 exonuclease to various structures was investigated using native electrophoretic mobility shift assays (EMSA) in the absence of the essential divalent metal cofactor. Binding of T5 exonuclease to either blunt-ended duplexes or single-stranded oligonucleotides could not be detected by EMSA. However, duplexes with 5'-overhangs, flaps and pseudo-Y structures showed decreased mobility with added T5 exonuclease. On binding to DNA the wild-type enzyme was rendered partially resistant to proteolysis, yielding a biologically active 31.5 kDa fragment. However, the protein-DNA complex remained susceptible to inactivation by p-hydroxymercuribenzoate (PHMB, a cysteine-specific modifying agent), suggesting that neither cysteine is intimately associated with substrate binding. Replacement of both cysteine residues of the molecule with serine did not greatly alter the catalytic or binding characteristics of the protein but did render it highly resistant to inhibition by PHMB.     

Specific inhibition of the E.coli RecBCD enzyme by Chi sequences in single-stranded oligodeoxyribonucleotides

RecBCD is an ATP-dependent helicase and exonuclease which generates 3′ single-stranded DNA (ssDNA) ends used by RecA for homologous recombination. The exonuclease activity is altered when RecBCD encounters a Chi sequence (5′-GCTGGTGG-3′) in double-stranded DNA (ds DNA), an event critical to the generation of the 3′-ssDNA. This study tests the effect of ssDNA oligonucleotides having a Chi sequence (Chi⁺) or a single base change that abolishes the Chi sequence (Chi^o), on the enzymatic activities of RecBCD. Our results show that a 14 and a 20mer with Chi⁺ in the center of the molecule inhibit the exonuclease and helicase activities of RecBCD to a greater extent than the corresponding Chi^o oligonucleotides. Oligonucleotides with the Chi sequence at one end, or the Chi sequence alone in an 8mer, failed to show Chi-specific inhibition of RecBCD. Thus, Chi recognition requires that Chi be flanked by DNA at either end. Further experiments indicated that the oligonucleotides inhibit RecBCD from binding to its dsDNA substrate. These results suggest that a specific site for Chi recognition exists on RecBCD, which binds Chi with greater affinity than a non-Chi sequence and is probably adjacent to non-specific DNA binding sites.     

Flap Endonuclease Activity of Gene 6 Exonuclease of Bacteriophage T7

Flap endonucleases remove flap structures generated during DNA replication. Gene 6 protein of bacteriophage T7 is a 5′–3′-exonuclease specific for dsDNA. Here we show that gene 6 protein also possesses a structure-specific endonuclease activity similar to known flap endonucleases. The flap endonuclease activity is less active relative to its exonuclease activity. The major cleavage by the endonuclease activity occurs at a position one nucleotide into the duplex region adjacent to a dsDNA-ssDNA junction. The efficiency of cleavage of the flap decreases with increasing length of the 5′-overhang. A 3′-single-stranded tail arising from the same end of the duplex as the 5′-tail inhibits gene 6 protein flap endonuclease activity. The released flap is not degraded further, but the exonuclease activity then proceeds to hydrolyze the 5′-terminal strand of the duplex. T7 gene 2.5 single-stranded DNA-binding protein stimulates the exonuclease and also the endonuclease activity. This stimulation is attributed to a specific interaction between the two proteins because Escherichia coli single-stranded DNA binding protein does not produce this stimulatory effect. The ability of gene 6 protein to remove 5′-terminal overhangs as well as to remove nucleotides from the 5′-termini enables it to effectively process the 5′-termini of Okazaki fragments before they are ligated.     

