Holliday Junction Resolvase Research Articles

Holliday junction resolvase RuvC targets biofilm eDNA and confers plant resistance to vascular pathogens.

A biofilm lifestyle is critical for bacterial pathogens to colonize and protect themselves from host immunity and antimicrobial chemicals in plants and animals. The formation and regulation mechanisms of phytobacterial biofilm are still obscure. Here we found that the protein Ralstonia solanacearum resistance to ultraviolet C (RuvC) is highly abundant in biofilm and positively regulates pathogenicity by controlling systemic movement in tomato xylem. RuvC protein accumulates at the later stage of biofilm development and specifically targets Holliday junction (HJ)-like structures to disrupt the biofilm extracellular DNA (eDNA) lattice, thus facilitating biofilm dispersal. Recombinant RuvC protein can resolve extracellular HJ to prevent bacterial biofilm formation. Heterologous expression of R. solanacearum or Xanthomonas oryzae pv. oryzae RuvC with plant secretion signal in tomato or rice confers resistance to bacterial wilt or bacterial blight disease, respectively. Plant chloroplast-localized HJ resolvase monokaryotic chloroplast 1 (MOC1), which shares structural similarity with bacterial RuvC, shows a strong inhibitory effect on bacterial biofilm formation. Relocalization of SlMOC1 to apoplast in tomato roots leads to increased resistance to bacterial wilt. Our novel finding reveals a critical pathogenesis mechanism of R. solanacearum and provides an efficient biotechnology strategy to improve plant resistance to bacterial vascular disease.

Mycobacterium smegmatis putative Holliday junction resolvases RuvC and RuvX play complementary roles in the processing of branched DNA structures

In eubacteria, Holliday junction (HJ) resolvases (HJRs) are crucial for faithful segregation of newly replicated chromosomes, homologous recombination and repair of stalled/collapsed DNA replication forks. However, compared with the Escherichia coli HJRs, little is known about their orthologs in mycobacterial species. A genome-wide analysis of Mycobacterium smegmatis identified two genes encoding putative HJRs, namely RuvC (MsRuvC) and RuvX (MsRuvX); but whether they play redundant, overlapping, or distinct roles remains unknown. Here, we reveal that MsRuvC exists as a homodimer while MsRuvX as a monomer in solution, and both showed high-binding affinity for branched DNAs compared with unbranched DNA species. Interestingly, the DNA cleavage specificities of MsRuvC and MsRuvX were found to be mutually exclusive: the former efficiently promotes HJ resolution, in a manner analogous to the E. coli RuvC, but does not cleave other branched DNA species; whereas the latter is a versatile DNase capable of cleaving a variety of branched DNA structures, including 3' and 5' flap DNA, splayed-arm DNA and double-stranded DNA with 3' and 5' overhangs but lacks the HJ resolution activity. Point mutations in the RNase H-like domains of MsRuvC and MsRuvX pinpointed critical residues required for their DNA cleavage activities, and also demonstrated uncoupling between DNA-binding and DNA cleavage activities. Unexpectedly, we found robust evidence that MsRuvX possesses a double-strand/single-strand junction-specific endonuclease and single-stranded DNA exonucleolytic activities. Combined, our findings highlight that the RuvC and RuvX DNases play distinct complementary, and not redundant, roles in the processing of branched DNA structures in M. smegmatis.

Germline Mutations of Holliday Junction Resolvase Genes in Multiple Primary Malignancies Involving Lung Cancer Lead to PARP Inhibitor Sensitization.

