Expression and Localization of GEN1 in Mouse Mammary Epithelial Cells

GEN1, a Holliday junction resolvase, is involved in homologous repair of DNA double strand break and in maintaining centrosome integrity. Although GEN1 mutants have been reported in breast cancer patients and cell lines, little is currently known about the functions of GEN1 in the development and oncogenic transformation of mammary gland. In the present study, we demonstrate that GEN1 expression is correlated with mammary epithelial cell proliferation, differentiation in various physiological stages as well as casein. By immunofluorescence analysis, the centrosomal association of GEN1 is confirmed in mammary epithelial cells. Additionally, GEN1 is likely involved in DNA damage response of breast cancer cell lines. These results suggest that GEN1 may play an important role in the development of mammary gland; its response upon DNA damage indicates that GEN1 gene alteration may contribute to breast cancer formation.     

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

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

The Structure-Specific Endonucleases MUS81 and SEND1 Are Essential for Telomere Stability in Arabidopsis

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.     

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

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

Mus81-Eme1 are essential components of a Holliday junction resolvase.

Mus81, a fission yeast protein related to the XPF subunit of ERCC1-XPF nucleotide excision repair endonuclease, is essential for meiosis and important for coping with stalled replication forks. These processes require resolution of X-shaped DNA structures known as Holliday junctions. We report that Mus81 and an associated protein Eme1 are components of an endonuclease that resolves Holliday junctions into linear duplex products. Mus81 and Eme1 are required during meiosis at a late step of meiotic recombination. The mus81 meiotic defect is rescued by expression of a bacterial Holliday junction resolvase. These findings constitute strong evidence that Mus81 and Eme1 are subunits of a nuclear Holliday junction resolvase.     

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.     

Partial suppression of the fission yeast rqh1(-) phenotype by expression of a bacterial Holliday junction resolvase

A key stage during homologous recombination is the processing of the Holliday junction, which determines the outcome of the recombination reaction. To dissect the pathways of Holliday junction processing in a eukaryote, we have targeted an Escherichia coli Holliday junction resolvase to the nuclei of fission yeast recombination-deficient mutants and analysed their phenotypes. The resolvase partially complements the UV and hydroxyurea hypersensitivity and associated aberrant mitoses of an rqh1(-) mutant. Rqh1 is a member of the RecQ subfamily of DNA helicases that control recombination particularly during S-phase. Significantly, overexpression of the resolvase in wild-type cells partly mimics the loss of viability, hyper-recombination and 'cut' phenotype of an rqh1(-) mutant. These results indicate that Holliday junctions form in wild-type cells that are normally removed in a non-recombinogenic way, possibly by Rqh1 catalysing their reverse branch migration. We propose that in the absence of Rqh1, replication fork arrest results in the accumulation of Holliday junctions, which can either impede sister chromatid segregation or lead to the formation of recombinants through Holliday junction resolution.     

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.     

The human Holliday junction resolvase GEN1 rescues the meiotic phenotype of a Schizosaccharomyces pombe mus81 mutant

A key step in meiotic recombination involves the nucleolytic resolution of Holliday junctions to generate crossovers. Although the enzyme that performs this function in human cells is presently unknown, recent studies led to the identification of the XPG-family endonuclease GEN1 that promotes Holliday junction resolution in vitro, suggesting that it may perform a related function in vivo. Here, we show that ectopic expression of GEN1 in fission yeast mus81Δ strains results in Holliday junction resolution and crossover formation during meiosis.     

Resolving branched DNA intermediates with structure-specific nucleases during replication in eukaryotes

Genome duplication requires that replication forks track the entire length of every chromosome. When complications occur, homologous recombination-mediated repair supports replication fork movement and recovery. This leads to physical connections between the nascent sister chromatids in the form of Holliday junctions and other branched DNA intermediates. A key role in the removal of these recombination intermediates falls to structure-specific nucleases such as the Holliday junction resolvase RuvC in Escherichia coli. RuvC is also known to cut branched DNA intermediates that originate directly from blocked replication forks, targeting them for origin-independent replication restart. In eukaryotes, multiple structure-specific nucleases, including Mus81–Mms4/MUS81–EME1, Yen1/GEN1, and Slx1–Slx4/SLX1–SLX4 (FANCP) have been implicated in the resolution of branched DNA intermediates. It is becoming increasingly clear that, as a group, they reflect the dual function of RuvC in cleaving recombination intermediates and failing replication forks to assist the DNA replication process.     