The herpes simplex virus type 1 alkaline nuclease and single-stranded DNA binding protein mediate strand exchange in vitro

The replication of herpes simplex virus type 1 (HSV-1) DNA is associated with a high degree of homologous recombination. While cellular enzymes may take part in mediating this recombination, we present evidence for an HSV-1-encoded recombinase activity. HSV-1 alkaline nuclease, encoded by the UL12 gene, is a 5'-->3' exonuclease that shares homology with Redalpha, commonly known as lambda exonuclease, an exonuclease required for homologous recombination by bacteriophage lambda. The HSV-1 single-stranded DNA binding protein ICP8 is an essential protein for HSV DNA replication and possesses single-stranded DNA annealing activities like the Redbeta synaptase component of the phage lambda recombinase. Here we show that UL12 and ICP8 work together to effect strand exchange much like the Red system of lambda. Purified UL12 protein and ICP8 mediated the complete exchange between a 7.25-kb M13mp18 linear double-stranded DNA molecule and circular single-stranded M13 DNA, forming a gapped circle and a displaced strand as final products. The optimal conditions for strand exchange were 1 mM MgCl(2), 40 mM NaCl, and pH 7.5. Stoichiometric amounts of ICP8 were required, and strand exchange did not depend on the nature of the double-stranded end. Nuclease-defective UL12 could not support this reaction. These data suggest that diverse DNA viruses appear to utilize an evolutionarily conserved recombination mechanism.     

Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

Background

SXT is an integrating conjugative element (ICE) originally isolated from Vibrio cholerae, the bacterial pathogen that causes cholera. It houses multiple antibiotic and heavy metal resistance genes on its ca. 100 kb circular double stranded DNA (dsDNA) genome, and functions as an effective vehicle for the horizontal transfer of resistance genes within susceptible bacterial populations. Here, we characterize the activities of an alkaline exonuclease (S066, SXT-Exo) and single strand annealing protein (S065, SXT-Bet) encoded on the SXT genetic element, which share significant sequence homology with Exo and Bet from bacteriophage lambda, respectively.

Results

SXT-Exo has the ability to degrade both linear dsDNA and single stranded DNA (ssDNA) molecules, but has no detectable endonuclease or nicking activities. Adopting a stable trimeric arrangement in solution, the exonuclease activities of SXT-Exo are optimal at pH 8.2 and essentially require Mn²⁺ or Mg²⁺ ions. Similar to lambda-Exo, SXT-Exo hydrolyzes dsDNA with 5'- to 3'-polarity in a highly processive manner, and digests DNA substrates with 5'-phosphorylated termini significantly more effectively than those lacking 5'-phosphate groups. Notably, the dsDNA exonuclease activities of both SXT-Exo and lambda-Exo are stimulated by the addition of lambda-Bet, SXT-Bet or a single strand DNA binding protein encoded on the SXT genetic element (S064, SXT-Ssb). When co-expressed in E. coli cells, SXT-Bet and SXT-Exo mediate homologous recombination between a PCR-generated dsDNA fragment and the chromosome, analogous to RecET and lambda-Bet/Exo.

Conclusions

The activities of the SXT-Exo protein are consistent with it having the ability to resect the ends of linearized dsDNA molecules, forming partially ssDNA substrates for the partnering SXT-Bet single strand annealing protein. As such, SXT-Exo and SXT-Bet may function together to repair or process SXT genetic elements within infected V. cholerae cells, through facilitating homologous DNA recombination events. The results presented here significantly extend our general understanding of the properties and activities of alkaline exonuclease and single strand annealing proteins of viral/bacteriophage origin, and will assist the rational development of bacterial recombineering systems.     

Sequence-specific fluorescent labeling of double-stranded DNA observed at the single molecule level

Fluorescent labeling of a short sequence of double-stranded DNA (dsDNA) was achieved by ligating a labeled dsDNA fragment to a stem–loop triplex forming oligonucleotide (TFO). After the TFO has wound around the target sequence by ligand-induced triple helix formation, its extremities hybridize to each other, leaving a dangling single-stranded sequence, which is then ligated to a fluorescent dsDNA fragment using T4 DNA ligase. A non-repeated 15 bp sequence present on lambda DNA was labeled and visualized by fluorescence microscopy after DNA combing. The label was found to be attached at a specific position located at 4.2 ± 0.5 kb from one end of the molecule, in agreement with the location of the target sequence for triple helix formation (4.4 kb from one end). In addition, an alternative combing process was noticed in which a DNA molecule becomes attached to the combing slide from the label rather than from one of its ends. The method described herein provides a new tool for the detection of very short sequences of dsDNA and offers various perspectives in the micromanipulation of single DNA molecules.