Holliday Junction Intermediates Research Articles

Functional Analysis of DNA Replication Fork Reversal Catalyzed by Mycobacterium tuberculosis RuvAB Proteins

Initially discovered in Escherichia coli, RuvAB proteins are ubiquitous in bacteria and play a dual role as molecular motor proteins responsible for branch migration of the Holliday junction(s) and reversal of stalled replication forks. Despite mounting genetic evidence for a crucial role of RuvA and RuvB proteins in reversal of stalled replication forks, the mechanistic aspects of this process are still not fully understood. Here, we elucidate the ability of Mycobacterium tuberculosis RuvAB (MtRuvAB) complex to catalyze the reversal of replication forks using a range of DNA replication fork substrates. Our studies show that MtRuvAB, unlike E. coli RuvAB, is able to drive replication fork reversal via the formation of Holliday junction intermediates, suggesting that RuvAB-catalyzed fork reversal involves concerted unwinding and annealing of nascent leading and lagging strands. We also demonstrate the reversal of replication forks carrying hemi-replicated DNA, indicating that MtRuvAB complex-catalyzed fork reversal is independent of symmetry at the fork junction. The fork reversal reaction catalyzed by MtRuvAB is coupled to ATP hydrolysis, is processive, and culminates in the formation of an extended reverse DNA arm. Notably, we found that sequence heterology failed to impede the fork reversal activity of MtRuvAB. We discuss the implications of these results in the context of recognition and processing of varied types of replication fork structures by RuvAB proteins.

Activation of XerCD-dif recombination by the FtsK DNA translocase

The FtsK translocase pumps dsDNA directionally at ∼5 kb/s and facilitates chromosome unlinking by activating XerCD site-specific recombination at dif, located in the replication terminus of the Escherichia coli chromosome. We show directly that the γ regulatory subdomain of FtsK activates XerD catalytic activity to generate Holliday junction intermediates that can then be resolved by XerC. Furthermore, we demonstrate that γ can activate XerCD-dif recombination in the absence of the translocase domain, when it is fused to XerCD, or added in isolation. In these cases the recombination products are topologically complex and would impair chromosome unlinking. We propose that FtsK translocation and activation of unlinking are normally coupled, with the translocation being essential for ensuring that the products of recombination are topologically unlinked, an essential feature of the role of FtsK in chromosome segregation.

Stoichiometric incorporation of base substitutions at specific sites in supercoiled DNA and supercoiled recombination intermediates

Supercoiled DNA is the relevant substrate for a large number of DNA transactions and has additionally been found to be a favorable form for delivering DNA and protein-DNA complexes to cells. We report here a facile method for stoichiometrically incorporating several different modifications at multiple, specific, and widely spaced sites in supercoiled DNA. The method is based upon generating an appropriately gapped circular DNA, starting from single-strand circular DNA from two phagemids with oppositely oriented origins of replication. The gapped circular DNA is annealed with labeled and unlabeled synthetic oligonucleotides to make a multiply nicked circle, which is covalently sealed and supercoiled. The method is efficient, robust and can be readily scaled up to produce large quantities of labeled supercoiled DNA for biochemical and structural studies. We have applied this method to generate dye-labeled supercoiled DNA with heteroduplex bubbles for a Förster resonance energy transfer (FRET) analysis of supercoiled Holliday junction intermediates in the λ integrative recombination reaction. We found that a higher-order structure revealed by FRET in the supercoiled Holliday junction intermediate is preserved in the linear recombination product. We suggest that in addition to studies on recombination complexes, these methods will be generally useful in other reactions and systems involving supercoiled DNA.