The incidence of multiple primary malignancies (MPM) involving lung cancer has increased in recent decades. There is an urgent need to clarify the genetic profile of such patients and explore more efficacious therapy for them. Peripheral blood samples from MPM involving patients with lung cancer were assessed by whole-exome sequencing (WES), and the identified variants were referenced for pathogenicity using the public available database. Pathway enrichment analysis of mutated genes was performed to identify the most relevant pathway. Next, the effects of mutations in relevant pathway on function and response to targeted drugs were verified by in vitro and in vivo experiments. Germline exomes of 71 patients diagnosed with MPM involving lung cancer were sequenced. Pathway enrichment analysis shows that the homologous recombination repair (HRR) pathway has the strongest correlation. Moreover, HRR genes, especially key Holliday junction resolvases (HJR) genes (GEN1, BLM, SXL4, and RMI1), were most frequently mutated, unlike the status in the samples from patients with lung cancer only. Next, we identified a total of seven mutations in HJR genes led to homologous recombination DNA repair deficiency and rendered lung cancer cells sensitive to PARP inhibitor treatment, both in vitro and in vivo. This is the first study to map the profile of germline mutations in patients with MPM involving lung cancer. This study may shed light on early prevention and novel targeted therapies for MPM involving patients with lung cancer with HJR mutations.

Classical and novel properties of Holliday junction resolvase SynRuvC from Synechocystis sp. PCC6803.

Cyanobacteria, which have a photoautotrophic lifestyle, are threatened by ultraviolet solar rays and the reactive oxygen species generated during photosynthesis. They can adapt to environmental conditions primarily because of their DNA damage response and repair mechanisms, notably an efficient homologous recombination repair system. However, research on double-strand break (DSB) repair pathways, including the Holliday junction (HJ) resolution process, in Synechocystis sp. PCC6803 is limited. Here, we report that SynRuvC from cyanobacteria Synechocystis sp. PCC6803 has classical HJ resolution activity. We investigated the structural specificity, sequence preference, and biochemical properties of SynRuvC. SynRuvC strongly preferred Mn2+ as a cofactor, and its cleavage site predominantly resides within the 5'-TG↓(G/A)-3' sequence. Interestingly, novel flap endonuclease and replication fork intermediate cleavage activities of SynRuvC were also determined, which distinguish it from other reported RuvCs. To explore the effect of SynRuvC on cell viability, we constructed a knockdown mutant and an overexpression strain of Synechocystis sp. PCC6803 (synruvCKD and synruvCOE) and assessed their survival under a variety of conditions. Knockdown of synruvC increased the sensitivity of cells to MMS, HU, and H2O2. The findings suggest that a novel RuvC family HJ resolvase SynRuvC is important in a variety of DNA repair processes and stress resistance in Synechocystis sp. PCC6803.

Method to generate Holliday junction recombination intermediates via RecA-mediated four-strand exchange

DNA molecules that contain single Holliday junctions have served as model substrates to investigate the pathway in which homologous recombination intermediates are processed. However, the preparation of DNA containing Holliday junctions in high yield remains a challenge. In this work, we used a nicking endonuclease to generate gapped DNA, from which α-structured DNA or figure-8 DNA were created via RecA-mediated reactions. The resulting DNA molecules were found to serve as good substrates for Holliday junction resolvases. The simplified method negates the requirement for radioactive labelling of DNA, making the generation of Holliday junction DNA more accessible to non-experts.

The RuvABC Holliday Junction Processing System Is Not Required for IS26-Mediated Targeted Conservative Cointegrate Formation.

