Drosophila MUS312 interacts with the nucleotide excision repair endonuclease MEI-9 to generate meiotic crossovers

MEI-9 is the Drosophila homolog of the human structure-specific DNA endonuclease XPF. Like XPF, MEI-9 functions in nucleotide excision repair and interstrand crosslink repair. MEI-9 is also required to generate meiotic crossovers, in a function thought to be associated with resolution of Holliday junction intermediates. We report here the identification of MUS312, a protein that physically interacts with MEI-9. We show that mutations in mus312 elicit a meiotic phenotype identical to that of mei-9 mutants. A missense mutation in mei-9 that disrupts the MEI-9-MUS312 interaction abolishes the meiotic function of mei-9 but does not affect the DNA repair functions of mei-9. We propose that MUS312 facilitates resolution of meiotic Holliday junction intermediates by MEI-9.     

Studies on crossover-specific mutants and the distribution of crossing over in Drosophila females

In Drosophila females, the majority of recombination events do not become crossovers and those that do occur are nonrandomly distributed. Furthermore, a group of Drosophila mutants specifically reduce crossing over, suggesting that crossovers depend on different gene products than noncrossovers. In mei-218 mutants, crossing over is reduced by approximately 90% while noncrossovers and the initiation of recombination remain unchanged. Importantly, the residual crossovers have a more random distribution than wild-type. It has been proposed that mei-218 has a role in establishing the crossover distribution by determining which recombination sites become crossovers. Surprisingly, a diverse group of genes, including those required for double strand break (DSB) formation or repair, have an effect on crossover distribution. Not all of these mutants, however, have a crossover-specific defect like mei-218 and it is not understood why some crossover-defective mutants alter the distribution of crossovers. Intragenic recombination experiments suggest that mei-218 is required for a molecular transition of the recombination intermediate late in the DSB repair pathway. We propose that the changes in crossover distribution in some crossover-defective mutants are a secondary consequence of the crossover reductions. This may be the activation of a regulatory system that ensures at least one crossover per chromosome, and which compensates for an absence of crossovers by attempting to generate them at random locations.     

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.     

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.     

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.     

Trans events associated with crossovers are revealed in the absence of mismatch repair genes in Saccharomyces cerevisiae

Genetic analysis of recombination in Saccharomyces cerevisiae has revealed products with structures not predicted by the double-strand break repair model of meiotic recombination. A particular type of recombinant containing trans heteroduplex DNA has been observed at two loci. Trans events were originally identified only in tetrads in which the non-Mendelian segregations were not associated with a crossover. Because of this, these events were proposed to have arisen from the unwinding of double Holliday junctions. Previous studies used palindromes, refractory to mismatch repair, as genetic markers whereas we have used a complementary approach of deleting mismatch repair proteins to identify heteroduplex DNA. We found that the markers occurred in trans and were associated with crossovers. In both mlh1Delta and msh2Delta strains, the frequency of trans events associated with a crossover exceeded that predicted from the random association of crossovers with noncrossover trans events. We propose two different models to account for trans events associated with crossovers and discuss the relevance to wild-type DSB repair.     

REC, Drosophila MCM8, drives formation of meiotic crossovers

Crossovers ensure the accurate segregation of homologous chromosomes from one another during meiosis. Here, we describe the identity and function of the Drosophila melanogaster gene recombination defective (rec), which is required for most meiotic crossing over. We show that rec encodes a member of the mini-chromosome maintenance (MCM) protein family. Six MCM proteins (MCM2–7) are essential for DNA replication and are found in all eukaryotes. REC is the Drosophila ortholog of the recently identified seventh member of this family, MCM8. Our phylogenetic analysis reveals the existence of yet another family member, MCM9, and shows that MCM8 and MCM9 arose early in eukaryotic evolution, though one or both have been lost in multiple eukaryotic lineages. Drosophila has lost MCM9 but retained MCM8, represented by REC. We used genetic and molecular methods to study the function of REC in meiotic recombination. Epistasis experiments suggest that REC acts after the Rad51 ortholog SPN-A but before the endonuclease MEI-9. Although crossovers are reduced by 95% in rec mutants, the frequency of noncrossover gene conversion is significantly increased. Interestingly, gene conversion tracts in rec mutants are about half the length of tracts in wild-type flies. To account for these phenotypes, we propose that REC facilitates repair synthesis during meiotic recombination. In the absence of REC, synthesis does not proceed far enough to allow formation of an intermediate that can give rise to crossovers, and recombination proceeds via synthesis-dependent strand annealing to generate only noncrossover products.     

