InteractiveFly: GeneBrief

recombination-defective: Biological Overview | References

BIOLOGICAL OVERVIEW

Crossovers ensure the accurate segregation of homologous chromosomes from one another during meiosis. This study describes the identity and function of the Drosophila gene recombination defective (rec), which is required for most meiotic crossing over. 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 (Matsubayashi, 2003) is the Drosophila ortholog of the recently identified seventh member of this family, MCM8. 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. Genetic and molecular methods were used 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, it is proposed 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 (Blanton, 2005).

Faithful segregation of homologous chromosomes in meiosis requires crossovers, which, in concert with sister chromatid cohesion, form the chiasmata that hold and orient homologs on the meiotic spindle. Crossovers are distributed nonrandomly between chromosomes, along each chromosome arm, and relative to one another, indicating that meiotic recombination is tightly regulated. One aspect of this regulation is the process that determines whether a recombination event becomes a crossover or a noncrossover (Blanton, 2005).

Models of meiotic recombination must account for the production of both crossovers and noncrossovers. Current models are derived from the double-strand break (DSB) repair model. In this model, recombination is initiated with a DSB on one chromatid. Resection of the 5' ends leaves 3' single-stranded overhangs. One of these overhanging ends invades a homologous, non-sister duplex and primes repair DNA synthesis. The strand displaced by the migrating synthesis bubble is captured by the other 3' overhang, which primes synthesis using the displaced strand as a template. Ligation of the newly synthesized ends to the resected 5' ends generates an intermediate with two Holliday junctions. This double Holliday junction (DHJ) intermediate is resolved by an unknown endonuclease to form either crossover or noncrossover products (Blanton, 2005).

Recent data from yeast has resulted in modification of this model. Allers and Lichten (2001) physically monitored formation of recombination intermediates and products in Saccharomyces cerevisiae using an ectopic recombination system and found that noncrossover products appear before DHJ intermediates. They proposed that noncrossovers arise not through a DHJ intermediate, but through synthesis-dependent strand annealing (SDSA). In SDSA, the nascent strand dissociates from the template and anneals to the other resected end. Trimming of any overhangs and filling in of any gaps, followed by ligation, results in noncrossover products. Subsequent genetic tests of this model in S. cerevisiae are consistent with most noncrossovers coming from SDSA, while the remainder are derived from a DHJ intermediate (Blanton, 2005).

These models also take into account the occurrence of gene conversion—nonreciprocal transfer of information from one duplex to another—that can be associated with both crossovers and noncrossovers. Possible origins of gene conversion during SDSA are illustrated in this paper. Heteroduplex DNA (hDNA), in which the two strands are derived from different parental molecules, is produced by both invasion of a single-stranded overhang into a homologous template and annealing of a newly synthesized strand to the other single-stranded overhang. Sequence differences between homologous chromosomes result in base/base mismatches and insertion/deletion heterologies in hDNA, and these can be recognized and repaired by the mismatch repair system. The product contains a region of sequence derived from the homologous chromosome, referred to as a gene conversion tract. If heterologies are not repaired, each strand will convey different genetic information to the haploid product of meiosis. Upon the first round of DNA replication and mitosis after fertilization or germination, these strands separate, resulting in the post-meiotic segregation (PMS) of parental alleles. PMS results in a mosaic individual, or, for unicellular eukaryotes, a sectored colony (Blanton, 2005).

Though it is more difficult to physically observe intermediates formed during meiotic recombination in Drosophila, a wealth of evidence indicates that recombination is also initiated by DSBs in this organism. MEI-W68, the Drosophila ortholog of Spo11, which catalyzes meiotic DSB formation in S. cerevisiae, is required to generate both crossovers and noncrossovers, and in mei-W68 mutants recombination is restored by treatment with ionizing radiation. Mutations in Drosophila genes required for strand invasion cause female sterility that is suppressed by mutation of mei-W68. Thus, the early steps in meiotic recombination appear to be similar between Drosophila and S. cerevisiae. In contrast, later stages of crossover production are different, since most crossovers in Drosophila require the XPF/Rad1 ortholog MEI-9, its binding partner ERCC1, and several novel proteins, including MUS-312 and MEI-218. In addition, it is not known whether noncrossovers in Drosophila are derived from a DHJ intermediate or SDSA, although SDSA is the most common pathway for repair of mitotic DSBs generated by transposable element excision (Blanton, 2005).