The insertion sequence IS26 plays a key role in the spread of antibiotic resistance genes in Gram-negative bacteria. IS26 and members of the IS26 family are able to use two distinct mechanisms to form cointegrates made up of two DNA molecules linked via directly oriented copies of the IS. The well-known copy-in (formerly replicative) reaction occurs at very low frequency, and the more recently discovered targeted conservative reaction, which joins two molecules that already include an IS, is substantially more efficient. Experimental evidence has indicated that, in the targeted conservative mode, the action of Tnp26, the IS26 transposase, is required only at one end. How the Holliday junction (HJ) intermediate generated by the Tnp26-catalyzed single-strand transfer is processed to form the cointegrate is not known. We recently proposed that branch migration and resolution via the RuvABC system may be needed to process the HJ; here, we have tested this hypothesis. In reactions between a wild-type and a mutant IS26, the presence of mismatched bases near one IS end impeded the use of that end. In addition, evidence of gene conversion, potentially consistent with branch migration, was detected in some of the cointegrates formed. However, the targeted conservative reaction occurred in strains that lacked the recG, ruvA, or ruvC genes. As the RuvC HJ resolvase is not required for targeted conservative cointegrate formation, the HJ intermediate formed by the action of Tnp26 must be resolved by an alternate route. IMPORTANCE In Gram-negative bacteria, the contribution of IS26 to the spread of antibiotic resistance and other genes that provide cells with an advantage under specific conditions far exceeds that of any other known insertion sequence. This is likely due to the unique mechanistic features of IS26 action, particularly its propensity to cause deletions of adjacent DNA segments and the ability of IS26 to use two distinct reaction modes for cointegrate formation. The high frequency of the unique targeted conservative reaction mode that occurs when both participating molecules include an IS26 is also key. Insights into the detailed mechanism of this reaction will help to shed light on how IS26 contributes to the diversification of the bacterial and plasmid genomes it is found in. These insights will apply more broadly to other members of the IS26 family found in Gram-positive as well as Gram-negative pathogens.

Structural and Functional Characterization of the Holliday Junction Resolvase RuvC from Deinococcus radiodurans.

Holliday junctions (HJs) are four-way DNA structures, which are an important intermediate in the process of homologous recombination. In most bacteria, HJs are cleaved by specific nucleases called RuvC resolvases at the end of homologous recombination. Deinococcus radiodurans is an extraordinary radiation-resistant bacterium and is known as an ideal model organism for elucidating DNA repair processes. Here, we described the biochemical properties and the crystal structure of RuvC from D. radiodurans (DrRuvC). DrRuvC exhibited an RNase H fold that belonged to the retroviral integrase family. Among many DNA substrates, DrRuvC specifically bound to HJ DNA and cleaved it. In particular, Mn2+ was the preferred bivalent metal co-factor for HJ cleavage, whereas high concentrations of Mg2+ inhibited the binding of DrRuvC to HJ. In addition, DrRuvC was crystallized and the crystals diffracted to 1.6 Å. The crystal structure of DrRuvC revealed essential amino acid sites for cleavage and binding activities, indicating that DrRuvC was a typical resolvase with a characteristic choice for metal co-factor.

SLX4 dampens MutSα-dependent mismatch repair.

The tumour suppressor SLX4 plays multiple roles in the maintenance of genome stability, acting as a scaffold for structure-specific endonucleases and other DNA repair proteins. It directly interacts with the mismatch repair (MMR) protein MSH2 but the significance of this interaction remained unknown until recent findings showing that MutSβ (MSH2-MSH3) stimulates in vitro the SLX4-dependent Holliday junction resolvase activity. Here, we characterize the mode of interaction between SLX4 and MSH2, which relies on an MSH2-interacting peptide (SHIP box) that drives interaction of SLX4 with both MutSβ and MutSα (MSH2-MSH6). While we show that this MSH2 binding domain is dispensable for the well-established role of SLX4 in interstrand crosslink repair, we find that it mediates inhibition of MutSα-dependent MMR by SLX4, unravelling an unanticipated function of SLX4.

First Characterization of a Hafnia Phage Reveals Extraordinarily Large Burst Size and Unusual Plaque Polymorphism.

