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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., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

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 Escherichia 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 dsDNA 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 ssDNA 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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Homologous recombination involves the formation of branched DNA molecules that may interfere with chromosome segregation. To resolve these persistent joint molecules, cells rely on the activation of structure-selective endonucleases (SSEs) during the late stages of the cell cycle. However, the premature activation of SSEs compromises genome integrity, due to untimely processing of replication and/or recombination intermediates. Here, we used a biochemical approach to show that the budding yeast SSEs Mus81 and Yen1 possess the ability to cleave the central recombination intermediate known as the displacement loop or D-loop. Moreover, we demonstrate that, consistently with previous genetic data, the simultaneous action of Mus81 and Yen1, followed by ligation, is sufficient to recreate the formation of a half-crossover precursor in vitro. Our results provide not only mechanistic explanation for the formation of a half-crossover, but also highlight the critical importance for precise regulation of these SSEs to prevent chromosomal rearrangements., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Holliday junction resolution is a crucial process in homologous recombination and DNA double-strand break repair. Complete Holliday junction resolution requires two stepwise incisions across the center of the junction, but the precise mechanism of metal ion-catalyzed Holliday junction cleavage remains elusive. Here, we perform a metal ion-triggered catalysis in crystals to investigate the mechanism of Holliday junction cleavage by MOC1. We capture the structures of MOC1 in complex with a nicked Holliday junction at various catalytic states, including the ground state, the one-metal ion binding state, and the two-metal ion binding state. Moreover, we also identify a third metal ion that may aid in the nucleophilic attack on the scissile phosphate. Further structural and biochemical analyses reveal a metal ion-mediated allosteric regulation between the two active sites, contributing to the enhancement of the second strand cleavage following the first strand cleavage, as well as the precise symmetric cleavage across the Holliday junction. Our work provides insights into the mechanism of metal ion-catalyzed Holliday junction resolution by MOC1, with implications for understanding how cells preserve genome integrity during the Holliday junction resolution phase., (© 2024. The Author(s).)

Purpose: 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., Experimental Design: 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., Results: 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., Conclusions: 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., (©2024 American Association for Cancer Research.)

The recently identified novel Holliday junction-resolving enzyme, termed Hjc_15-6, activity investigation results imply DNA cleavage by Hjc_15-6 in a manner that potentially enhances the molecular self-assembly that may be exploited for creating DNA-networks and nanostructures. The study also demonstrates Pwo DNA polymerase acting in combination with Hjc_15-6 capability to produce large amounts of DNA that transforms into large DNA-network structures even without DNA template and primers. Furthermore, it is demonstrated that Hjc_15-6 prefers Holliday junction oligonucleotides as compared to Y-shaped oligonucleotides as well as efficiently cleaves typical branched products from isothermal DNA amplification of both linear and circular DNA templates amplified by phi29-like DNA polymerase. The assembly of large DNA network structures was observed in real time, by transmission electron microscopy, on negative stained grids that were freshly prepared, and also on the same grids after incubation for 4 days under constant cooling. Hence, Hjc_15-6 is a promising molecular tool for efficient production of various DNA origamis that may be implemented for a wide range of applications such as within medical biomaterials, catalytic materials, molecular devices and biosensors., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Josefin Ahlqvist reports financial support was provided by European Union. There is no conflict of interest for any of the authors., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Congenital anomalies of the kidney and urinary tract (CAKUT) have been attributed to genetic and environmental factors. However, monogenic and copy number variations cannot sufficiently explain the cause of the majority of CAKUT cases. Multiple genes through various modes of inheritance may lead to CAKUT pathogenesis. We previously showed that Robo2 and Gen1 coregulated the germination of ureteral buds (UB), significantly increasing CAKUT incidence. Furthermore, MAPK/ERK pathway activation is the central mechanism of these two genes. Thus, we explored the effect of the MAPK/ERK inhibitor U0126 in the CAKUT phenotype in Robo2 PB/+ Gen1 PB/+ mice. Intraperitoneal injection of U0126 during pregnancy prevented the development of the CAKUT phenotype in Robo2 PB/+ Gen1 PB/+ mice. Additionally, a single dose of 30 mg/kg U0126 on day 10.5 embryos (E10.5) was most effective for reducing CAKUT incidence and ectopic UB outgrowth in Robo2 PB/+ Gen1 PB/+ mice. Furthermore, embryonic kidney mesenchymal levels of p-ERK were significantly decreased on day E11.5 after U0126 treatment, along with decreased cell proliferation index PHH3 and ETV5 expression. Collectively, Gen1 and Robo2 exacerbated the CAKUT phenotype in Robo2 PB/+ Gen1 PB/+ mice through the MAPK/ERK pathway, increasing proliferation and ectopic UB outgrowth., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Resolution of Holliday junctions is a critical intermediate step of homologous recombination in which junctions are processed by junction-resolving endonucleases. Although binding and cleavage are well understood, the question remains how the enzymes locate their substrate within long duplex DNA. Here we track fluorescent dimers of endonuclease I on DNA, presenting the complete single-molecule reaction trajectory for a junction-resolving enzyme finding and cleaving a Holliday junction. We show that the enzyme binds remotely to dsDNA and then undergoes 1D diffusion. Upon encountering a four-way junction, a catalytically-impaired mutant remains bound at that point. An active enzyme, however, cleaves the junction after a few seconds. Quantitative analysis provides a comprehensive description of the facilitated diffusion mechanism. We show that the eukaryotic junction-resolving enzyme GEN1 also undergoes facilitated diffusion on dsDNA until it becomes located at a junction, so that the general resolution trajectory is probably applicable to many junction resolving enzymes., (© 2022. The Author(s).)