In Drosophila, mutagenesis screens have been used to identify many novel genes required for meiotic recombination. The gene recombination defective (rec) was identified more than 25 years ago by Grell (1984) in an ethyl methanesulfonate (EMS) screen for temperature-sensitive recombination mutants. Her preliminary characterization of two null alleles showed that rec mutants have high levels of chromosome nondisjunction and reduced fertility, both indicative of homologous chromosome segregation defects. Since these mutants are able to pair homologous chromosomes normally, but exhibit a severe reduction in crossing over, Grell concluded that rec is involved in generating meiotic crossovers (Blanton, 2005).

To gain insight into the function of the REC protein in meiotic recombination, rec was molecularly and it was found to encode the Drosophila ortholog of MCM8. The eukaryotic mini-chromosome maintenance (MCM) family of proteins contains six members (MCM2-7) that form a heterohexameric helicase required for replication. Though MCM2-7 are essential in all eukaryotes, rec mutants are viable, and no function has been found for REC outside of meiosis. To explore the defect in meiotic recombination further, the distribution of crossing over was examined in rec mutants and it was found that residual crossovers are distributed abnormally. This finding, coupled with epistasis analysis, suggests that REC might act at an intermediate step in recombination. Further insight into the function of REC comes from the finding that the frequency of noncrossovers is substantially increased in rec mutants, and that these noncrossover events have significantly shorter gene conversion tracts than those of wild-type females. Based on these phenotypes and the structural similarity between REC and MCM proteins, it is proposed that REC facilitates processive repair DNA synthesis, and is a prerequisite for formation of the DHJ intermediate during meiotic recombination. In the absence of REC, recombination proceeds through SDSA to generate noncrossovers (Blanton, 2005).

Understanding how crossovers form is crucial to understanding the mechanisms eukaryotes use to faithfully pass half of their genetic information to the next generation. In Drosophila, many components of the meiotic recombination pathway have been identified, but a complete picture of the process has yet to emerge. This paper describes the molecular and genetic characterization of an important participant in this pathway -- REC, the Drosophila homolog of MCM8, giving new insight into requirements for crossover formation (Blanton, 2005).

The data support a model in which REC acts at an intermediate step of meiotic recombination. REC is not required for pre-meiotic S phase because homologous chromosomes in rec mutant females form normal synaptonemal complex, indicative of complete replication of genomic DNA. The finding that rec mutant females have about twice the normal number of noncrossover gene conversions indicates that initiation of recombination is not impaired in rec mutants; rather, very few DSBs are repaired as crossovers. The data suggest that REC functions after strand invasion, since females mutant for both rec and spn-A, which encodes the Rad51 ortholog, phenocopy spn-A single mutants. Based on the distribution of residual crossovers in rec mutants and in mei-9; rec double mutants, it is likely that REC does not function with MEI-9 at resolution but acts at some previous step (Blanton, 2005).

Normally, some recombination events become crossovers and some become noncrossovers. An increase in noncrossovers would occur if the crossover pathway were blocked so that most or all events followed the noncrossover pathway. In the ry intragenic recombination assay, noncrossover gene conversions are recovered only if they span a mutant site and convert that site to the wild-type sequence. In contrast, a crossover can be recovered if it occurs anywhere between the two mutations, as long as it generates a wild-type chromosome. Based on conversion tract lengths and the distance between the two mutations, it is expected that many of the crossovers recovered would not be detected if they instead became noncrossovers, because they would not contain a conversion tract long enough to span a mutant site. The increase in noncrossovers that was observed in rec mutants, therefore, appears to be more than expected from this simple interpretation. A possible explanation for the increased frequency of noncrossovers in rec mutants comes from a hypothesis proposed by Bhagat (2004), who suggested that crossover distribution is disrupted as the result of a feedback mechanism that ensures one crossover per chromosome. The proposed feedback mechanism senses some intermediate in the crossover pathway (e.g., the DHJ structure). In mutants in which this intermediate does not form, a signal causes the cell to initiate additional recombination events to ensure that a crossover is obtained. These initiations may occur outside the normal constraints, leading to a disruption of the normal distribution and an apparent polar reduction in crossing over. According to this model, rec mutants are impaired in formation of some crucial intermediate leading to crossovers. As a result, more recombination events are initiated, but most of these still become noncrossovers. Thus, the frequency of noncrossovers is elevated, and the crossovers that are produced do not follow the normal distribution (Blanton, 2005).