The Caenorhabditis elegans Homolog of Gen1/Yen1 Resolvases Links DNA Damage Signaling to DNA Double-Strand Break Repair

DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR), which can involve Holliday junction (HJ) intermediates that are ultimately resolved by nucleolytic enzymes. An N-terminal fragment of human GEN1 has recently been shown to act as a Holliday junction resolvase, but little is known about the role of GEN-1 in vivo. Holliday junction resolution signifies the completion of DNA repair, a step that may be coupled to signaling proteins that regulate cell cycle progression in response to DNA damage. Using forward genetic approaches, we identified a Caenorhabditis elegans dual function DNA double-strand break repair and DNA damage signaling protein orthologous to the human GEN1 Holliday junction resolving enzyme. GEN-1 has biochemical activities related to the human enzyme and facilitates repair of DNA double-strand breaks, but is not essential for DNA double-strand break repair during meiotic recombination. Mutational analysis reveals that the DNA damage-signaling function of GEN-1 is separable from its role in DNA repair. GEN-1 promotes germ cell cycle arrest and apoptosis via a pathway that acts in parallel to the canonical DNA damage response pathway mediated by RPA loading, CHK1 activation, and CEP-1/p53–mediated apoptosis induction. Furthermore, GEN-1 acts redundantly with the 9-1-1 complex to ensure genome stability. Our study suggests that GEN-1 might act as a dual function Holliday junction resolvase that may coordinate DNA damage signaling with a late step in DNA double-strand break repair.

An Antimicrobial Peptide That Targets DNA Repair Intermediates In Vitro Inhibits Salmonella Growth within Murine Macrophages

The hexapeptide WRWYCR was previously identified on the basis of its ability to inhibit bacteriophage lambda integrase-mediated recombination by trapping and preventing resolution of the Holliday junction intermediate. This peptide inhibits several unrelated DNA repair enzymes that bind to and process Holliday junctions and branched DNA substrates. WRWYCR and its d stereoisomer, wrwycr, are bactericidal against both Gram-positive and Gram-negative bacteria, causing the accumulation of DNA breaks, chromosome segregation defects, and the filamentation of cells. DNA repair is a novel target of antibiotics. In the present study, we examined the ability of the peptides to inhibit the growth of Salmonella in mammalian cells. J774A.1 macrophage-like cells and murine peritoneal macrophages were infected with Salmonella enterica serovar Typhimurium and grown in the presence or absence of peptide. We found that peptide wrwycr reduced the number of Salmonella cells recovered after 24 h growth in J774A.1 cells by 100 to 1,000 times, depending on the multiplicity of infection. The peptide also inhibited Salmonella growth in peritoneal macrophages, and although higher doses were required, these were not toxic to the host cells. The apparent lower level of potency of the peptide paralleled the lower level of replication of Salmonella and the lower level of permeation of the peptide in the peritoneal macrophages than in the J774.1 cells. Treatment with peptide wrwycr elicited the SOS response in a significant fraction of the intracellular bacteria, as would be expected if the mechanism of bacterial killing was the same in pure culture and in host cells. These results represent a proof of principle of the antimicrobial activities of compounds that target DNA repair.

Peptide wrwycr Inhibits the Excision of Several Prophages and Traps Holliday Junctions inside Bacteria

Peptide inhibitors of phage lambda site-specific recombination were previously isolated by screening synthetic combinatorial peptide libraries. These inhibitors cause the accumulation of complexes between the recombinase and the Holliday junction intermediate of several highly divergent tyrosine recombinases. Peptide WRWYCR and its d-amino acid derivative bind to the center of protein-free junctions and prevent their resolution either by site-specific recombinases or by junction resolvases or helicases. With lesser affinity, the peptides also bind to branched DNA molecules that mimic replication forks. The peptides are bactericidal to both gram-positive and gram-negative bacteria, presumably because they can interfere with DNA repair and with chromosome dimer resolution by the XerC and XerD tyrosine recombinases. In order to test the correspondence between their mechanism in vivo and in vitro, we have tested and shown peptide wrwycr's ability to inhibit the excision of several prophages (lambda, P22, Gifsy-1, Gifsy-2, Fels-1, Fels-2) and to trap Holliday junction intermediates of phage lambda site-specific recombination in vivo. In addition, we found that the peptide inhibits replication of the Salmonella prophage Fels-1 while integrated in the chromosome. These findings further support the proposed mechanistic basis for the antimicrobial activity of the peptide and its use as a tool to dissect strand exchange-dependent DNA repair within cells.