During the development of humoral immunity, activated B lymphocytes undergo vigorous proliferative, transcriptional, metabolic, and DNA remodeling activities; hence, their genomes are constantly exposed to an onslaught of genotoxic agents and processes. Branched DNA intermediates generated during replication and recombinational repair pose genomic threats if left unresolved and so, they must be eliminated by structure-selective endonucleases to preserve the integrity of these DNA transactions for the faithful duplication and propagation of genetic information. To investigate the role of two such enzymes, GEN1 and MUS81, in B cell biology, we established B-cell conditional knockout mouse models and found that deletion of GEN1 and MUS81 in early B-cell precursors abrogates the development and maturation of B-lineage cells while the loss of these enzymes in mature B cells inhibit the generation of robust germinal centers. Upon activation, these double-null mature B lymphocytes fail to proliferate and survive while exhibiting transcriptional signatures of p53 signaling, apoptosis, and type I interferon response. Metaphase spreads of these endonuclease-deficient cells showed severe and diverse chromosomal abnormalities, including a preponderance of chromosome breaks, consistent with a defect in resolving recombination intermediates. These observations underscore the pivotal roles of GEN1 and MUS81 in safeguarding the genome to ensure the proper development and proliferation of B lymphocytes., Competing Interests: KF, LF, RS, WY, AM, WA, JP, JC No competing interests declared, (© 2022, Fernandez, Feeney et al.)

Holliday junction is the key homologous recombination intermediate, resolved by structure-selective endonucleases (SSEs). SLX1 is the most promiscuous SSE of the GIY-YIG nuclease superfamily. In fungi and animals, SLX1 nuclease activity relies on a non-enzymatic partner, SLX4, but no SLX1-SLX4 like complex has ever been characterized in plants. Plants exhibit specialized DNA repair and recombination machinery. Based on sequence similarity with the GIY-YIG nuclease domain of SLX1 proteins from fungi and animals, At-HIGLE was identified to be a possible SLX1 like nuclease from plants. Here, we elucidated the crystal structure of the At-HIGLE nuclease domain from Arabidopsis thaliana, establishing it as a member of the SLX1-lineage of the GIY-YIG superfamily with structural changes in DNA interacting regions. We show that At-HIGLE can process branched-DNA molecules without an SLX4 like protein. Unlike fungal SLX1, At-HIGLE exists as a catalytically active homodimer capable of generating two coordinated nicks during HJ resolution. Truncating the extended C-terminal region of At-HIGLE increases its catalytic activity, changes the nicking pattern, and monomerizes At-HIGLE. Overall, we elucidated the first structure of a plant SLX1-lineage protein, showed its HJ resolving activity independent of any regulatory protein, and identified an in-built novel regulatory mechanism engaging its C-terminal region., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

This study describes the production, characterization and structure determination of a novel Holliday junction-resolving enzyme. The enzyme, termed Hjc_15-6, is encoded in the genome of phage Tth15-6, which infects Thermus thermophilus. Hjc_15-6 was heterologously produced in Escherichia coli and high yields of soluble and biologically active recombinant enzyme were obtained in both complex and defined media. Amino-acid sequence and structure comparison suggested that the enzyme belongs to a group of enzymes classified as archaeal Holliday junction-resolving enzymes, which are typically divalent metal ion-binding dimers that are able to cleave X-shaped dsDNA-Holliday junctions (Hjs). The crystal structure of Hjc_15-6 was determined to 2.5 Å resolution using the selenomethionine single-wavelength anomalous dispersion method. To our knowledge, this is the first crystal structure of an Hj-resolving enzyme originating from a bacteriophage that can be classified as an archaeal type of Hj-resolving enzyme. As such, it represents a new fold for Hj-resolving enzymes from phages. Characterization of the structure of Hjc_15-6 suggests that it may form a dimer, or even a homodimer of dimers, and activity studies show endonuclease activity towards Hjs. Furthermore, based on sequence analysis it is proposed that Hjc_15-6 has a three-part catalytic motif corresponding to E-SD-EVK, and this motif may be common among other Hj-resolving enzymes originating from thermophilic bacteriophages., (open access.)

Congenital anomalies of the kidney and urinary tract (CAKUT) are a family of often-concurrent diseases with various anatomical spectra. Null-mutant Gen1 mice frequently develop multiple urinary phenotypes, most commonly duplex kidneys, and are ideal subjects for research on ectopic budding in CAKUT development. The upper and lower kidney poles of the Gen1 PB/PB mouse were examined by histology, immunofluorescence, and immunohistochemistry. The newborn Gen1 PB/PB mouse lower poles were significantly more hypoplastic than the corresponding upper poles, with significantly fewer glomeruli. On embryonic day 14.5, immediately before first urine formation, the upper pole kidney was already larger than the lower pole kidney. In vivo and in vitro, embryonic kidney upper poles had more ureteric buds than lower poles. Gen1 PB/PB embryos exhibited ectopic ureteric buds, usually near the original budding site, occasionally far away, or, rarely, derived from the primary budding site. Therefore, ectopia of the ureteric buds is the core of CAKUT formation. Further studies will be needed to investigate the regulatory roles of these genes in initial ureteric budding and subsequent ontogenesis during metanephros development., Competing Interests: Declaration of competing interest This study has no competing financial interests exist., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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 Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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 Δ pina was observed at low temperatures (cold sensitivity). When deletion of the HJ resolvase genes was combined, Δ pina Δ hjc and Δ pina Δ hje exhibited severe cold sensitivity. Δ hjm 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.

DNA lesions that impede fork progression cause replisome stalling and threaten genome stability. Bacillus subtilis RecA, at a lesion-containing gap, interacts with and facilitates DisA pausing at these branched intermediates. Paused DisA suppresses its synthesis of the essential c-di-AMP messenger. The RuvAB-RecU resolvasome branch migrates and resolves formed Holliday junctions (HJ). We show that DisA prevents DNA degradation. DisA, which interacts with RuvB, binds branched structures, and reduces the RuvAB DNA-dependent ATPase activity. DisA pre-bound to HJ DNA limits RuvAB and RecU activities, but such inhibition does not occur if the RuvAB- or RecU-HJ DNA complexes are pre-formed. RuvAB or RecU pre-bound to HJ DNA strongly inhibits DisA-mediated synthesis of c-di-AMP, and indirectly blocks cell proliferation. We propose that DisA limits RuvAB-mediated fork remodeling and RecU-mediated HJ cleavage to provide time for damage removal and replication restart in order to preserve genome integrity.