A unique lytic phage infecting Hafnia paralvei was isolated and identified. Hafnia phage Ca belongs to the family Autographiviridae, possessing an icosahedral head with a diameter of 55 nm and a short non-contractile tail. Unusually, the burst size of Hafnia phage Ca of 10,292 ± 1,097 plaque-forming units (PFUs)/cell is much larger than other dsDNA phages reported before. Compared to the genome of the related phage, Hafnia phage Ca genome contains extra genes including DNA mimic ocr, dGTP triphosphohydrolase inhibitor, endonuclease, endonuclease VII, and HNH homing endonuclease gene. Extraordinarily, the phage developed different sizes of plaques when a single plaque was picked out and inoculated on a double-layer Luria broth agar plate with its host. Furthermore, varied packaging tightness for the tails of Hafnia phage Ca was observed (tail length: 4.35–45.92 nm). Most of the tails appeared to be like a cone with appendages, some were dot-like, bun-like, table tennis racket handle-like, and ponytail-like. Although the complete genome of Hafnia phage Ca is 40,286 bp, an incomplete genome with a deletion of a 397-bp fragment, containing one ORF predicted as HNH homing endonuclease gene (HEG), was also found by high throughput sequencing. Most of the genome of the virus particles in large plaques is complete (>98%), while most of the genome of the virus particles in small plaques is incomplete (>98%), and the abundance of both of them in medium-sized plaques is similar (complete, 40%; incomplete, 60%). In an experiment to see if the phage could be protective to brocade carps intramuscularly injected with H. paralvei LY-23 and phage Ca, the protection rate of Hafnia phage Ca to brocade carp (Cyprinus aka Koi) against H. paralvei was 33.38% (0.01 < p < 0.05). This study highlights some new insights into the peculiar biological and genomic characteristics of phage.

Genetic Study of Four Candidate Holliday Junction Processing Proteins in the Thermophilic Crenarchaeon Sulfolobus acidocaldarius.

Homologous recombination (HR) is thought to be important for the repair of stalled replication forks in hyperthermophilic archaea. Previous biochemical studies identified two branch migration helicases (Hjm and PINA) and two Holliday junction (HJ) resolvases (Hjc and Hje) as HJ-processing proteins; however, due to the lack of genetic evidence, it is still unclear whether these proteins are actually involved in HR in vivo and how their functional relation is associated with the process. To address the above questions, we constructed hjc-, hje-, hjm-, and pina single-knockout strains and double-knockout strains of the thermophilic crenarchaeon Sulfolobus acidocaldarius and characterized the mutant phenotypes. Notably, we succeeded in isolating the hjm- and/or pina-deleted strains, suggesting that the functions of Hjm and PINA are not essential for cellular growth in this archaeon, as they were previously thought to be essential. Growth retardation in Δpina was observed at low temperatures (cold sensitivity). When deletion of the HJ resolvase genes was combined, Δpina Δhjc and Δpina Δhje exhibited severe cold sensitivity. Δhjm exhibited severe sensitivity to interstrand crosslinkers, suggesting that Hjm is involved in repairing stalled replication forks, as previously demonstrated in euryarchaea. Our findings suggest that the function of PINA and HJ resolvases is functionally related at lower temperatures to support robust cellular growth, and Hjm is important for the repair of stalled replication forks in vivo.

Structural and Mechanistic Insight into CRISPR-Cas9 Inhibition by Anti-CRISPR Protein AcrIIC4Hpa

Phages, plasmids, and other mobile genetic elements express inhibitors of CRISPR-Cas immune systems, known as anti-CRISPR proteins, to protect themselves from targeted destruction. These anti-CRISPR proteins have been shown to function through very diverse mechanisms. In this work we investigate the activity of an anti-CRISPR isolated from a prophage in Haemophilus parainfluenzae that blocks CRISPR-Cas9 DNA cleavage activity. We determine the three-dimensional crystal structure of AcrIIC4Hpa and show that it binds to the Cas9 Recognition Domain. This binding does not prevent the Cas9-anti-CRISPR complex from interacting with target DNA but does inhibit DNA cleavage. AcrIIC4Hpa likely acts by blocking the conformational changes that allow the HNH and RuvC endonuclease domains to contact the DNA sites to be nicked.

Canonical and novel non-canonical activities of the Holliday junction resolvase Yen1.