Cytoplasmic Localization and Evolutionary Conservation of MEI-218, a Protein Required for Meiotic Crossing-over in Drosophila

During Drosophila oogenesis, the oocyte is formed within a 16-cell cyst immediately after four incomplete cell divisions. One of the primary events in oocyte development is meiotic recombination. Here, we report the intracellular localization of the MEI-218 protein that is specifically required for meiotic crossing-over. To understand the role of mei-218 in meiosis and to study the regulation of genes required for meiotic recombination, we characterized the expression pattern of its RNA and protein. Furthermore, we cloned and sequenced mei-218 from two other Drosophila species. The mei-218 RNA and protein have a similar expression pattern, appearing first in early meiotic prophase and then rapidly disappearing as prophase is completed. This pattern corresponds to a specific appearance of the mei-218 gene product in the region of the ovary where meiotic prophase occurs. Although mei-218 is required for 95% of all crossovers, the protein is found exclusively in the cytoplasm. Based on these results, we suggest that mei-218 does not have a direct role in recombination but rather regulates other factors required for the production of crossovers. We propose that mei-218 is a molecular link between oocyte differentiation and meiosis.     

Drosophila PCH2 is required for a pachytene checkpoint that monitors double-strand-break-independent events leading to meiotic crossover formation

During meiosis, programmed DNA double-strand breaks (DSBs) are repaired to create at least one crossover per chromosome arm. Crossovers mature into chiasmata, which hold and orient the homologous chromosomes on the meiotic spindle to ensure proper segregation at meiosis I. This process is usually monitored by one or more checkpoints that ensure that DSBs are repaired prior to the meiotic divisions. We show here that mutations in Drosophila genes required to process DSBs into crossovers delay two important steps in meiotic progression: a chromatin-remodeling process associated with DSB formation and the final steps of oocyte selection. Consistent with the hypothesis that a checkpoint has been activated, the delays in meiotic progression are suppressed by a mutation in the Drosophila homolog of pch2. The PCH2-dependent delays also require proteins thought to regulate the number and distribution of crossovers, suggesting that this checkpoint monitors events leading to crossover formation. Surprisingly, two lines of evidence suggest that the PCH2-dependent checkpoint does not reflect the accumulation of unprocessed recombination intermediates: the delays in meiotic progression do not depend on DSB formation or on mei-41, the Drosophila ATR homolog, which is required for the checkpoint response to unrepaired DSBs. We propose that the sites and/or conditions required to promote crossovers are established independently of DSB formation early in meiotic prophase. Furthermore, the PCH2-dependent checkpoint is activated by these events and pachytene progression is delayed until the DSB repair complexes required to generate crossovers are assembled. Interestingly, PCH2-dependent delays in prophase may allow additional crossovers to form.     

mei-41 is required for precocious anaphase in Drosophila females

This paper reports on a new role for mei-41 in cell cycle control during meiosis. This function is revealed by the requirement of mei-41 for the precocious anaphase observed in crossover-defective mutants. Normally in Drosophila oocytes, tension on the meiotic spindle causes a metaphase I arrest. This tension results because crossovers, and the resulting chiasmata, hold homologs together that are being pulled by kinetochore microtobules toward opposite spindle poles. In the absence of tension, such as in a recombination-defective mutant, metaphase arrest is not observed and meiosis proceeds through the two divisions. Here we show that in some recombination-defective mutants, the precocious anaphase requires the mei-41 gene product. For example, metaphase arrest is not observed in mei-218 mutants because of the severe reduction in crossing over. In mei-41 mei-218 double mutants, however, metaphase arrest was restored. The effect of mei-41 is dependent on double-strand break formation. Thus, in mutants that fail to initiate meiotic recombination the absence of mei-41 has no effect. Received: 15 October 1999; in revised form: 9 December 1999 / Accepted: 13 December 1999     

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.     