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 Mg 2+ 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/Mg 2+ 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Homologous recombination forms and resolves an entangled DNA Holliday Junction (HJ) crucial for achieving genetic reshuffling and genome repair. To maintain genomic integrity, specialized resolvase enzymes cleave the entangled DNA into two discrete DNA molecules. However, it is unclear how two similar stacking isomers are distinguished, and how a cognate sequence is found and recognized to achieve accurate recombination. We here use single-molecule fluorescence observation and cluster analysis to examine how prototypic bacterial resolvase RuvC singles out two of the four HJ strands and achieves sequence-specific cleavage. We find that RuvC first exploits, then constrains the dynamics of intrinsic HJ isomer exchange at a sampled branch position to direct cleavage toward the catalytically competent HJ conformation and sequence, thus controlling recombination output at minimal energetic cost. Our model of rapid DNA scanning followed by 'snap-locking' of a cognate sequence is strikingly consistent with the conformational proofreading of other DNA-modifying enzymes., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Holliday junctions are four-way DNA structures that may arise during meiotic recombination, double-strand break repair, or postreplicative repair by the reciprocal exchange of single strands between two DNA molecules. Given their ability to effectively bridge two sister chromatids or homologous chromosomes, cells have implemented various pathways to ensure their timely removal. One of them is the nucleolytic processing of the Holliday junctions by specialized structure-selective endonucleases termed resolvases, which sever the connection between the linked molecules. These Holliday junction resolvases are essential tools of the DNA damage repair machinery to ensure accurate chromosomal segregation, whose activities can be modulated by posttranslational modifications like phosphorylation. Here, we describe a protocol to purify S. cerevisiae Yen1 resolvase in two different phosphorylation states (high and low) and to set up a biochemical assay to compare their ability to process a synthetic, oligonucleotide-based Holliday junction structures.

MutSα and MutSβ play important roles in DNA mismatch repair and are linked to inheritable cancers and degenerative disorders. Here, we show that MSH2 and MSH3, the two components of MutSβ, bind SLX4 protein, a scaffold for the assembly of the SLX1-SLX4-MUS81-EME1-XPF-ERCC1 (SMX) trinuclease complex. SMX promotes the resolution of Holliday junctions (HJs), which are intermediates in homologous recombinational repair. We find that MutSβ binds HJs and stimulates their resolution by SLX1-SLX4 or SMX in reactions dependent upon direct interactions between MutSβ and SLX4. In contrast, MutSα does not stimulate HJ resolution. MSH3-depleted cells exhibit reduced sister chromatid exchanges and elevated levels of homologous recombination ultrafine bridges (HR-UFBs) at mitosis, consistent with defects in the processing of recombination intermediates. These results demonstrate a role for MutSβ in addition to its established role in the pathogenic expansion of CAG/CTG trinucleotide repeats, which is causative of myotonic dystrophy and Huntington's disease., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The Holliday junction, a four-way DNA structure, is an important intermediate of homologous recombination. Proper Holliday junction resolution is critical to complete the recombination process. In most bacterial cells, the Holliday junction cleavage is mainly performed by a specific endonuclease RuvC. Here, we describe the biochemical properties and the crystal structure of RuvC from an opportunistic pathogen, Pseudomonas aeruginosa (PaRuvC). PaRuvC specifically binds to the Holliday junction DNA and preferentially cleaves it at the consensus 5'-TTC-3'. PaRuvC uses Mg 2+ as the preferred divalent metal cofactor for Holliday junction cleavage and its optimum pH is 8.0-9.0. Elevated temperatures (37-60 °C) boost the catalytic activity, but temperatures higher than 53 °C reduce the protein stability. The crystal structure of PaRuvC determined at 2.4 Å and mutagenesis analysis reveal key residues involved in the dimer formation, substrate binding and catalysis. Our results are expected to provide useful information to combat antibiotic resistance of Pseudomonas aeruginosa by targeting its homologous recombination system., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Limited genetic factors were uncovered for the development of congenital anomalies of the kidney and urinary tract (CAKUT). We previously reported that a Holliday junction resolvase Gen1 was essential for early metanephric development in mice. This comprehensive follow-up study focused on the roles of Gen1 in late metanephric development. We found that Gen1 mutation impaired the late development of both kidney and urinary tract. In vivo and ex-vivo kidney primordia culture confirmed decreased ureteric bud branching in Gen1 mutants, which consequently caused hypoplasia. We also observed abnormal urinary tract development. Programmed apoptosis at the end of nephric duct disappeared in Gen1 mutants, which caused abnormal ureter-bladder connections, leading to vesicoureteral reflux (VUR) or ureterovesical junction obstruction (UVJO). Mechanistically, RNA-seq analysis proved that Gen1 mutation impaired the expression of multiple regulatory genes for the metanephric development, including Six2 . Taken together, our study provides more insight into the roles of Gen1 in the development of the kidney and urinary tract, which may have potential clinical significance in the treatment and/or prevention of CAKUT., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Extracellular DNA (eDNA) is a critical component of the extracellular matrix of bacterial biofilms that protects the resident bacteria from environmental hazards, which includes imparting significantly greater resistance to antibiotics and host immune effectors. eDNA is organized into a lattice-like structure, stabilized by the DNABII family of proteins, known to have high affinity and specificity for Holliday junctions (HJs). Accordingly, we demonstrated that the branched eDNA structures present within the biofilms formed by NTHI in the middle ear of the chinchilla in an experimental otitis media model, and in sputum samples recovered from cystic fibrosis patients that contain multiple mixed bacterial species, possess an HJ-like configuration. Next, we showed that the prototypic Escherichia coli HJ-specific DNA-binding protein RuvA could be functionally exchanged for DNABII proteins in the stabilization of biofilms formed by 3 diverse human pathogens, uropathogenic E. coli , nontypeable Haemophilus influenzae, and Staphylococcus epidermidis Importantly, while replacement of DNABII proteins within the NTHI biofilm matrix with RuvA was shown to retain similar mechanical properties when compared to the control NTHI biofilm structure, we also demonstrated that biofilm eDNA matrices stabilized by RuvA could be subsequently undermined upon addition of the HJ resolvase complex, RuvABC, which resulted in significant biofilm disruption. Collectively, our data suggested that nature has recapitulated a functional equivalent of the HJ recombination intermediate to maintain the structural integrity of bacterial biofilms., Competing Interests: The authors declare no competing interest.