Promiscuous and lineage-specific roles of cell cycle regulators in haematopoiesis

DNA double strand breaks are efficiently repaired by homologous recombination. One of the last steps of this process is resolution of Holliday junctions that are formed at the sites of genetic exchange between homologous DNA. Although various resolvases with Holliday junctions processing activity have been identified in bacteriophages, bacteria and archaebacteria, eukaryotic resolvases have been elusive. Recent biochemical evidence has revealed that RAD51C and XRCC3, members of the RAD51-like protein family, are involved in Holliday junction resolution in mammalian cells. However, purified recombinant RAD51C and XRCC3 proteins have not shown any Holliday junction resolution activity. In addition, these proteins did not reveal the presence of a nuclease domain, which raises doubts about their ability to function as a resolvase. Furthermore, oocytes from infertile Rad51C mutant mice exhibit precocious separation of sister chromatids at metaphase II, a phenotype that reflects a defect in sister chromatid cohesion, not a lack of Holliday junction resolution. Here we discuss a model to explain how a Holliday junction resolution defect can lead to sister chromatid separation in mouse oocytes. We also describe other recent in vitro and in vivo evidence supporting a late role for RAD51C in homologous recombination in mammalian cells, which is likely to be resolution of the Holliday junction.     

DNA Binding and Cleavage by the Fowlpox Virus Resolvase

The first steps of poxvirus DNA synthesis yield concatemeric arrays of covalently linked genomes. The virus-encoded Holliday junction resolvase is required to process concatemers into unit-length genomes for packaging. Previous studies of the vaccinia virus resolvase have been problematic due to poor protein solubility. We found that fowlpox virus resolvase was much more tractable. Fowlpox resolvase formed complexes with a variety of branched DNA substrates, but not linear DNA, and had the highest affinity for a Holliday junction substrate, illustrating a previously unappreciated affinity for Holliday junctions over other substrates. The cleavage activity was monitored in fixed time assays, showing that, as with vaccinia resolvase, the fowlpox enzyme could cleave a wide array of branched DNA substrates. Single turnover kinetic analysis revealed the Holliday junction substrate was cleaved 90-fold faster than a splayed duplex substrate containing a single to double strand transition. Multiple turnover kinetic analysis, however, showed that the cleavage step was not limiting for the full reaction cycle. Cleavage by resolvase was also tightly coupled at symmetrical positions across the junction, and coupling required the complete Holliday junction structure. Last, we found that cleavage of an extruded cruciform yielded a product, which after treatment with ligase, had the properties expected for covalently closed DNA hairpin ends, as is seen for poxvirus genome monomers. These findings provide a tractable poxvirus resolvase usable for the development of small molecule inhibitors.Poxvirus DNA replication is proposed to proceed by a “rolling hairpin” mechanism to yield linear concatemers, in which genomes are arranged in mostly head-to-head and tail-to tail orientation (Fig. 1, step 1) (1). The terminal sequences at each junction form an inverted repeat, which can be extruded to form a cruciform structure (step 2) (2). Cleavage of the resulting Holliday junctions on each end frees the monomer genome from the concatemer (step 3). The nicks left behind after resolution of the Holliday junction can then be ligated, yielding the hairpin DNA ends characteristic of poxviruses (step 4).Open in a separate windowFIGURE 1.Role of poxvirus resolvase during viral replication. Black lines indicate single DNA strands. Half-arrows indicate repeated sequences. Small arrows indicate resolvase cleavage sites. 1) Poxvirus genome replication yields concatemers; 2) inverted repeat sequences at concatemer junctions extrude to form cruciform structures; 3) Holliday junction cleavage by resolvase at cruciform structures yields unit-length genomes with preserved hairpin ends; 4) ligase seals nicks to yield mature genome monomers.The vaccinia virus resolvase gene, A22R, was first recognized in bioinformatic surveys to encode a member of the RNase H superfamily of polynucleotide phosphotransfer enzymes (3). These enzymes catalyze attack of a hydroxyl group on a phosphodiester bond, thereby supporting a variety of nuclease or DNA joining reactions. Garcia et al. (3) purified recombinant vaccinia resolvase and showed that it displayed cleaving activity on model Holliday junctions. They also generated a conditional A22R recombinant vaccinia virus and showed that in the absence of A22R expression, vaccinia failed to replicate and concatemer junctions accumulated, indicating that A22 resolvase indeed is required for concatemer resolution in vivo (4). Subsequent studies by Garcia et al. (5) and Culyba et al. (6) showed that vaccinia resolvase had little sequence specificity, and that cleavage yielded a 3′-hydroxyl group suitable for subsequent DNA ligation. Culyba et al. (7) also showed that several further branched DNA molecules could be cleaved by vaccinia resolvase, establishing that the enzyme could potentially process a variety of branched DNA forms expected to arise during recombination or replication, suggesting possible additional roles for poxvirus resolvase.Progress in studying poxvirus resolvase has been limited by the poor solubility of the purified vaccinia protein. For example, in Garcia et al. (5), the vaccinia resolvase was fused to maltose-binding protein to improve solubility, but consequently the properties of the maltose-binding protein portion of the fusion must be considered in interpreting the results. Pilot studies from our laboratory showed that the insolubility and low activity of the vaccinia virus resolvase precluded its use in high-throughput screens for inhibitors (data not shown).In an effort to identify a more tractable poxvirus resolvase protein, we attempted to clone four other poxvirus resolvase genes and purify the gene products after overexpression in bacteria. We found that the fowlpox resolvase was much more soluble and active than the others tested. Analysis of cleavage revealed that a wide range of branched DNA forms were substrates, paralleling results with vaccinia resolvase and establishing that these activities are a conserved property of poxvirus resolvases. Binding analysis on these same DNA forms also revealed a strict specificity for branched DNA, with the highest affinity binding for the Holliday junction, suggesting that DNA binding specificity is the major discriminatory mechanism for DNA cleavage activity. Kinetic analysis was feasible with fowlpox resolvase, allowing us to show that the first-order rate constant for strand cleavage under single turnover conditions is 90-fold greater for a Holliday junction substrate than for a splayed duplex substrate. However, this rate constant was not limiting for the Holliday junction under multiple turnover conditions, where the rate of strand cleavage is 1.9-fold slower for the Holliday junction than for the splayed duplex. Last, we show that fowlpox resolvase cleavage at Holliday junctions is coupled, so that nicking on one strand also promoted nicking on the strand located across the junction from it. These studies indicate that fowlpox resolvase is well suited to in vitro analysis and suggests approaches to high-throughput screening for resolvase inhibitors.     