The defect in rec mutants is not limited to an increased production of noncrossovers at the apparent expense of crossovers. Noncrossover gene conversion tract length is significantly reduced in rec mutants. This could result from a defect in generating hDNA or a defect in repairing hDNA. Defects in repair of hDNA result in PMS of markers within the heteroduplex tract. PMS was not detected in any of the events from wild-type or rec mutant females. Thus, rec mutants are not defective in repair of hDNA; rather, formation of hDNA may be compromised (Blanton, 2005).

The length of hDNA can be affected by the extent of strand invasion and the amount of repair synthesis. In S. cerevisiae, the Mer3 helicase has been shown in vitro to stimulate Rad51-mediated strand invasion (Mazina, 2004). As in rec mutants, mutations in the gene that encodes Mer3 cause a reduction in the frequency of crossovers and an increase in the frequency of noncrossovers (Nakagawa, 1999). However, in physical assays mer3 mutants are defective in the transition from DSB to strand invasion intermediate. The data suggest that Drosophila REC acts after strand invasion, so the notion is not favored that REC performs a function similar to that of Mer3. Furthermore, based on the similarity of REC to replicative MCMs, it is thought plausible that rec mutants have shorter conversion tracts because repair synthesis is diminished (Blanton, 2005).

What is the relationship between reduced repair synthesis and decreased crossing over in rec mutants? In S. cerevisiae, crossovers are believed to arise through resolution of the DHJ intermediate. Although this process can also give rise to noncrossovers, most noncrossovers are thought to arise through SDSA. There is evidence in S. cerevisiae that formation of a DHJ intermediate requires more repair synthesis than SDSA. If this is also the case in Drosophila, then decreased repair synthesis would increase the probability that a meiotic DSB will be repaired through SDSA instead of the DHJ pathway (Blanton, 2005).

A model is proposed in which REC drives crossover formation by acting at the repair synthesis step of meiotic recombination. In the absence of REC, synthesis does not proceed far enough to allow second-end capture and formation of the DHJ intermediate, resulting in a deficit of crossovers. Noncrossovers may still be formed through SDSA. There are two versions of this model. First, REC may facilitate repair synthesis at all sites of recombination. In this version, noncrossovers may normally arise through the DHJ pathway or the SDSA pathway, but in rec mutants the SDSA pathway is favored; the decrease in gene conversion tract length in rec mutants reflects an overall decrease in repair synthesis. Alternatively, REC may facilitate synthesis only at those recombination sites designated to become DHJ intermediates . In this version of the model, sites lacking REC in wild-type flies undergo SDSA. The decrease in mean tract length in rec mutants is due to loss of those noncrossovers that would have arisen via a DHJ intermediate (Blanton, 2005).

The data do not indicate whether noncrossovers in wild-type flies arise through SDSA, DHJ, or a combination of the two. In Drosophila, SDSA is a primary pathway for DSB repair in nonmeiotic cells. It may be that SDSA is the 'default' pathway for recombinational repair of DSBs, and that meiosis-specific modifications promote formation of DHJs to allow crossing over. REC does not appear to play a role in SDSA in nonmeiotic cells), and therefore REC may be a component of the meiosis-specific modifications to DSB repair in Drosophila. To better understand the role of REC and the process of meiotic recombination, it will be important to determine the source of noncrossover recombinants in wild-type females (Blanton, 2005).

REC, a new member of the MCM-related protein family, is required for meiotic recombination in Drosophila

rec mutations result in an extremely low level of recombination and a high frequency of primary non-disjunction in the female meiosis of Drosophila. This study demonstrates that the rec gene encodes a novel protein related to the mini-chromosome maintenance (MCM) proteins. Six MCM proteins (MCM2-7) are conserved in eukaryotic genomes, and they function as heterohexamers in the initiation and progression of mitotic DNA replication. Three rec alleles, rec¹, rec² and rec³, were found to possess mutations within this gene, and P element-mediated germline transformation with a wild-type rec cDNA fully rescued the rec mutant phenotypes. The 885 amino acid REC protein has an MCM domain in the middle of its sequence and, like MCM2, 4, 6 and 7, REC contains a putative Zn-finger motif. Phylogenetic analyses revealed that REC is distantly related to the six conserved MCM proteins. Database searches reveal that there are candidates for orthologs of REC in other higher eukaryotes, including human. Whether rec is involved in DNA repair in the mitotic division after the DNA damage caused by methylmethane sulfonate (MMS) or by X-rays was assessed. These analyses suggest that the rec gene has no, or only a minor, role in DNA repair and recombination in somatic cells (Matsubayashi, 2003).