The Holliday junction (HJ) is a key intermediate during homologous recombination and DNA double-strand break repair. Timely HJ resolution by resolvases is critical for maintaining genome stability. The mechanisms underlying sequence-specific substrate recognition and cleavage by resolvases remain elusive. The monokaryotic chloroplast 1 protein (MOC1) specifically cleaves four-way DNA junctions in a sequence-specific manner. Here, we report the crystal structures of MOC1 from Zea mays, alone or bound to HJ DNA. MOC1 uses a unique β-hairpin to embrace the DNA junction. A base-recognition motif specifically interacts with the junction center, inducing base flipping and pseudobase-pair formation at the strand-exchanging points. Structures of MOC1 bound to HJ and different metal ions support a two-metal ion catalysis mechanism. Further molecular dynamics simulations and biochemical analyses reveal a communication between specific substrate recognition and metal ion-dependent catalysis. Our study thus provides a mechanism for how a resolvase turns substrate specificity into catalytic efficiency.

Long Interspersed Elements (LINEs), also known as non-LTR retrotransposons, encode a multifunctional protein that reverse transcribes its mRNA into DNA at the site of insertion by target primed reverse transcription. The second half of the integration reaction remains very poorly understood. Second-strand DNA cleavage and second-strand DNA synthesis were investigated in vitro using purified components from a site-specific restriction-like endonuclease (RLE) bearing LINE. DNA structure was shown to be a critical component of second-strand DNA cleavage. A hitherto unknown and unexplored integration intermediate, an open '4-way' DNA junction, was recognized by the element protein and cleaved in a Holliday junction resolvase-like reaction. Cleavage of the 4-way junction resulted in a natural primer-template pairing used for second-strand DNA synthesis. A new model for RLE LINE integration is presented., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Resolution of branched DNA structures is pivotal for repair of stalled replication forks and meiotic recombination intermediates. The Yen1 nuclease cleaves both Holliday junctions and replication forks. We show that Yen1 interacts physically with Uls1, a suggested SUMO-targeted ubiquitin ligase that also contains a SWI/SNF-family ATPase-domain. Yen1 is SUMO-modified in its noncatalytic carboxyl terminus and DNA damage induces SUMOylation. SUMO-modification of Yen1 strengthens the interaction to Uls1, and mutations in SUMO interaction motifs in Uls1 weakens the interaction. However, Uls1 does not regulate the steady-state level of SUMO-modified Yen1 or chromatin-associated Yen1. In addition, SUMO-modification of Yen1 does not affect the catalytic activity in vitro. Consistent with a shared function for Uls1 and Yen1, mutations in both genes display similar phenotypes. Both uls1 and yen1 display negative genetic interactions with the alternative HJ-cleaving nuclease Mus81, manifested both in hypersensitivity to DNA damaging agents and in meiotic defects. Point mutations in ULS1 (uls1K975R and uls1C1330S, C1333S) predicted to inactivate the ATPase and ubiquitin ligase activities, respectively, are as defective as the null allele, indicating that both functions of Uls1 are essential. A micrococcal nuclease sequencing experiment showed that Uls1 had minimal effects on global nucleosome positioning/occupancy. Moreover, increased gene dosage of YEN1 partially alleviates the mus81 uls1 sensitivity to DNA damage. We suggest a preliminary model in which Uls1 acts in the same pathway as Yen1 to resolve branched DNA structures., Competing Interests: The authors have declared that no competing interests exist.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the leading cause of end-stage renal disease in children. Our group has discovered that Holliday Junction resolvase gene Gen1 is a potential candidate gene for CAKUT. Gen1 mutant mice showed CAKUT phenotypes similar to those observed in retinoic acid (RA)-deficient models. The expression of Raldh2, which encodes the key enzyme in RA synthesis, was reduced in Gen1 mutant metanephros through RNA sequencing. By real-time reverse transcription-PCR, the expression of both Raldh2 and downstream Ret was reduced in embryonic day (E) 11.5 Gen1 mutant ureters and E13.5 kidneys, and expression of RA receptor alpha was decreased in E13.5 Gen1 mutant ureters and kidneys. Further studies showed that all-trans retinoic acid (ATRA) rescued solitary kidney phenotype and improved ureteric branching; ATRA should be administered after ureteric budding to avoid increasing the incidence of ectopic budding in Gen1 mutants. Luciferase intensity of RA response element was lower in CHO-K1 cells transfected with Gen1 siRNA than in those transfected with scrambled RNA, and this inhibitory effect could be reversed by ATRA. These findings indicate that Gen1 mutation can result in renal malformation through RA signaling and Gen1-loss-induced CAKUT can be partly rescued by ATRA.

Holliday junction (HJ) resolution by resolving enzymes is essential for chromosome segregation and recombination-mediated DNA repair. HJs undergo two types of structural dynamics that determine the outcome of recombination: conformer exchange between two isoforms and branch migration. However, it is unknown how the preferred branch point and conformer are achieved between enzyme binding and HJ resolution given the extensive binding interactions seen in static crystal structures. Single-molecule fluorescence resonance energy transfer analysis of resolving enzymes from bacteriophages (T7 endonuclease I), bacteria (RuvC), fungi (GEN1) and humans (hMus81-Eme1) showed that both types of HJ dynamics still occur after enzyme binding. These dimeric enzymes use their multivalent interactions to achieve this, going through a partially dissociated intermediate in which the HJ undergoes nearly unencumbered dynamics. This evolutionarily conserved property of HJ resolving enzymes provides previously unappreciated insight on how junction resolution, conformer exchange and branch migration may be coordinated.