Yen1 and GEN1 are members of the Rad2/XPG family of nucleases that were identified as the first canonical nuclear Holliday junction (HJ) resolvases in budding yeast and humans due to their ability to introduce two symmetric, coordinated incisions on opposite strands of the HJ, yielding nicked DNA products that could be readily ligated. While GEN1 has been extensively characterized in vitro, much less is known about the biochemistry of Yen1. Here, we have performed the first in-depth characterization of purified Yen1. We confirmed that Yen1 resembles GEN1 in many aspects, including range of substrates targeted, position of most incisions they produce or the increase in the first incision rate by assembly of a dimer on a HJ, despite minor differences. However, we demonstrate that Yen1 is endowed with additional nuclease activities, like a nick-specific 5′-3′ exonuclease or HJ arm-chopping that could apparently blur its classification as a canonical HJ resolvase. Despite this, we show that Yen1 fulfils the requirements of a canonical HJ resolvase and hypothesize that its wider array of nuclease activities might contribute to its function in the removal of persistent recombination or replication intermediates.

The Cdc14 Phosphatase Controls Resolution of Recombination Intermediates and Crossover Formation during Meiosis.

Meiotic defects derived from incorrect DNA repair during gametogenesis can lead to mutations, aneuploidies and infertility. The coordinated resolution of meiotic recombination intermediates is required for crossover formation, ultimately necessary for the accurate completion of both rounds of chromosome segregation. Numerous master kinases orchestrate the correct assembly and activity of the repair machinery. Although much less is known, the reversal of phosphorylation events in meiosis must also be key to coordinate the timing and functionality of repair enzymes. Cdc14 is a crucial phosphatase required for the dephosphorylation of multiple CDK1 targets in many eukaryotes. Mutations that inactivate this phosphatase lead to meiotic failure, but until now it was unknown if Cdc14 plays a direct role in meiotic recombination. Here, we show that the elimination of Cdc14 leads to severe defects in the processing and resolution of recombination intermediates, causing a drastic depletion in crossovers when other repair pathways are compromised. We also show that Cdc14 is required for the correct activity and localization of the Holliday Junction resolvase Yen1/GEN1. We reveal that Cdc14 regulates Yen1 activity from meiosis I onwards, and this function is essential for crossover resolution in the absence of other repair pathways. We also demonstrate that Cdc14 and Yen1 are required to safeguard sister chromatid segregation during the second meiotic division, a late action that is independent of the earlier role in crossover formation. Thus, this work uncovers previously undescribed functions of the evolutionary conserved Cdc14 phosphatase in the regulation of meiotic recombination.

Dissecting the RecA-(In)dependent Response to Mitomycin C in Mycobacterium tuberculosis Using Transcriptional Profiling and Proteomics Analyses.

Mycobacteria exploit at least two independent global systems in response to DNA damage: the LexA/RecA-dependent SOS response and the PafBC-regulated pathway. Intracellular pathogens, such as Mycobacterium tuberculosis, are exposed to oxidative and nitrosative stress during the course of infection while residing inside host macrophages. The current understanding of RecA-independent responses to DNA damage is based on the saprophytic model of Mycobacterium smegmatis, a free-living and nonpathogenic mycobacterium. The aim of the present study was to identify elements of RecA-independent responses to DNA damage in pathogenic intracellular mycobacteria. With the help of global transcriptional profiling, we were able to dissect RecA-dependent and RecA-independent pathways. We profiled the DNA damage responses of an M. tuberculosis strain lacking the recA gene, a strain with an undetectable level of the PafBC regulatory system, and a strain with both systems tuned down simultaneously. RNA-Seq profiling was correlated with the evaluation of cell survival in response to DNA damage to estimate the relevance of each system to the overall sensitivity to genotoxic agents. We also carried out whole-cell proteomics analysis of the M. tuberculosis strains in response to mitomycin C. This approach highlighted that LexA, a well-defined key element of the SOS system, is proteolytically inactivated during RecA-dependent DNA repair, which we found to be transcriptionally repressed in response to DNA-damaging agents in the absence of RecA. Proteomics profiling revealed that AlkB was significantly overproduced in the ΔrecA pafBCCRISPRi/dCas9 strain and that Holliday junction resolvase RuvX was a DNA damage response factor that was significantly upregulated regardless of the presence of functional RecA and PafBC systems, thus falling into a third category of DNA damage factors: RecA- and PafBC-independent. While invisible to the mass spectrometer, the genes encoding alkA, dnaB, and dnaE2 were significantly overexpressed in the ΔrecA pafBCCRISPRi/dCas9 strain at the transcript level.