Mycobacterium tuberculosis RuvA Induces Two Distinct Types of Structural Distortions between the Homologous and Heterologous Holliday Junctions

A central step in the process of homologous genetic recombination is the strand exchange between two homologous DNA molecules, leading to the formation of the Holliday junction intermediate. Several lines of evidence, both in vitro and in vivo, suggest a concerted role for the Escherichia coli RuvABC protein complex in the process of branch migration and the resolution of the Holliday junctions. A number of investigations have examined the role of RuvA protein in branch migration of the Holliday junction in conjunction with its natural cellular partner, RuvB. However, it remains unclear whether the RuvABC protein complex or its individual subunits function differently in the context of DNA repair and homologous recombination. In this study, we have specifically investigated the function of RuvA protein using Holliday junctions containing either homologous or heterologous arms. Our data show that Mycobacterium tuberculosis ruvA complements E. coli DeltaruvA mutants for survival to genotoxic stress caused by different DNA-damaging agents, and the purified RuvA protein binds HJ in preference to any other substrates. Strikingly, our analysis revealed two distinct types of structural distortions caused by M. tuberculosis RuvA between the homologous and heterologous Holliday junctions. We interpret these data as evidence that local distortion of base pairing in the arms of homologous Holliday junctions by RuvA might augment branch migration catalyzed by RuvB. The biological significance of two modes of structural distortion caused by M. tuberculosis RuvA and the implications for its role in DNA repair and homologous recombination are discussed.

The Arabidopsis BLAP75/Rmi1 Homologue Plays Crucial Roles in Meiotic Double-Strand Break Repair

In human cells and in Saccharomyces cerevisiae, BLAP75/Rmi1 acts together with BLM/Sgs1 and TopoIIIα/Top3 to maintain genome stability by limiting crossover (CO) formation in favour of NCO events, probably through the dissolution of double Holliday junction intermediates (dHJ). So far, very limited data is available on the involvement of these complexes in meiotic DNA repair. In this paper, we present the first meiotic study of a member of the BLAP75 family through characterisation of the Arabidopsis thaliana homologue. In A. thaliana blap75 mutants, meiotic recombination is initiated, and recombination progresses until the formation of bivalent-like structures, even in the absence of ZMM proteins. However, chromosome fragmentation can be detected as soon as metaphase I and is drastic at anaphase I, while no second meiotic division is observed. Using genetic and imunolocalisation studies, we showed that these defects reflect a role of A. thaliana BLAP75 in meiotic double-strand break (DSB) repair—that it acts after the invasion step mediated by RAD51 and associated proteins and that it is necessary to repair meiotic DSBs onto sister chromatids as well as onto the homologous chromosome. In conclusion, our results show for the first time that BLAP75/Rmi1 is a key protein of the meiotic homologous recombination machinery. In A. thaliana, we found that this protein is dispensable for homologous chromosome recognition and synapsis but necessary for the repair of meiotic DSBs. Furthermore, in the absence of BLAP75, bivalent formation can happen even in the absence of ZMM proteins, showing that in blap75 mutants, recombination intermediates exist that are stable enough to form bivalent structures, even when ZMM are absent.

Amino-terminal domain interactions of λ integrase on arm-type DNA

In contrast to the other tyrosine recombinase family members, integrase protein (Int) of bacteriophage λ has an additional amino-terminal domain that binds to “arm-type” DNA sequences distant from those involved in strand exchange. The homomeric interaction between neighboring amino-terminal domains of Int is contributed by R30-D71 salt-bridge in a non-equivalent manner on Holliday-junction intermediates. In this report, R30 and D71 residues were investigated in regard to Int’s cooperative binding to “arm-type” DNA and the attenuating function of “arm-type” DNA. The results suggest the electrostatic interaction between residues 30 and 71 is dependent on “arm-type” DNA and contributes the “selective” inhibition of catalytic activity of λ Int by “arm-type” DNA.