GEN1 is a member of the FEN/EXO family of structure-selective nucleases that cleave 1 nt 3' to a variety of branchpoints. For each, the H2TH motif binds a monovalent ion and plays an important role in binding one helical arm of the substrates. We investigate here the importance of this metal ion on substrate specificity and GEN1 structure. In the presence of K+ ions the substrate specificity is wider than in Na+, yet four-way junctions remain the preferred substrate. In a combination of K+ and Mg2+ second strand cleavage is accelerated 17-fold, ensuring bilateral cleavage of the junction. We have solved crystal structures of Chaetomium thermophilum GEN1 with Cs+, K+ and Na+ bound. With bound Cs+ the loop of the H2TH motif extends toward the active site so that D199 coordinates a Mg2+, buttressed by an interaction of the adjacent Y200. With the lighter ions bound the H2TH loop changes conformation and retracts away from the active site. We hypothesize this conformational change might play a role in second strand cleavage acceleration.

Topoisomerase 3α, a class I topoisomerase, consists of a TOPRIM domain, an active centre and a variable number of zinc-finger domains (ZFDs) at the C-terminus, in multicellular organisms. Whereas the functions of the TOPRIM domain and the active centre are known, the specific role of the ZFDs is still obscure. In contrast to mammals where a knockout of TOP3α leads to lethality, we found that CRISPR/Cas induced mutants in Arabidopsis are viable but show growth retardation and meiotic defects, which can be reversed by the expression of the complete protein. However, complementation with AtTOP3α missing either the TOPRIM-domain or carrying a mutation of the catalytic tyrosine of the active centre leads to embryo lethality. Surprisingly, this phenotype can be overcome by the simultaneous removal of the ZFDs from the protein. In combination with a mutation of the nuclease AtMUS81, the TOP3α knockout proved to be also embryo lethal. Here, expression of TOP3α without ZFDs, and in particular without the conserved ZFD T1, leads to only a partly complementation in root growth-in contrast to the complete protein, that restores root length to mus81-1 mutant level. Expressing the E. coli resolvase RusA in this background, which is able to process Holliday junction (HJ)-like recombination intermediates, we could rescue this root growth defect. Considering all these results, we conclude that the ZFD T1 is specifically required for targeting the topoisomerase activity to HJ like recombination intermediates to enable their processing. In the case of an inactivated enzyme, this leads to cell death due to the masking of these intermediates, hindering their resolution by MUS81., Competing Interests: The authors have declared that no competing interests exist.

During meiosis, crossover recombination promotes the establishment of physical connections between homologous chromosomes, enabling their bipolar segregation. To ensure that persistent recombination intermediates are disengaged prior to the completion of meiosis, the Yen1(GEN1) resolvase is strictly activated at the onset of anaphase II. Whether controlled activation of Yen1 is important for meiotic crossing-over is unknown. Here, we show that CDK-mediated phosphorylation of Yen1 averts its pervasive recruitment to recombination intermediates during prophase I. Yen1 mutants that are refractory to phosphorylation resolve DNA joint molecules prematurely and form crossovers independently of MutLγ, the central crossover resolvase during meiosis. Despite bypassing the requirement for MutLγ in joint molecule processing and promoting crossover-specific resolution, unrestrained Yen1 impairs the spatial distribution of crossover events, genome-wide. Thus, active suppression of Yen1 function, and by inference also of Mus81-Mms4(EME1) and Slx1-Slx4(BTBD12) resolvases, avoids precocious resolution of recombination intermediates to enable meiotic crossover patterning., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cells maintain a small arsenal of resolving functions to process and eliminate complex DNA intermediates that result as a consequence of homologous recombination and distressed replication. Ordinarily the homologous recombination system serves as a high-fidelity mechanism to restore the integrity of a damaged genome, but in the absence of the appropriate resolving function it can turn DNA intermediates resulting from replication stress into pathological forms that are toxic to cells. Here we have investigated how the nucleases Mus81 and Gen1 and the helicase Blm contribute to survival after DNA damage or replication stress in Ustilago maydis cells with crippled yet homologous recombination-proficient forms of Brh2, the BRCA2 ortholog and primary Rad51 mediator. We found collaboration among the factors. Notable were three findings. First, the ability of Gen1 to rescue hydroxyurea sensitivity of dysfunctional Blm requires the absence of Mus81. Second, the response of mutants defective in Blm and Gen1 to hydroxyurea challenge is markedly similar suggesting cooperation of these factors in the same pathway. Third, the repair proficiency of Brh2 mutant variants deleted of its N-terminal DNA binding region requires not only Rad52 but also Gen1 and Mus81. We suggest these factors comprise a subpathway for channeling repair when Brh2 is compromised in its interplay with DNA., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Congenital anomalies of the kidney and urinary tract (CAKUT) are among the most common developmental defects in humans. Despite of several known CAKUT-related loci ( HNF1B , PAX2 , EYA1 , etc.), the genetic etiology of CAKUT remains to be elucidated for most patients. In this study, we report that disruption of the Holliday Junction resolvase gene Gen1 leads to renal agenesis, duplex kidney, hydronephrosis, and vesicoureteral reflux (VUR) in mice. GEN1 interacts with SIX1 and enhances the transcriptional activity of SIX1/EYA1, a key regulatory complex of the GDNF morphogen. Gen1 mutation impairs Grem1 and Gdnf expression, resulting in excessive ureteric bud formation and defective ureteric bud branching during early kidney development. These results revealed an unidentified role of GEN1 in kidney development and suggested its contribution to CAKUT., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Break-induced replication (BIR) is a DNA double-strand break repair pathway that leads to genomic instabilities similar to those observed in cancer. BIR proceeds by a migrating bubble where asynchrony between leading and lagging strand synthesis leads to accumulation of long single-stranded DNA (ssDNA). It remains unknown how this ssDNA is prevented from unscheduled pairing with the template, which can lead to genomic instability. Here, we propose that uncontrolled Rad51 binding to this ssDNA promotes formation of toxic joint molecules that are counteracted by Srs2. First, Srs2 dislodges Rad51 from ssDNA preventing promiscuous strand invasions. Second, it dismantles toxic intermediates that have already formed. Rare survivors in the absence of Srs2 rely on structure-specific endonucleases, Mus81 and Yen1, that resolve toxic joint-molecules. Overall, we uncover a new feature of BIR and propose that tight control of ssDNA accumulated during this process is essential to prevent its channeling into toxic structures threatening cell viability.

