Heteroduplex DNA in meiotic recombination in Drosophila mei-9 mutants

Meiotic recombination gives rise to crossovers, which are required in most organisms for the faithful segregation of homologous chromosomes during meiotic cell division. Characterization of crossover-defective mutants has contributed much to our understanding of the molecular mechanism of crossover formation. We report here a molecular analysis of recombination in a Drosophila melanogaster crossover-defective mutant, mei-9. In the absence of mei-9 activity, postmeiotic segregation associated with noncrossovers occurs at the expense of crossover products, suggesting that the underlying meiotic function for MEI-9 is in crossover formation rather than mismatch repair. In support of this, analysis of the arrangement of heteroduplex DNA in the postmeiotic segregation products reveals different patterns from those observed in Drosophila Msh6 mutants, which are mismatch-repair defective. This analysis also provides evidence that the double-strand break repair model applies to meiotic recombination in Drosophila. Our results support a model in which MEI-9 nicks Holliday junctions to generate crossovers during meiotic recombination, and, in the absence of MEI-9 activity, the double Holliday junction intermediate instead undergoes dissolution to generate noncrossover products in which heteroduplex is unrepaired.     

Drosophila hold'em is required for a subset of meiotic crossovers and interacts with the dna repair endonuclease complex subunits MEI-9 and ERCC1

Three Drosophila proteins, ERCC1, MUS312, and MEI-9, function in a complex proposed to resolve double-Holliday-junction intermediates into crossovers during meiosis. We report here the characterization of hold'em (hdm), whose protein product belongs to a single-strand-DNA-binding superfamily of proteins. Mutations in hdm result in reduced meiotic crossover formation and sensitivity to the DNA-damaging agent methyl methanesulfonate. Furthermore, HDM physically interacts with both MEI-9 and ERCC1 in a yeast two-hybrid assay. We conclude that HDM, MEI-9, MUS312, and ERCC1 form a complex that resolves meiotic recombination intermediates into crossovers.     

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.     

Mutational analysis of the Drosophila DNA repair and recombination gene mei-9

Drosophila mei-9 is essential for several DNA repair and recombination pathways, including nucleotide excision repair (NER), interstrand crosslink repair, and meiotic recombination. To better understand the role of MEI-9 in these processes, we characterized 10 unique mutant alleles of mei-9. These include a P-element insertion that disrupts repair functions but not the meiotic function; three nonsense mutations, one of which has nearly wild-type levels of protein; three missense mutations, one of which disrupts the meiotic function but not repair functions; two small in-frame deletions; and one frameshift.     

Holliday junctions in the eukaryotic nucleus: resolution in sight?

The Holliday junction is a key recombination intermediate whose resolution generates crossovers. Interplay between recombination, repair and replication has moved the Holliday junction to the center stage of nuclear DNA metabolism. Holliday junction resolvases in the eukaryotic nucleus have long eluded identification. The endonucleases Mus81/Mms4-Eme1 and XPF-MEI-9/MUS312 are structurally related to the archaeal resolvase Hjc and were found to be involved in crossover formation in budding yeast and flies, respectively. Although these endonucleases might represent one class of eukaryotic resolvases, their substrate preference opens up the possibility that junctions other than classical Holliday junctions might contribute to crossovers. Holliday junction resolution to non-crossover products can also be achieved topologically, for example, by the action of RecQ-like DNA helicases combined with topoisomerase III.     

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.     

Drosophila ERCC1 is required for a subset of MEI-9-dependent meiotic crossovers

Drosophila MEI-9 is the catalytic subunit of a DNA structure-specific endonuclease required for nucleotide excision repair (NER). The enzymatic activity of this endonuclease during NER requires the presence of a second, noncatalytic subunit called ERCC1. In addition to its role in NER, MEI-9 is required for the generation of most meiotic crossovers. To better understand the role of MEI-9 in crossover formation, we report here the characterization of the Drosophila Ercc1 gene. We created an Ercc1 mutant through homologous gene targeting. We find that Ercc1 mutants are identical to mei-9 mutants in sensitivity to DNA-damaging agents, but have a less severe reduction in the number of meiotic crossovers. MEI-9 protein levels are reduced in Ercc1 mutants; however, overexpression of MEI-9 is not sufficient to restore meiotic crossing over in Ercc1 mutants. We conclude that MEI-9 can generate some meiotic crossovers in an ERCC1-independent manner.     