DNA double strand break repair and crossing over mediated by RuvABC resolvase and RecG translocase

DNA double-strand breaks threaten the stability of the genome, and yet are induced deliberately during meiosis in order to provoke homologous recombination and generate the crossovers needed to promote faithful chromosome transmission. Crossovers are secured via biased resolution of the double Holliday junction intermediates formed when both ends of the broken chromosome engage an intact homologue. To investigate whether the enzymes catalysing branch migration and resolution of Holliday junctions are directed to favour production of either crossover or noncrossover products, we engineered a genetic system based on DNA breakage induced by the I-SceI endonuclease to detect analogous exchanges in Escherichia coli where the enzymology of recombination is more fully understood. Analysis of the recombinants generated revealed approximately equal numbers of crossover and noncrossover products, regardless of whether repair is mediated via RecG, RuvABC, or the RusA resolvase. We conclude that there little or no control of crossing over at the level of Holliday junction resolution. Thus, if similar resolvases function during meiosis, additional factors must act to bias recombination in favour of crossover progeny.

Synthesis-dependent strand annealing in meiosis.

Recent studies led to the proposal that meiotic gene conversion can result after transient engagement of the donor chromatid and subsequent DNA synthesis-dependent strand annealing (SDSA). Double Holliday junction (dHJ) intermediates were previously proposed to form both reciprocal crossover recombinants (COs) and noncrossover recombinants (NCOs); however, dHJs are now thought to give rise mainly to COs, with SDSA forming most or all NCOs. To test this model in Saccharomyces cerevisiae, we constructed a random spore system in which it is possible to identify a subset of NCO recombinants that can readily be accounted for by SDSA, but not by dHJ-mediated recombination. The diagnostic class of recombinants is one in which two markers on opposite sides of a double-strand break site are converted, without conversion of an intervening heterologous insertion located on the donor chromatid. This diagnostic class represents 26% of selected NCO recombinants. Tetrad analysis using the same markers provided additional evidence that SDSA is a major pathway for NCO gene conversion in meiosis.

The Bacteroides NBU1 Integrase Performs a Homology-independent Strand Exchange to Form a Holliday Junction Intermediate

The Bacteroides mobilizable transposon NBU1 uses an integrase (IntN1) that is a tyrosine recombinase for its integration and excision from the host chromosome. Previously we showed that IntN1 makes 7-bp staggered cuts within the NBU1 att sites, and certain mismatches within the crossover region of the attN1 site (G(-2)C attN1) or the chromosomal target site (C(-3)G attBT1-1) enhanced the in vivo integration efficiency. Here we describe an in vitro integration system for NBU1. We used nicked substrates and a Holliday junction trapping peptide to show that NBU1 integration proceeds via formation of a Holliday junction intermediate that is formed by exchange of bottom strands. Some mismatches next to the first strand exchange site (in reactions with C(-3)G attBT1-1 or G(-2)C attN1 with their wild-type partner site) not only allowed formation of the Holliday junction intermediate but also increased the rate of recombinant formation. The second strand exchange appears to be homology-dependent. IntN1 is the only tyrosine recombinase known to catalyze a reaction that is more efficient in the presence of mismatches and where the first strand exchange is homology-independent. The possible mechanisms by which the mismatches stimulate recombination are discussed.

New Peptide Inhibitors of Type IB Topoisomerases: Similarities and Differences Vis-a-vis Inhibitors of Tyrosine Recombinases

Topoisomerases relieve topological tension in DNA by breaking and rejoining DNA phosphodiester bonds. Type IB topoisomerases such as vaccinia topoisomerase (vTopo) and human topoisomerase I are structurally and mechanistically similar to the tyrosine recombinase family of enzymes, which includes bacteriophage lambda Integrase (Int). Previously, our laboratory identified peptide inhibitors of Int from a synthetic peptide combinatorial library. The most potent of these peptides also inhibit vTopo. Here, we used the same mixture-based screening procedure to identify peptide inhibitors directly against vTopo using a plasmid relaxation assay. The two most potent new peptides identified, WYCRCK and KCCRCK, inhibit plasmid relaxation, DNA cleavage and Holliday junction (HJ) resolution mediated by vTopo. The peptides tested bind double-stranded DNA at high concentrations but do not appear to displace the enzyme from its DNA substrate. WYCRCK binds specifically to HJ and perturbs the central base-pairing. This peptide also accumulates HJ intermediates when it inhibits Int-mediated recombination, whereas KCCRCK does not. Interestingly, WYCRCK shares four amino acids with a peptide identified against Int, WRWYCR. The octapeptide WRWYCRCK, containing amino acids from both hexapeptides, is more potent than either against vTopo. All peptides are less potent against the type IA Escherichia coli topoisomerase I or against restriction endonucleases. Like the Int-inhibitory peptide WRWYCR, WYCRCK binds to HJs, and both inhibit junction resolution by vTopo. Our results suggest that the newly identified WYCRCK and peptide WRWYCR interact with a distorted DNA intermediate arising during vTopo-mediated catalysis, or interfere with specific interactions between vTopo and DNA.