Human GEN1 and yeast Yen1 are endonucleases with the ability to cleave Holliday junctions (HJs), which are proposed intermediates in recombination. In vivo, GEN1 and Yen1 function secondarily to Mus81, which has weak activity on intact HJs. We show that the genetic relationship is reversed in Drosophila, with Gen mutants having more severe defects than mus81 mutants. In vitro, DmGen, like HsGEN1, efficiently cleaves HJs, 5΄ flaps, splayed arms, and replication fork structures. We find that the cleavage rates for 5΄ flaps are significantly higher than those for HJs for both DmGen and HsGEN1, even in vast excess of enzyme over substrate. Kinetic studies suggest that the difference in cleavage rates results from a slow, rate-limiting conformational change prior to HJ cleavage: formation of a productive dimer on the HJ. Despite the stark difference in vivo that Drosophila uses Gen over Mus81 and humans use MUS81 over GEN1, we find the in vitro activities of DmGen and HsGEN1 to be strikingly similar. These findings suggest that simpler branched structures may be more important substrates for Gen orthologs in vivo, and highlight the utility of using the Drosophila model system to further understand these enzymes., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Holliday junctions, four-stranded DNA structures formed during homologous recombination, are disentangled by resolvases that have been found in prokaryotes and eukaryotes but not in plant organelles. Here, we identify monokaryotic chloroplast 1 (MOC1) as a Holliday junction resolvase in chloroplasts by analyzing a green alga Chlamydomonas reinhardtii mutant defective in chloroplast nucleoid (DNA-protein complex) segregation. MOC1 is structurally similar to a bacterial Holliday junction resolvase, resistance to ultraviolet (Ruv) C, and genetically conserved among green plants. Reduced or no expression of MOC1 in Arabidopsis thaliana leads to growth defects and aberrant chloroplast nucleoid segregation. In vitro biochemical analysis and high-speed atomic force microscopic analysis revealed that A. thaliana MOC 1 (AtMOC1) binds and cleaves the core of Holliday junctions symmetrically. MOC1 may mediate chloroplast nucleoid segregation in green plants by resolving Holliday junctions., (Copyright © 2017, American Association for the Advancement of Science.)

Holliday junction (HJ) is a hallmark intermediate in DNA recombination and must be processed by dissolution (for double HJ) or resolution to ensure genome stability. Although HJ resolvases have been identified in all domains of life, there is a long-standing effort to search in prokaryotes and eukarya for proteins promoting HJ migration. Here, we report the structural and functional characterization of a novel ATPase, Sulfolobus islandicusPilT N-terminal-domain-containing ATPase (SisPINA), encoded by the gene adjacent to the resolvase Hjc coding gene. PINA is conserved in archaea and vital for S. islandicus viability. Purified SisPINA forms hexameric rings in the crystalline state and in solution, similar to the HJ migration helicase RuvB in Gram-negative bacteria. Structural analysis suggests that ATP binding and hydrolysis cause conformational changes in SisPINA to drive branch migration. Further studies reveal that SisPINA interacts with SisHjc and coordinates HJ migration and cleavage., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Holliday junction-resolving enzymes are nucleases that are highly specific for the structure of the junction, to which they bind in dimeric form. Two symmetrically disposed cleavages are made. These are not simultaneous, but the second cleavage is accelerated relative to the first, so ensuring that bilateral cleavage occurs during the lifetime of the DNA-protein complex. In eukaryotic cells there are two known junction-resolving activities. GEN1 is similar to enzymes from lower organisms. A crystallographic structure of a fungal GEN1 bound to the product of resolution has been determined. These complexes are dimerized within the crystal lattice such that the strands of the products may be simply reconnected to form a junction. These structures suggest a trajectory for the resolution process., (© 2016 Federation of European Biochemical Societies.)

Holliday junction (HJ) resolving enzyme RecU is involved in DNA repair and recombination. We have determined the crystal structure of inactive mutant (D88N) of RecU from Bacillus subtilis in complex with a 12 base palindromic DNA fragment at a resolution of 3.2 Å. This structure shows the stalk region and the essential N-terminal region (NTR) previously unseen in our DNA unbound structure. The flexible nature of the NTR in solution was confirmed using SAXS. Thermofluor studies performed to assess the stability of RecU in complex with the arms of an HJ indicate that it confers stability. Further, we performed molecular dynamics (MD) simulations of wild type and an NTR deletion variant of RecU, with and without HJ. The NTR is observed to be highly flexible in simulations of the unbound RecU, in agreement with SAXS observations. These simulations revealed domain dynamics of RecU and their role in the formation of complex with HJ. The MD simulations also elucidate key roles of the NTR, stalk region, and breathing motion of RecU in the formation of the reactive state., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Genetic recombination provides an important mechanism for the repair of DNA double-strand breaks. Homologous pairing and strand exchange lead to the formation of DNA intermediates, in which sister chromatids or homologous chromosomes are covalently linked by four-way Holliday junctions (HJs). Depending on the type of recombination reaction that takes place, intermediates may have single or double HJs, and their resolution is essential for proper chromosome segregation. In mitotic cells, double HJs are primarily dissolved by the BLM helicase-TopoisomeraseIIIα-RMI1-RMI2 (BTR) complex, whereas single HJs (and double HJs that have escaped the attention of BTR) are resolved by structure-selective endonucleases known as HJ resolvases. These enzymes are ubiquitous in nature, because they are present in bacteriophage, bacteria, archaea, and simple and complex eukaryotes. The human HJ resolvase GEN1 is a member of the XPG/Rad2 family of 5'-flap endonucleases. Biochemical studies of GEN1 revealed that it cleaves synthetic DNA substrates containing a single HJ by a mechanism similar to that shown by the prototypic HJ resolvase, Escherichia coli RuvC protein, but it is unclear whether these substrates fully recapitulate the properties of recombination intermediates that arise within a physiological context. Here, we show that GEN1 efficiently cleaves both single and double HJs contained within large recombination intermediates. Moreover, we find that GEN1 exhibits a weak sequence preference for incision between two G residues that reside in a T-rich region of DNA. These results contrast with those obtained with RuvC, which exhibits a strict requirement for the consensus sequence 5'- A / T TT G / C -3'., Competing Interests: The authors declare no conflict of interest.