Joint Molecule Resolution Requires the Redundant Activities of MUS-81 and XPF-1 during Caenorhabditis elegans Meiosis

The generation and resolution of joint molecule recombination intermediates is required to ensure bipolar chromosome segregation during meiosis. During wild type meiosis in Caenorhabditis elegans, SPO-11-generated double stranded breaks are resolved to generate a single crossover per bivalent and the remaining recombination intermediates are resolved as noncrossovers. We discovered that early recombination intermediates are limited by the C. elegans BLM ortholog, HIM-6, and in the absence of HIM-6 by the structure specific endonuclease MUS-81. In the absence of both MUS-81 and HIM-6, recombination intermediates persist, leading to chromosome breakage at diakinesis and inviable embryos. MUS-81 has an additional role in resolving late recombination intermediates in C. elegans. mus-81 mutants exhibited reduced crossover recombination frequencies suggesting that MUS-81 is required to generate a subset of meiotic crossovers. Similarly, the Mus81-related endonuclease XPF-1 is also required for a subset of meiotic crossovers. Although C. elegans gen-1 mutants have no detectable meiotic defect either alone or in combination with him-6, mus-81 or xpf-1 mutations, mus-81;xpf-1 double mutants are synthetic lethal. While mus-81;xpf-1 double mutants are proficient for the processing of early recombination intermediates, they exhibit defects in the post-pachytene chromosome reorganization and the asymmetric disassembly of the synaptonemal complex, presumably triggered by crossovers or crossover precursors. Consistent with a defect in resolving late recombination intermediates, mus-81; xpf-1 diakinetic bivalents are aberrant with fine DNA bridges visible between two distinct DAPI staining bodies. We were able to suppress the aberrant bivalent phenotype by microinjection of activated human GEN1 protein, which can cleave Holliday junctions, suggesting that the DNA bridges in mus-81; xpf-1 diakinetic oocytes are unresolved Holliday junctions. We propose that the MUS-81 and XPF-1 endonucleases act redundantly to process late recombination intermediates to form crossovers during C. elegans meiosis.     

Meiotic recombination in Drosophila Msh6 mutants yields discontinuous gene conversion tracts

Crossovers (COs) generated through meiotic recombination are important for the correct segregation of homologous chromosomes during meiosis. Several models describing the molecular mechanism of meiotic recombination have been proposed. These models differ in the arrangement of heteroduplex DNA (hDNA) in recombination intermediates. Heterologies in hDNA are usually repaired prior to the recovery of recombination products, thereby obscuring information about the arrangement of hDNA. To examine hDNA in meiotic recombination in Drosophila melanogaster, we sought to block hDNA repair by conducting recombination assays in a mutant defective in mismatch repair (MMR). We generated mutations in the MMR gene Msh6 and analyzed recombination between highly polymorphic homologous chromosomes. We found that hDNA often goes unrepaired during meiotic recombination in an Msh6 mutant, leading to high levels of postmeiotic segregation; however, hDNA and gene conversion tracts are frequently discontinuous, with multiple transitions between gene conversion, restoration, and unrepaired hDNA. We suggest that these discontinuities reflect the activity of a short-patch repair system that operates when canonical MMR is defective.     

Multiple barriers to nonhomologous DNA end joining during meiosis in Drosophila

Repair of meiotic double-strand breaks (DSBs) uses the homolog and recombination to yield crossovers while alternative pathways such as nonhomologous end joining (NHEJ) are suppressed. Our results indicate that NHEJ is blocked at two steps of DSB repair during meiotic prophase: first by the activity of the MCM-like protein MEI-218, which is required for crossover formation, and, second, by Rad51-related proteins SPN-B (XRCC3) and SPN-D (RAD51C), which physically interact and promote homologous recombination (HR). We further show that the MCM-like proteins also promote the activity of the DSB repair checkpoint pathway, indicating an early requirement for these proteins in DSB processing. We propose that when a meiotic DSB is formed in the absence of both MEI-218 and SPN-B or SPN-D, a DSB substrate is generated that can enter the NHEJ repair pathway. Indeed, due to its high error rate, multiple barriers may have evolved to prevent NHEJ activity during meiosis.     

Eliminating Both Canonical and Short-Patch Mismatch Repair in Drosophila melanogaster Suggests a New Meiotic Recombination Model