Mrc1 and Srs2 are major actors in the regulation of spontaneous crossover

In vegetative cells, most recombination intermediates are metabolized without an association with a crossover (CO). The avoidance of COs allows for repair and prevents genomic rearrangements, potentially deleterious if the sequences involved are at ectopic locations. We have designed a system that permits to screen spontaneous intragenic recombination events in Saccharomyces cerevisiae and to investigate the CO outcome in different genetic contexts. We have analyzed the CO outcome in the absence of the Srs2 and Sgs1 helicases, DNA damage checkpoint proteins as well as in a mutant proliferating cell nuclear antigen (PCNA) and found that they all contribute to genome stability. Remarkably high effects on COs are mediated by srs2Delta, mrc1Delta and a pol30-RR mutation in PCNA. Our results support the view that Mrc1 plays a specific role in DNA replication, promoting the Srs2 recruitment to PCNA independently of checkpoint signaling. Srs2 would prevent formation of double Holliday junctions (dHJs) and thus CO formation. Sgs1 also negatively regulates CO formation but through a different process that resolves dHJs to yield non-CO products.

Involvement of Topoisomerase III in Telomere-Telomere Recombination

Telomere maintenance is required for chromosome stability, and telomeres are typically replicated by the action of telomerase. In both mammalian tumor and yeast cells that lack telomerase, telomeres are maintained by an alternative (ALT) recombination mechanism. In yeast, Sgs1p and its associated type IA topoisomerase, Top3p, may work coordinately in removing Holliday junction intermediates from a crossover-producing recombination pathway. Previous studies have also indicated that Sgs1 helicase acts in a telomere recombination pathway. Here we show that topoisomerase III is involved in telomere-telomere recombination. The recovery of telomere recombination-dependent survivors in a telomerase-minus yeast strain was dependent on Top3p catalytic activity. Moreover, the RIF1 and RIF2 genes are required for the establishment of TOP3/SGS1-dependent telomere-telomere recombination. In human Saos-2 ALT cells, human topoisomerase IIIalpha (hTOP3alpha) also contributes to telomere recombination. Strikingly, the telomerase activity is clearly enhanced in surviving si-hTOP3alpha Saos-2 ALT cells. Altogether, the present results suggest a potential role for hTOP3alpha in dissociating telomeric structures in telomerase-deficient cells, providing therapeutic implications in human tumors.

DNA repair, a novel antibacterial target: Holliday junction‐trapping peptides induce DNA damage and chromosome segregation defects

Holliday junction intermediates arise in several central pathways of DNA repair, replication fork restart, and site-specific recombination catalysed by tyrosine recombinases. Previously identified hexapeptide inhibitors of phage lambda integrase-mediated recombination block the resolution of Holliday junction intermediates in vitro and thereby inhibit recombination, but have no DNA cleavage activity themselves. The most potent peptides are specific for the branched DNA structure itself, as opposed to the integrase complex. Based on this activity, the peptides inhibit several unrelated Holliday junction-processing enzymes in vitro, including the RecG helicase and RuvABC junction resolvase complex. We have found that some of these hexapeptides are potent bactericidal antimicrobials, effective against both Gm+ and Gm- bacteria. Using epifluorescence microscopy and flow cytometry, we have characterized extensively the physiology of bacterial cells treated with these peptides. The hexapeptides cause DNA segregation abnormalities, filamentation and DNA damage. Damage caused by the peptides induces the SOS response, and is synergistic with damage caused by UV and mitomycin C. Our results are consistent with the model that the hexapeptides affect DNA targets that arise during recombination-dependent repair. We propose that the peptides trap intermediates in the repair of collapsed replication forks, preventing repair and resulting in bacterial death. Inhibition of DNA repair constitutes a novel target of antibiotic therapy. The peptides affect targets that arise in multiple pathways, and as expected, are quite resistant to the development of spontaneous antibiotic resistance.