Novel insights into ATP-Stimulated Cleavage of branched DNA and RNA Substrates through Structure-Guided Studies of the Holliday Junction Resolvase RuvX

Much of our understanding of the homologous recombination (HR) machinery hinges on studies using Escherichia coli as a model organism. Interestingly enough, studies on the HR machinery in different bacterial species casts doubt on the universality of the E. coli paradigm. The human pathogen Mycobacterium tuberculosis encodes two Holliday junction (HJ)‐resolvase paralogues, namely RuvC and RuvX; however, insights into their structural features and functional relevance is still limited. Here, we report on structure-guided functional studies of the M. tuberculosis RuvX HJ resolvase (MtRuvX). The crystalline MtRuvX is a dimer in the asymmetric unit, and each monomer has a RNAse H fold vis-à-vis RuvC-like nucleases. Interestingly, MtRuvX also contains some unique features, including the residues essential for ATP binding/coordination of Mg2+ ions. Indeed, MtRuvX exhibited an intrinsic, robust ATPase activity, which was further accentuated by DNA cofactors. Structure-guided substitutions of single residues at the ATP binding/Mg2+coordination sites while markedly attenuating the ATPase activity completely abrogated HJ cleavage, indicating an unanticipated relationship between ATP hydrolysis and DNA cleavage. However, the affinity of ATPase-deficient mutants for the HJ was not impaired. Contrary to RuvC, MtRuvX exhibits relaxed substrate specificity, cleaving a variety of branched DNA/RNA substrates. Notably, ATP hydrolysis plays a regulatory role, rendering MtRuvX from a canonical HJ resolvase to a DNA/RNA non-sequence specific endonuclease, indicating a link between HJ resolvase and nucleic acid metabolism. These findings provide novel insights into the structure and dual-functional activities of MtRuvX, and suggest that it may play an important role in DNA/RNA metabolism.

DNA polymerase theta suppresses mitotic crossing over.

Polymerase theta-mediated end joining (TMEJ) is a chromosome break repair pathway that is able to rescue the lethality associated with the loss of proteins involved in early steps in homologous recombination (e.g., BRCA1/2). This is due to the ability of polymerase theta (Pol θ) to use resected, 3' single stranded DNA tails to repair chromosome breaks. These resected DNA tails are also the starting substrate for homologous recombination. However, it remains unknown if TMEJ can compensate for the loss of proteins involved in more downstream steps during homologous recombination. Here we show that the Holliday junction resolvases SLX4 and GEN1 are required for viability in the absence of Pol θ in Drosophila melanogaster, and lack of all three proteins results in high levels of apoptosis. Flies deficient in Pol θ and SLX4 are extremely sensitive to DNA damaging agents, and mammalian cells require either Pol θ or SLX4 to survive. Our results suggest that TMEJ and Holliday junction formation/resolution share a common DNA substrate, likely a homologous recombination intermediate, that when left unrepaired leads to cell death. One major consequence of Holliday junction resolution by SLX4 and GEN1 is cancer-causing loss of heterozygosity due to mitotic crossing over. We measured mitotic crossovers in flies after a Cas9-induced chromosome break, and observed that this mutagenic form of repair is increased in the absence of Pol θ. This demonstrates that TMEJ can function upstream of the Holiday junction resolvases to protect cells from loss of heterozygosity. Our work argues that Pol θ can thus compensate for the loss of the Holliday junction resolvases by using homologous recombination intermediates, suppressing mitotic crossing over and preserving the genomic stability of cells.