In most meiotic systems, recombination is essential to form connections between homologs that ensure their accurate segregation from one another. Meiotic recombination is initiated by DNA double-strand breaks that are repaired using the homologous chromosome as a template. Studies of recombination in budding yeast have led to a model in which most early repair intermediates are disassembled to produce noncrossovers. Selected repair events are stabilized so they can proceed to form double-Holliday junction (dHJ) intermediates, which are subsequently resolved into crossovers. This model is supported in yeast by physical isolation of recombination intermediates, but the extent to which it pertains to animals is unknown. We sought to test this model in Drosophila melanogaster by analyzing patterns of heteroduplex DNA (hDNA) in recombination products. Previous attempts to do this have relied on knocking out the canonical mismatch repair (MMR) pathway, but in both yeast and Drosophila the resulting recombination products are complex and difficult to interpret. We show that, in Drosophila, this complexity results from a secondary, short-patch MMR pathway that requires nucleotide excision repair. Knocking out both canonical and short-patch MMR reveals hDNA patterns that reveal that many noncrossovers arise after both ends of the break have engaged with the homolog. Patterns of hDNA in crossovers could be explained by biased resolution of a dHJ; however, considering the noncrossover and crossover results together suggests a model in which a two-end engagement intermediate with unligated HJs can be disassembled by a helicase to a produce noncrossover or nicked by a nuclease to produce a crossover. While some aspects of this model are similar to the model from budding yeast, production of both noncrossovers and crossovers from a single, late intermediate is a fundamental difference that has important implications for crossover control.     

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.     

The Effect of mei-41 on Rdna Redundancy in DROSOPHILA MELANOGASTER

The recombination and repair defective mutant, mei-41, exhibits three rather striking effects on the genetic properties and chromosomal stability of rDNA in Drosophila. First, mei-41 inhibits rDNA magnification. However, mei-9, another recombination and repair defective mutation has no similar effect. This indicates that magnification requires some, but not all, of the gene products necessary for meiotic exchange. Second, under magnifying conditions, mei-41 induces interchanges between the X rDNA and either arm of the Ybb- chromosome. These interchanges occur at high frequency and are independent of rDNA orientation. Third, in mei-41 bb+/Ybb+ males, bobbed mutants in the X, but not the Y, also arise at high frequency. Evidence suggests that these events involve the rDNA type I insertion. The recombination and repair defective properties of mei-41 together with our results regarding its unusual and specific effects involving rDNA are explained in a simple model that has general implications for chromosome structure.     

mus309 mutation, defective in DNA double-strand break repair, affects intergenic but not intragenic meiotic recombination in Drosophila melanogaster

The effect was investigated of the hypomorphic DNA double-strand break repair, notably synthesis-dependent strand annealing, deficient mutation mus309 on the third chromosome of Drosophila melanogaster on intergenic and intragenic meiotic recombination in the X chromosome. The results showed that the mutation significantly increases the frequency of intergenic crossing over in two of three gene intervals of the X chromosome studied. Interestingly the increase was most prevalent in the tip of the X chromosome where crossovers normally are least frequent per physical map unit length. In particular crossing over interference was also affected, indicating that the effect of the mus309 mutation involves preconditions of crossing over but not the event of crossing over itself. On the other hand, the results also show that most probably the mutation does not have any effect on intragenic recombination, i.e. gene conversion. These results are fully consistent with the present molecular models of meiotic crossing over initiated by double-strand breaks of DNA followed by formation of a single-end-invasion intermediate, or D-loop, which is subsequently processed to generate either crossover or non-crossover products involving formation of a double Holliday junction. In particular the results suggest that the mus309 gene is involved in resolution of the D-loop, thereby affecting the choice between double-strand-break repair (DSBR) and synthesis-dependent strand annealing (SDSA) pathways of meiotic recombination.     

mei-38 is required for chromosome segregation during meiosis in Drosophila females

Meiotic chromosome segregation occurs in Drosophila oocytes on an acentrosomal spindle, which raises interesting questions regarding spindle assembly and function. One is how to organize a bipolar spindle without microtubule organizing centers at the poles. Another question is how to orient the chromosomes without kinetochore capture of microtubules that grow from the poles. We have characterized the mei-38 gene in Drosophila and found it may be required for chromosome organization within the karyosome. Nondisjunction of homologous chromosomes occurs in mei-38 mutants primarily at the first meiotic division in females but not in males where centrosomes are present. Most meiotic spindles in mei-38 oocytes are bipolar but poorly organized, and the chromosomes appear disorganized at metaphase. mei-38 encodes a novel protein that is conserved in the Diptera and may be a member of a multigene family. Mei-38 was previously identified (as ssp1) due to a role in mitotic spindle assembly in a Drosophila cell line. MEI-38 protein localizes to a specific population of spindle microtubules, appearing to be excluded from the overlap of interpolar microtubules in the central spindle. We suggest MEI-38 is required for the stability of parallel microtubules, including the kinetochore microtubules.