The archaeal Xpf/Mus81/FANCM homolog Hef and the Holliday junction resolvase Hjc define alternative pathways that are essential for cell viability in Haloferax volcanii

The XPF/MUS81 family of endonucleases is found in eukaryotes and archaea, in the former they play a critical role in DNA repair and replication fork restart. Hef is a XPF/MUS81 family member found in Euryarchaea and is related to the Fanconi anemia protein FANCM. We have studied the role of Hef in the euryarchaeon Haloferax volcanii. Unlike Xpf in eukaryotes, Hef is not involved in nucleotide excision repair; instead, this function is encoded by the uvrABC genes. Similarly, deletion of hef confers only moderate sensitivity to DNA crosslinking agents, whereas mutation of FANCM in leads to hypersensitivity in eukaryotes. However, Hef is essential for cell viability when the Holliday junction resolvase Hjc is absent, and both the helicase and nuclease activities of Hef are indispensable. By contrast, single mutants of hjc and hef display no significant defects in growth or homologous recombination. This suggests that Hef and Hjc are redundant for the resolution of recombination intermediates, and that Hef is the functional homolog of eukaryotic Mus81. Furthermore, deletion of hef in a recombination-deficient ΔradA background is highly deleterious but deletion of hjc has no effect. Therefore, Hjc acts exclusively in homologous recombination whereas Hef, in addition to its role in resolving recombination intermediates, can act in a pathway that avoids the use of homologous recombination. We propose that Hef and Hjc provide alternative means to restart stalled DNA replication forks.     

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.     

SLX-1 Is Required for Maintaining Genomic Integrity and Promoting Meiotic Noncrossovers in the Caenorhabditis elegans Germline

Although the SLX4 complex, which includes structure-specific nucleases such as XPF, MUS81, and SLX1, plays important roles in the repair of several kinds of DNA damage, the function of SLX1 in the germline remains unknown. Here we characterized the endonuclease activities of the Caenorhabditis elegans SLX-1-HIM-18/SLX-4 complex co-purified from human 293T cells and determined SLX-1 germline function via analysis of slx-1(tm2644) mutants. SLX-1 shows a HIM-18/SLX-4-dependent endonuclease activity toward replication forks, 5'-flaps, and Holliday junctions. slx-1 mutants exhibit hypersensitivity to UV, nitrogen mustard, and camptothecin, but not gamma irradiation. Consistent with a role in DNA repair, recombination intermediates accumulate in both mitotic and meiotic germ cells in slx-1 mutants. Importantly, meiotic crossover distribution, but not crossover frequency, is altered on chromosomes in slx-1 mutants compared to wild type. This alteration is not due to changes in either the levels or distribution of double-strand breaks (DSBs) along chromosomes. We propose that SLX-1 is required for repair at stalled or collapsed replication forks, interstrand crosslink repair, and nucleotide excision repair during mitosis. Moreover, we hypothesize that SLX-1 regulates the crossover landscape during meiosis by acting as a noncrossover-promoting factor in a subset of DSBs.     

Drosophila FANCM Helicase Prevents Spontaneous Mitotic Crossovers Generated by the MUS81 and SLX1 Nucleases

Several helicases function during repair of double-strand breaks and handling of blocked or stalled replication forks to promote pathways that prevent formation of crossovers. Among these are the Bloom syndrome helicase BLM and the Fanconi anemia group M (FANCM) helicase. To better understand functions of these helicases, we compared phenotypes of Drosophila melanogaster Blm and Fancm mutants. As previously reported for BLM, FANCM has roles in responding to several types of DNA damage in preventing mitotic and meiotic crossovers and in promoting the synthesis-dependent strand annealing pathway for repair of a double-strand gap. In most assays, the phenotype of Fancm mutants is less severe than that of Blm mutants, and the phenotype of Blm Fancm double mutants is more severe than either single mutant, indicating both overlapping and unique functions. It is thought that mitotic crossovers arise when structure-selective nucleases cleave DNA intermediates that would normally be unwound or disassembled by these helicases. When BLM is absent, three nucleases believed to function as Holliday junction resolvases—MUS81-MMS4, MUS312-SLX1, and GEN—become essential. In contrast, no single resolvase is essential in mutants lacking FANCM, although simultaneous loss of GEN and either of the others is lethal in Fancm mutants. Since Fancm mutants can tolerate loss of a single resolvase, we were able to show that spontaneous mitotic crossovers that occur when FANCM is missing are dependent on MUS312 and either MUS81 or SLX1.     

The Drosophila Meiotic Recombination Gene Mei-9 Encodes a Homologue of the Yeast Excision Repair Protein Rad1

Meiotic recombination and DNA repair are mediated by overlapping sets of genes. In the yeast Saccharomyces cerevisiae, many genes required to repair DNA double-strand breaks are also required for meiotic recombination. In contrast, mutations in genes required for nucleotide excision repair (NER) have no detectable effects on meiotic recombination in S. cerevisiae. The Drosophila melanogaster mei-9 gene is unique among known recombination genes in that it is required for both meiotic recombination and NER. We have analyzed the mei-9 gene at the molecular level and found that it encodes a homologue of the S. cerevisiae excision repair protein Rad1, the probable homologue of mammalian XPF/ERCC4. Hence, the predominant process of meiotic recombination in Drosophila proceeds through a pathway that is at least partially distinct from that of S. cerevisiae, in that it requires an NER protein. The biochemical properties of the Rad1 protein allow us to explain the observation that mei-9 mutants suppress reciprocal exchange without suppressing the frequency of gene conversion.     

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.     