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

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

Functional overlap between the structure-specific nucleases Yen1 and Mus81-Mms4 for DNA-damage repair in S. cerevisiae

In eukaryotic cells, multiple DNA repair mechanisms respond to a wide variety of DNA lesions. Homologous recombination-dependent repair provides a pathway for dealing with DNA double-strand breaks and replication fork demise. A key step in this process is the resolution of recombination intermediates such as Holliday junctions (HJs). Recently, nucleases from yeast (Yen1) and human cells (GEN1) were identified that can resolve HJ intermediates, in a manner analogous to the E. coli HJ resolvase RuvC. Here, we have analyzed the role of Yen1 in DNA repair in S. cerevisiae, and show that while yen1Δ mutants are repair-proficient, yen1Δ mus81Δ double mutants are exquisitely sensitive to a variety of DNA-damaging agents that disturb replication fork progression. This phenotype is dependent upon RAD52, indicating that toxic recombination intermediates accumulate in the absence of Yen1 and Mus81. After MMS treatment, yen1Δ mus81Δ double mutants arrest with a G2 DNA content and unsegregated chromosomes. These findings indicate that Yen1 can act upon recombination/repair intermediates that arise in MUS81-defective cells following replication fork damage.     

Critical Functions of Rpa3/Ssb3 in S-Phase DNA Damage Responses in Fission Yeast

Replication Protein A (RPA) is a heterotrimeric, single-stranded DNA (ssDNA)–binding complex required for DNA replication and repair, homologous recombination, DNA damage checkpoint signaling, and telomere maintenance. Whilst the larger RPA subunits, Rpa1 and Rpa2, have essential interactions with ssDNA, the molecular functions of the smallest subunit Rpa3 are unknown. Here, we investigate the Rpa3 ortholog Ssb3 in Schizosaccharomyces pombe and find that it is dispensable for cell viability, checkpoint signaling, RPA foci formation, and meiosis. However, increased spontaneous Rad11^Rpa1 and Rad22^Rad52 nuclear foci in ssb3Δ cells indicate genome maintenance defects. Moreover, Ssb3 is required for resistance to genotoxins that disrupt DNA replication. Genetic interaction studies indicate that Ssb3 has a close functional relationship with the Mms1-Mms22 protein complex, which is required for survival after DNA damage in S-phase, and with the mitotic functions of Mus81-Eme1 Holliday junction resolvase that is required for recovery from replication fork collapse. From these studies we propose that Ssb3 plays a critical role in mediating RPA functions that are required for repair or tolerance of DNA lesions in S-phase. Rpa3 orthologs in humans and other species may have a similar function.     