One can speculate that REC may perform a comparable role in meiotic DNA replication to that of ordinary MCMs in mitotic DNA replication. Recent research on meiosis has focused on the link between premeiotic DNA replication and recombination. In this respect, it is interesting that the temperature-sensitive period of rec³ (a temperature-sensitive allele) coincides with that of premeiotic S phase (Grell, 1978). It has not yet been determined whether or not premeiotic DNA replication occurs in the rec mutants. However, the following facts suggest that failure of premeiotic DNA replication in the rec mutants is an unlikely scenario: (1) electron microscope analyses of the premeiotic stage of the rec mutant failed to reveal any anomalies, including any in synaptonemal complex structure; (2) oogenesis is completed normally; (3) the oocytes develop normally except for a lack of recombination. An alternative explanation is that rec has a meiosis-specific accessory function during premeiotic DNA replication stage, and that this function is essential for the subsequent recombination process but non-essential for replication itself. This idea is consistent with the observation that rec has no or only a minor role in somatic DNA repair and recombination. In lower eukaryotes, several lines of evidence have indicated that meiosis-specific events, such as recombination and reductional chromosome segregation, are linked or coupled to premeiotic DNA replication. These studies have suggested that the premeiotic DNA replication process includes functions besides replication itself that are specialized for subsequent meiotic events, such as recombination. The rec gene might perform such meiosis-specific but unknown functions during premeiotic replication. Since the first event after DNA replication might be the formation of programmed double- strand breaks (DSBs), it would be interesting to know whether DSBs are formed or not in the rec mutants (Matsubayashi, 2003).

Differential requirements for MCM proteins in DNA replication in Drosophila S2 cells

The MCM2-7 proteins are crucial components of the pre replication complex (preRC) in eukaryotes. Since they are significantly more abundant than other preRC components, it was of interest to determining whether the entire cellular content was necessary for DNA replication in vivo. A systematic depletion of the MCM proteins in Drosophila S2 cells was performed using dsRNA-interference. Reducing MCM2-6 levels by >95%-99% had no significant effect on cell cycle distribution or viability. Depletion of MCM7 however caused an S-phase arrest. MCM2-7 depletion produced no change in the number of replication forks as measured by PCNA loading. MCM8 was also depleted. This caused a 30% reduction in fork number, but no significant effect on cell cycle distribution or viability. No additive effects were observed by co-depleting MCM8 and MCM5. These studies suggest that, in agreement with what has previously been observed for Xenopus in vitro, not all of the cellular content of MCM2-6 proteins is needed for normal cell cycling. They also reveal an unexpected unique role for MCM7. Finally they suggest that MCM8 has a role in DNA replication in S2 cells (Crevel, 2007; Full text of article).

Search PubMed for articles about Drosophila Rec

Allers, T. and Lichten, M. (2001) Differential timing and control of noncrossover and crossover recombination during meiosis. Cell 106: 47-57. PubMed ID: 11461701

Bhagat, R., Manheim, E. A., Sherizen, D. E. and McKim, K. S. (2004). Studies on crossover-specific mutants and the distribution of crossing over in Drosophila females. Cytogenet Genome Res 107: 160-171. PubMed ID: 15467361

Blanton, H. L., Radford, S. J., McMahan, S., Kearney, H. M., Ibrahim, J. G. and Sekelsky, J. (2005). REC, Drosophila MCM8, drives formation of meiotic crossovers. PLoS Genet. 1(3): e40. PubMed ID: 16189551

Crevel, G., et al. (2007). Differential requirements for MCM proteins in DNA replication in Drosophila S2 cells. PLoS One 2(9): e833. PubMed ID: 17786205

Grell, R. F. (1984). Time of recombination in the Drosophila melanogaster oocyte. III. Selection and characterization of temperature-sensitive and -insensitive recombination-deficient alleles in Drosophila. Genetics 108: 435-443

Mazina, O. M., et al. (2004). Saccharomyces cerevisiae Mer3 helicase stimulates 3'-5' heteroduplex extension by Rad51; implications for crossover control in meiotic recombination. Cell 117: 47-56. PubMed ID: 15066281

Matsubayashi, H., Yamamoto, M. T. (2003). REC, a new member of the MCM-related protein family, is required for meiotic recombination in Drosophila. Genes Genet. Syst. 78: 363-371. PubMed ID: 14676427

Nakagawa, T. and Ogawa, H. (1999). The Saccharomyces cerevisiae MER3 gene, encoding a novel helicase-like protein, is required for crossover control in meiosis. EMBO J. 18: 5714-5723. PubMed ID: 10523314

date revised: 2 January 2023

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