Processing of intermediates in recombination and DNA repair: identification of a new endonuclease that specifically cleaves Holliday junctions.

The formation and subsequent resolution of Holliday junctions are critical stages in recombination. We describe a new Escherichia coli endonuclease that resolves Holliday intermediates by junction cleavage. The 14 kDa Rus protein binds DNA containing a synthetic four-way junction (X-DNA) and introduces symmetrical cuts in two strands to give nicked duplex products. Rus also processes Holliday intermediates made by RecA into products that are characteristic of junction resolution. The cleavage activity on X-DNA is remarkably similar to that of RuvC. Both proteins preferentially cut the same two strands at the same location. Increased expression of Rus suppresses the DNA repair and recombination defects of ruvA, ruvB and ruvC mutants. We conclude that all ruv strains are defective in junction cleavage, and discuss pathways for Holliday junction resolution by RuvAB, RuvC, RecG and Rus.     

Three structure-selective endonucleases are essential in the absence of BLM helicase in Drosophila

DNA repair mechanisms in mitotically proliferating cells avoid generating crossovers, which can contribute to genome instability. Most models for the production of crossovers involve an intermediate with one or more four-stranded Holliday junctions (HJs), which are resolved into duplex molecules through cleavage by specialized endonucleases. In vitro studies have implicated three nuclear enzymes in HJ resolution: MUS81-EME1/Mms4, GEN1/Yen1, and SLX4-SLX1. The Bloom syndrome helicase, BLM, plays key roles in preventing mitotic crossover, either by blocking the formation of HJ intermediates or by removing HJs without cleavage. Saccharomyces cerevisiae mutants that lack Sgs1 (the BLM ortholog) and either Mus81-Mms4 or Slx4-Slx1 are inviable, but mutants that lack Sgs1 and Yen1 are viable. The current view is that Yen1 serves primarily as a backup to Mus81-Mms4. Previous studies with Drosophila melanogaster showed that, as in yeast, loss of both DmBLM and MUS81 or MUS312 (the ortholog of SLX4) is lethal. We have now recovered and analyzed mutations in Drosophila Gen. As in yeast, there is some redundancy between Gen and mus81; however, in contrast to the case in yeast, GEN plays a more predominant role in responding to DNA damage than MUS81-MMS4. Furthermore, loss of DmBLM and GEN leads to lethality early in development. We present a comparison of phenotypes occurring in double mutants that lack DmBLM and either MUS81, GEN, or MUS312, including chromosome instability and deficiencies in cell proliferation. Our studies of synthetic lethality provide insights into the multiple functions of DmBLM and how various endonucleases may function when DmBLM is absent.     

Nucleotide excision repair endonuclease genes in Drosophila melanogaster

Nucleotide excision repair (NER) is the primary pathway for the removal of ultraviolet light-induced damage and bulky adducts from DNA in eukaryotes. During NER, the helix is unwound around the damaged site, and incisions are made on the 5' and 3' sides, to release an oligonucleotide carrying the lesion. Repair synthesis can then proceed, using the intact strand as a template. The incisions flanking the lesion are catalyzed by different structure-specific endonucleases. The 5' incision is made by a heterodimer of XPF and ERCC1 (Rad1p-Rad10p in Saccharomyces cerevisiae), and the 3' incision is made by XPG (Rad2p in S. cerevisiae). We previously showed that the Drosophila XPF homologue is encoded by the meiotic recombination gene mei-9. We report here the identification of the genes encoding the XPG and ERCC1 homologues (XPG(Dm) and ERCC1(Dm)). XPG(Dm) is encoded by the mus201 gene; we found frameshift mutations predicted to produce truncated XPG(Dm) proteins in each of two mus201 alleles. These mutations cause defects in nucleotide excision repair and hypersensitivity to alkylating agents and ultraviolet light, but do not cause hypersensitivity to ionizing radiation and do not impair viability or fertility. ERCC1(Dm) interacts strongly in a yeast two-hybrid assay with MEI-9, indicative of the presumed requirement for these polypeptides to dimerize to form the functional endonuclease. The Drosophila Ercc1 gene maps to polytene region 51D1-2. The nucleotide excision repair gene mus210 maps nearby (51E-F) but is distinct from Ercc1.     

Differential timing and control of noncrossover and crossover recombination during meiosis.

Unitary models of meiotic recombination postulate that a central intermediate containing Holliday junctions is resolved to generate either noncrossover or crossover recombinants, both of which contain heteroduplex DNA. Contrary to this expectation, we find that during meiosis in Saccharomyces cerevisiae, noncrossover heteroduplex products are formed at the same time as Holliday junction intermediates. Crossovers appear later, when these intermediates are resolved. Furthermore, noncrossover and crossover recombination are regulated differently. ndt80 mutants arrest in meiosis with unresolved Holliday junction intermediates and very few crossovers, while noncrossover heteroduplex products are formed at normal levels and with normal timing. These results suggest that crossovers are formed by resolution of Holliday junction intermediates, while most noncrossover recombinants arise by a different, earlier pathway.     

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.