Cells have evolved mechanisms to protect, restart and repair perturbed replication forks, allowing full genome duplication, even under replication stress. Interrogating the interplay between nuclease-helicase Dna2 and Holliday junction (HJ) resolvase Yen1, we find the Dna2 helicase activity acts parallel to homologous recombination (HR) in promoting DNA replication and chromosome detachment at mitosis after replication fork stalling. Yen1, but not the HJ resolvases Slx1-Slx4 and Mus81-Mms4, safeguards chromosome segregation by removing replication intermediates that escape Dna2. Post-replicative DNA damage checkpoint activation in Dna2 helicase-defective cells causes terminal G2/M arrest by precluding Yen1-dependent repair, whose activation requires progression into anaphase. These findings explain the exquisite replication stress sensitivity of Dna2 helicase-defective cells, and identify a non-canonical role for Yen1 in the processing of replication intermediates that is distinct from HJ resolution. The involvement of Dna2 helicase activity in completing replication may have implications for DNA2-associated pathologies, including cancer and Seckel syndrome.

Resolution of the Holliday junction (HJ) is essential for homologous recombination and DNA repair. In Saccharomyces cerevisiae, HJ resolvase Yen1 and the Mus81-Mms4 complex are redundant in DNA damage repair. In cultured mammalian cells, such redundancy also exists between Yen1 ortholog GEN1 and the Mus81-Mms1 ortholog MUS81-EME1. In this report, we further tested if GEN1 and EME1 redundantly affect HJ-related physiological processes in mice. We found that combined homozygous mutations of Gen1 and Eme1 led to synthetic lethality during early embryonic stages. Homozygous Gen1 mutations did not cause DNA repair deficiency in mouse embryonic fibroblast (MEF) cells, but made heterozygous Eme1 mutant MEFs more sensitive to various DNA-damaging reagents. Gen1 mutations also reduced the meiotic recombination efficiency in Eme1 mutant mice. These results suggest that Gen1 and Eme1 play redundant roles in DNA repair and meiotic recombination in vivo.

Mitochondrial DNA is susceptible to the action of reactive oxygen species generated by the reactions of oxidative phosphorylation. Homologous recombination is one of the mechanisms providing integrity of the mitochondrial genome. Some proteins that take part in this process in budding yeast mitochondria have been identified. These include Abf2p, the major protein of the mt-nucleoid that specifically binds cruciform DNA, and Cce1p - Holliday junction resolvase. Here we show that Abf2p does not significantly affect either binding of Cce1p to branched DNA or rate and specificity of Holliday junction resolution. These data suggest the existence of an alternative homologous recombination pathway in yeast mitochondria.

To identify new proteins required for faithful meiotic chromosome segregation, we screened a Schizosaccharomyces pombe deletion mutant library and found that deletion of the dbl2 gene led to missegregation of chromosomes during meiosis. Analyses of both live and fixed cells showed that dbl2Δ mutant cells frequently failed to segregate homologous chromosomes to opposite poles during meiosis I. Removing Rec12 (Spo11 homolog) to eliminate meiotic DNA double-strand breaks (DSBs) suppressed the segregation defect in dbl2Δ cells, indicating that Dbl2 acts after the initiation of meiotic recombination. Analyses of DSBs and Holliday junctions revealed no significant defect in their formation or processing in dbl2Δ mutant cells, although some Rec12-dependent DNA joint molecules persisted late in meiosis. Failure to segregate chromosomes in the absence of Dbl2 correlated with persistent Rad51 foci, and deletion of rad51 or genes encoding Rad51 mediators also suppressed the segregation defect of dbl2Δ. Formation of foci of Fbh1, an F-box helicase that efficiently dismantles Rad51-DNA filaments, was impaired in dbl2Δ cells. Our results suggest that Dbl2 is a novel regulator of Fbh1 and thereby Rad51-dependent DSB repair required for proper meiotic chromosome segregation and viable sex cell formation. The wide conservation of these proteins suggests that our results apply to many species.