Processing of joint molecule intermediates by structure-selective endonucleases during homologous recombination in eukaryotes

Homologous recombination is required for maintaining genomic integrity by functioning in high-fidelity repair of DNA double-strand breaks and other complex lesions, replication fork support, and meiotic chromosome segregation. Joint DNA molecules are key intermediates in recombination and their differential processing determines whether the genetic outcome is a crossover or non-crossover event. The Holliday model of recombination highlights the resolution of four-way DNA joint molecules, termed Holliday junctions, and the bacterial Holliday junction resolvase RuvC set the paradigm for the mechanism of crossover formation. In eukaryotes, much effort has been invested in identifying the eukaryotic equivalent of bacterial RuvC, leading to the discovery of a number of DNA endonucleases, including Mus81?CMms4/EME1, Slx1?CSlx4/BTBD12/MUS312, XPF?CERCC1, and Yen1/GEN1. These nucleases exert different selectivity for various DNA joint molecules, including Holliday junctions. Their mutant phenotypes and distinct species-specific characteristics expose a surprisingly complex system of joint molecule processing. In an attempt to reconcile the biochemical and genetic data, we propose that nicked junctions constitute important in vivo recombination intermediates whose processing determines the efficiency and outcome (crossover/non-crossover) of homologous recombination.     

Branch migration and Holliday junction resolution catalyzed by activities from mammalian cells

During homologous recombination, DNA strand exchange leads to Holliday junction formation. The movement, or branch migration, of this junction along DNA extends the length of the heteroduplex joint. In prokaryotes, branch migration and Holliday junction resolution are catalyzed by the RuvA and RuvB proteins, which form a complex with RuvC resolvase to form a "resolvasome". Mammalian cell-free extracts have now been fractionated to reveal analogous activities. An ATP-dependent branch migration activity, which migrates junctions through >2700 bp, cofractionates with the Holliday junction resolvase during several chromatographic steps. Together, the two activities promote concerted branch migration/resolution reactions similar to those catalyzed by E. coli RuvABC, highlighting the preservation of this essential pathway in recombination and DNA repair from prokaryotes to mammals.     

The structure of Escherichia coli RusA endonuclease reveals a new Holliday junction DNA binding fold

Holliday junction resolution performed by a variety of structure-specific endonucleases is a key step in DNA recombination and repair. It is believed that all resolvases carry out their reaction chemistries in a similar fashion, utilizing a divalent cation to facilitate the hydrolysis of the phosphodiester backbone of the DNA, but their architecture varies. To date, with the exception of bacteriophage T4 endonuclease VII, each of the known resolvase enzyme structures has been categorized into one of two families: the integrases and the nucleases. We have now determined the structure of the Escherichia coli RusA Holliday junction resolvase, which reveals a fourth structural class for these enzymes. The structure suggests that dimer formation is essential for Mg(2+) cation binding and hence catalysis and that like the other resolvases, RusA distorts its Holliday junction target upon binding. Key residues identified by mutagenesis experiments are well positioned to interact with the DNA.     

Role of RAD51C and XRCC3 in genetic recombination and DNA repair

In germ line cells, recombination is required for gene reassortment and proper chromosome segregation at meiosis, whereas in somatic cells it provides an important mechanism for the repair of DNA double-strand breaks. Five proteins (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) that share homology with RAD51 recombinase and are known as the RAD51 paralogs are important for recombinational repair, as paralog-defective cell lines exhibit spontaneous chromosomal aberrations, defective DNA repair, and reduced gene targeting. The paralogs form two distinct protein complexes, RAD51B-RAD51C-RAD51D-XRCC2 and RAD51C-XRCC3, but their precise cellular roles remain unknown. Here, we show that, like MLH1, RAD51C localized to mouse meiotic chromosomes at pachytene/diplotene. Using immunoprecipitation and gel filtration analyses, we found that Holliday junction resolvase activity associated tightly and co-eluted with the 80-kDa RAD51C-XRCC3 complex. Taken together, these data indicate that the RAD51C-XRCC3-associated Holliday junction resolvase complex associates with crossovers and may play an essential role in the resolution of recombination intermediates prior to chromosome segregation.