DNA recombination and repair in Wolbachia: RecA and related proteins.

Wolbachia is an obligate intracellular bacterium that has undergone extensive genomic streamlining in its arthropod and nematode hosts. Because the gene encoding the bacterial DNA recombination/repair protein RecA is not essential in Escherichia coli, abundant expression of this protein in a mosquito cell line persistently infected with Wolbachia strain wStri was unexpected. However, RecA's role in the lytic cycle of bacteriophage lambda provides an explanation for retention of recA in strains known to encode lambda-like WO prophages. To examine DNA recombination/repair capacities in Wolbachia, a systematic examination of RecA and related proteins in complete or nearly complete Wolbachia genomes from supergroups A, B, C, D, E, F, J and S was undertaken. Genes encoding proteins including RecA, RecF, RecO, RecR, RecG and Holliday junction resolvases RuvA, RuvB and RuvC are uniformly absent from Wolbachia in supergroup C and have reduced representation in supergroups D and J, suggesting that recombination and repair activities are compromised in nematode-associated Wolbachia, relative to strains that infect arthropods. An exception is filarial Wolbachia strain wMhie, assigned to supergroup F, which occurs in a nematode host from a poikilothermic lizard. Genes encoding LexA and error-prone polymerases are absent from all Wolbachia genomes, suggesting that the SOS functions induced by RecA-mediated activation of LexA do not occur, despite retention of genes encoding a few proteins that respond to LexA induction in E. coli. Three independent E. coli accessions converge on a single Wolbachia UvrD helicase, which interacts with mismatch repair proteins MutS and MutL, encoded in nearly all Wolbachia genomes. With the exception of MutL, which has been mapped to a eukaryotic association module in Phage WO, proteins involved in recombination/repair are uniformly represented by single protein annotations. Putative phage-encoded MutL proteins are restricted to Wolbachia supergroups A and B and show higher amino acid identity than chromosomally encoded MutL orthologs. This analysis underscores differences between nematode and arthropod-associated Wolbachia and describes aspects of DNA metabolism that potentially impact development of procedures for transformation and genetic manipulation of Wolbachia.

Recombinational repair in the absence of holliday junction resolvases in E. coli.

Cells possess two major DNA damage tolerance pathways that allow them to duplicate their genomes despite the presence of replication blocking lesions: translesion synthesis (TLS) and daughter strand gap repair (DSGR). The TLS pathway involves specialized DNA polymerases that are able to synthesize past DNA lesions while DSGR relies on Recombinational Repair (RR). At least two mechanisms are associated with RR: Homologous Recombination (HR) and RecA Mediated Excision Repair (RAMER). While HR and RAMER both depend on RecFOR and RecA, only the HR mechanism should involve Holliday Junctions (HJs) resolvase reactions. In this study we investigated the role of HJ resolvases, RuvC, TopIII and RusA on the balance between RAMER and HR in E. coli MG1655 derivatives. Using UV survival measurements, we first clearly establish that, in this genetic background, topB and ruvC define two distinct pathways of HJ resolution. We observed that a recA mutant is much more sensitive to UV than the ruvC topB double mutant which is deficient in HR because of its failure to resolve HJs. This difference is independent of RAMER, the SOS system, RusA, and the three TLS DNA polymerases, and may be accounted for by Double Strand Break repair mechanisms such as Synthesis Dependent Strand Annealing, Single Strand Annealing, or Break Induced Replication, which are independent of HJ resolvases. We then used a plasmid-based assay, in which RR is triggered by a single blocking lesion present on a plasmid molecule, to establish that while HR requires topB, ruvC or rusA, RAMER is independent of these genes and, as expected, requires a functional UvrABC excinuclease. Surprisingly, analysis of the RR events in a strain devoid of HJ resolvases reveals that the UvrABC dependent repair of the single lesion present on the plasmid molecule can generate an excision track potentially extending to dozens of nucleotides.