P53 Monitors Replication Fork Regression by Binding to “Chickenfoot” Intermediates

The tumor suppressor protein, p53, utilizes multiple mechanisms to ensure faithful transmission of the genome including regulation of DNA replication, repair, and recombination. Monitoring these pathways may involve direct binding of p53 to the DNA intermediates of these processes. In this study, we generated templates resembling stalled replication forks and utilized electron microscopy to examine p53 interactions with these substrates. Our results show that p53 bound with high affinity to the junction of stalled forks, whereas two cancer-derived p53 mutants showed weak binding. Additionally, some of the templates were rearranged to form "chickenfoot" structures in the presence of p53. These were mostly formed due to p53 trapping intermediates of spontaneous fork regression; however, in a small population, the protein appeared to be promoting their formation. Collectively, these results demonstrate the importance of sequence-independent binding in p53-mediated maintenance of genomic integrity.

Determinants of Product Topology in a Hybrid Cre-Tn 3 Resolvase Site-specific Recombination System

Many natural DNA site-specific recombination systems achieve directionality and/or selectivity by making recombinants with a specific DNA topology. This property requires that the DNA architecture of the synapse and the mechanism of strand exchange are both under strict control. Previously we reported that Tn3 resolvase-mediated synapsis of the accessory binding sites from the Tn3 recombination site res can impose topological selectivity on Cre/loxP recombination. Here, we show that the topology of these reactions is profoundly affected by subtle changes in the hybrid recombination site les. Reversing the orientation of loxP relative to the res accessory sequence, or adding 4 bp to the DNA between loxP and the accessory sequence, can switch between two-noded and four-noded catenane products. By analysing Holliday junction intermediates, we show that the innate bias in the order of strand exchanges at loxP is maintained despite the changes in topology. We conclude that a specific synaptic structure formed by resolvase and the res accessory sequences permits Cre to align the adjoining loxP sites in several distinct ways, and that resolvase-mediated intertwining of the accessory sequences may be less than has been assumed previously.

Observing spontaneous branch migration of Holliday junctions one step at a time

Genetic recombination occurs between homologous DNA molecules via a four-way (Holliday) junction intermediate. This ancient and ubiquitous process is important for the repair of double-stranded breaks, the restart of stalled replication forks, and the creation of genetic diversity. Once formed, the four-way junction alone can undergo the stepwise exchange of base pairs known as spontaneous branch migration. Conventional ensemble assays, useful for finding average migration rates over long sequences, have been unable to examine the affect of sequence and structure on the migration process. Here, we present a single-molecule spontaneous branch migration assay with single-base pair resolution in a study of individual DNA junctions that can undergo one step of migration. Junctions exhibit markedly different dynamics of exchange between stacking conformers depending on the point of strand exchange, allowing the moment at which branch migration occurs to be detected. The free energy landscape of spontaneous branch migration is found to be highly nonuniform and governed by two types of sequence-dependent barriers, with unmediated local migration being up to 10 times more rapid than the previously deduced average rate.

MicroCommentary: Smarter than the average phage

SummaryThe seventh cholera pandemic emerged in the poorer nations of the world towards the end of the 20th century and continues to kill thousands of people per year. The causative agent of cholera, the Gram‐negative bacterium Vibrio cholera, is only pathogenic when it contains a lysogenic bacteriophage, CTXφ, that encodes the toxin responsible for inducing massive fluid loss from the human host. Site‐specific integration of CTXφ into chromosome I of V. cholera occurs at a site, dif, that is normally required for resolution of chromosome dimers generated by homologous recombination. An article in this issue of Molecular Microbiology reports the analysis of interactions between two host encoded recombinases, XerC and XerD, and the recombination sites involved in lysogeny. Surprisingly, recombination between the CTXφattP site and the chromosomal dif site requires additional recombinase binding sites, downstream from the positions of strand exchange, which might play an architectural role. The positions of strand cleavage also differ significantly between the two sites, suggesting a novel recombination mechanism that implicates additional host factors in resolution of the Holliday junction intermediate.