Poxviruses replicate their linear genomes by forming concatemers that must be resolved into monomeric units to produce new virions. A viral resolvase cleaves DNA four-way junctions extruded at the concatemer junctions to produce monomeric genomes. This cleavage reaction is required for viral replication, so the resolvase is an attractive target for small molecule inhibitors. To provide a platform for understanding resolvase mechanism and designing inhibitors, we have determined the crystal structure of the canarypox virus (CPV) resolvase. CPV resolvase is dimer of RNase H superfamily domains related to Escherichia coli RuvC, with an active site lined by highly conserved acidic residues that bind metal ions. There are several intriguing structural differences between resolvase and RuvC, and a model of the CPV resolvase·Holliday junction complex provides insights into the consequences of these differences, including a plausible explanation for the weak sequence specificity exhibited by the poxvirus enzymes. The model also explains why the poxvirus resolvases are more promiscuous than RuvC, cleaving a variety of branched, bulged, and flap-containing substrates. Based on the unique active site structure observed for CPV resolvase, we have carried out a series of experiments to test divalent ion usage and preferences. We find that the two resolvase metal binding sites have different preferences for Mg(2+) versus Mn(2+) Optimal resolvase activity is maintained with 5 μm Mn(2+) and 100 μm Mg(2+), concentrations that are well below those required for either metal alone. Together, our findings provide biochemical insights and structural models that will facilitate studying poxvirus replication and the search for efficient poxvirus inhibitors., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The Mycobacterium tuberculosis genome possesses homologues of the ruvC and yqgF genes that encode putative Holliday junction (HJ) resolvases. However, their gene expression profiles and enzymatic properties have not been experimentally defined. Here we report that expression of ruvC and yqgF is induced in response to DNA damage. Protein-DNA interaction assays with purified M. tuberculosis RuvC (MtRuvC) and YqgF (MtRuvX) revealed that both associate preferentially with HJ DNA, albeit with differing affinities. Although both MtRuvC and MtRuvX cleaved HJ DNA in vitro, the latter displayed robust HJ resolution activity by symmetrically related, paired incisions. MtRuvX showed a higher binding affinity for the HJ structure over other branched recombination and replication intermediates. An MtRuvX(D28N) mutation, eliminating one of the highly conserved catalytic residues in this class of endonucleases, dramatically reduced its ability to cleave HJ DNA. Furthermore, a unique cysteine (C38) fulfils a crucial role in HJ cleavage, consistent with disulfide-bond mediated dimerization being essential for MtRuvX activity. In contrast, E. coli YqgF is monomeric and exhibits no branched DNA binding or cleavage activity. These results fit with a functional modification of YqgF in M. tuberculosis so that it can act as a dimeric HJ resolvase analogous to that of RuvC., (© 2016 John Wiley & Sons Ltd.)

Structure-specific endonucleases act to repair potentially toxic structures produced by recombination and DNA replication, ensuring proper segregation of the genetic material to daughter cells during mitosis and meiosis. Arabidopsis thaliana has two putative homologs of the resolvase (structure-specific endonuclease): GEN1/Yen1. Knockout of resolvase genes GEN1 and SEND1, individually or together, has no detectable effect on growth, fertility, or sensitivity to DNA damage. However, combined absence of the endonucleases MUS81 and SEND1 results in severe developmental defects, spontaneous cell death, and genome instability. A similar effect is not seen in mus81 gen1 plants, which develop normally and are fertile. Absence of RAD51 does not rescue mus81 send1, pointing to roles of these proteins in DNA replication rather than DNA break repair. The enrichment of S-phase histone γ-H2AX foci and a striking loss of telomeric DNA in mus81 send1 further support this interpretation. SEND1 has at most a minor role in resolution of the Holliday junction but acts as an essential backup to MUS81 for resolution of toxic replication structures to ensure genome stability and to maintain telomere integrity., (© 2016 American Society of Plant Biologists. All rights reserved.)

We present the crystal structure of the junction-resolving enzyme GEN1 bound to DNA at 2.5 Å resolution. The structure of the GEN1 protein reveals it to have an elaborated FEN-XPG family fold that is modified for its role in four-way junction resolution. The functional unit in the crystal is a monomer of active GEN1 bound to the product of resolution cleavage, with an extensive DNA binding interface for both helical arms. Within the crystal lattice, a GEN1 dimer interface juxtaposes two products, whereby they can be reconnected into a four-way junction, the structure of which agrees with that determined in solution. The reconnection requires some opening of the DNA structure at the center, in agreement with permanganate probing and 2-aminopurine fluorescence. The structure shows that a relaxation of the DNA structure accompanies cleavage, suggesting how second-strand cleavage is accelerated to ensure productive resolution of the junction., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Holliday junctions (HJs) that physically link sister chromatids or homologous chromosomes are formed as intermediates during DNA repair by homologous recombination. Persistent recombination intermediates are acted upon by structure-selective endonucleases that are required for proper chromosome segregation at mitosis. Here, we have purified full-length human GEN1 protein and show that it promotes Holliday junction resolution by a mechanism that is analogous to that exhibited by the prototypic HJ resolvase E. coli RuvC. We find that GEN1 cleaves HJs by a nick and counter-nick mechanism involving dual co-ordinated incisions that lead to the formation of ligatable nicked duplex products. As observed with RuvC, cleavage of the first strand is rate limiting, while second strand cleavage is rapid. In contrast to RuvC, however, GEN1 is largely monomeric in solution, but dimerizes on the HJ. Using HJs containing non-cleavable phosphorothioate-containing linkages in one strand, we show that the two incisions can be uncoupled and that the first nick occurs upon GEN1 dimerization at the junction. These results indicate that the mechanism of HJ resolution is largely conserved from bacteria to man, despite a lack of sequence homology between the resolvases., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

DNA lesions caused by UV radiation are highly recombinogenic. In wild-type cells, the recombinogenic effect of UV partially reflects the processing of UV-induced pyrimidine dimers into DNA gaps or breaks by the enzymes of the nucleotide excision repair (NER) pathway. In this study, we show that unprocessed pyrimidine dimers also potently induce recombination between homologs. In NER-deficient rad14 diploid strains, we demonstrate that unexcised pyrimidine dimers stimulate crossovers, noncrossovers, and break-induced replication events. The same dose of UV is about six-fold more recombinogenic in a repair-deficient strain than in a repair-proficient strain. We also examined the roles of several genes involved in the processing of UV-induced damage in NER-deficient cells. We found that the resolvase Mus81p is required for most of the UV-induced inter-homolog recombination events. This requirement likely reflects the Mus81p-associated cleavage of dimer-blocked replication forks. The error-free post-replication repair pathway mediated by Mms2p suppresses dimer-induced recombination between homologs, possibly by channeling replication-blocking lesions into recombination between sister chromatids.