Genetic evidence for the involvement of mismatch repair proteins, PMS2 and MLH3, in a late step of homologous recombination

Homologous recombination (HR) repairs DNA double-strand breaks using intact homologous sequences as template DNA. Broken DNA and intact homologous sequences form joint molecules (JMs), including Holliday junctions (HJs), as HR intermediates. HJs are resolved to form crossover and noncrossover products. A mismatch repair factor, MLH3 endonuclease, produces the majority of crossovers during meiotic HR, but it remains elusive whether mismatch repair factors promote HR in nonmeiotic cells. We disrupted genes encoding the MLH3 and PMS2 endonucleases in the human B cell line, TK6, generating null MLH3-/- and PMS2-/- mutant cells. We also inserted point mutations into the endonuclease motif of MLH3 and PMS2 genes, generating endonuclease death MLH3DN/DN and PMS2EK/EK cells. MLH3-/- and MLH3DN/DN cells showed a very similar phenotype, a 2.5-fold decrease in the frequency of heteroallelic HR-dependent repair of restriction enzyme-induced double-strand breaks. PMS2-/- and PMS2EK/EK cells showed a phenotype very similar to that of the MLH3 mutants. These data indicate that MLH3 and PMS2 promote HR as an endonuclease. The MLH3DN/DN and PMS2EK/EK mutations had an additive effect on the heteroallelic HR. MLH3DN/DN/PMS2EK/EK cells showed normal kinetics of γ-irradiation-induced Rad51 foci but a significant delay in the resolution of Rad51 foci and a 3-fold decrease in the number of cisplatin-induced sister chromatid exchanges. The ectopic expression of the Gen1 HJ re-solvase partially reversed the defective heteroallelic HR of MLH3DN/DN/PMS2EK/EK cells. Taken together, we propose that MLH3 and PMS2 promote HR as endonucleases, most likely by processing JMs in mammalian somatic cells.

Holliday Junction Resolvase MOC1 Maintains Plastid and Mitochondrial Genome Integrity in Algae and Bryophytes.

When DNA double-strand breaks occur, four-stranded DNA structures called Holliday junctions (HJs) form during homologous recombination. Because HJs connect homologous DNA by a covalent link, resolution of HJ is crucial to terminate homologous recombination and segregate the pair of DNA molecules faithfully. We recently identified Monokaryotic Chloroplast1 (MOC1) as a plastid DNA HJ resolvase in algae and plants. Although Cruciform cutting endonuclease1 (CCE1) was identified as a mitochondrial DNA HJ resolvase in yeasts, homologs or other mitochondrial HJ resolvases have not been identified in other eukaryotes. Here, we demonstrate that MOC1 depletion in the green alga Chlamydomonas reinhardtii and the moss Physcomitrella patens induced ectopic recombination between short dispersed repeats in ptDNA. In addition, MOC1 depletion disorganized thylakoid membranes in plastids. In some land plant lineages, such as the moss P. patens, a liverwort and a fern, MOC1 dually targeted to plastids and mitochondria. Moreover, mitochondrial targeting of MOC1 was also predicted in charophyte algae and some land plant species. Besides causing instability of plastid DNA, MOC1 depletion in P. patens induced short dispersed repeat-mediated ectopic recombination in mitochondrial DNA and disorganized cristae in mitochondria. Similar phenotypes in plastids and mitochondria were previously observed in mutants of plastid-targeted (RECA2) and mitochondrion-targeted (RECA1) recombinases, respectively. These results suggest that MOC1 functions in the double-strand break repair in which a recombinase generates HJs and MOC1 resolves HJs in mitochondria of some lineages of algae and plants as well as in plastids in algae and plants.