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pch2: Biological Overview | References

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. This study shows 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 conserved AAA+ ATPase 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. It is proposed 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 (Joyce, 2009a).

Meiotic crossovers promote genetic variation and mature into chiasmata, which hold the homologous chromosomes together at metaphase I and direct their segregation at anaphase I. In the absence of chiasmata, homologs may segregate randomly, resulting in aneuploidy, which can lead to infertility, severe developmental consequences, or lethality. Therefore, it is not surprising that crossover formation is a tightly regulated process. The formation of crossovers depends on the repair of programmed DNA double-strand breaks (DSBs) through homologous recombination (McKim, 1998; Keeney, 2001). DSBs are believed to be catalyzed by the Spo11 protein, a suspected paralog of a type II topoisomerase from archaebacteria. DSBs that do not become crossovers are repaired as noncrossovers, often referred to as 'gene conversions' (Joyce, 2009a).

The mechanism for repairing DSBs to generate crossovers during meiotic prophase probably involves some kind of double Holliday junction, an intermediate in genetic recombination involving a mobile junction between four strands of DNA. By contrast, noncrossovers can be generated by a combination of repair pathways such as synthesis-dependent strand annealing. The meiotic DSB repair program involves proteins specialized for the generation of crossovers as well as generic DSB repair proteins. In Drosophila, the former group of 'crossover proteins' have been identified by mutations that cause reductions in the frequency of crossovers but not noncrossovers (reviewed in Mehrotra, 2007). The latter group includes proteins such as members of the Rad51 family, required to repair all DSBs (Joyce, 2009a).

Drosophila genes required for crossing over have been divided into two general classes: precondition and exchange genes. The distinction between the precondition and exchange classes has been based mainly on the effects of mutations on the distribution of crossovers. The few crossovers observed in the progeny of females homozygous for precondition mutants show an altered distribution, while the few crossovers generated by mothers homozygous for exchange mutants show a relatively normal distribution. Therefore, precondition genes may have a role in establishing the crossover distribution, while exchange genes are required later to carry out the reaction that generates crossovers (Joyce, 2009a and references therein).

Meiotic DSB repair in Drosophila is monitored by at least one checkpoint. When there is a defect in repairing meiotic DSBs in Drosophila females, the ATR/MEI-41-dependent DSB repair checkpoint is activated (see Drosophila Mei-41), resulting in a variety of developmental defects, including the failure of the oocyte to establish dorsal-ventral polarity. This checkpoint pathway may also have a more direct role in DSB repair since mutations in the mei-41 gene cause a reduction in crossing over. In budding yeast, checkpoint proteins may also have a role in determining whether repair occurs using the sister chromatid or the homolog (Joyce, 2009a).

Evidence for a new meiotic prophase checkpoint has been found in Drosophila females. Mutations in DSB repair genes and exchange genes cause delays in two meiotic events: a chromatin-remodeling response to DSBs and oocyte selection. Both of these phenotypes may be a consequence of a general delay in pachytene progression, suggestive of an activated checkpoint. Surprisingly, the delay in pachytene progression in DSB repair and exchange mutants is independent of DSB formation but requires precondition genes like mei-218 and rec. This suggests that the checkpoint is not the canonical DSB-repair checkpoint that depends on ATR/MEI-41. Instead, it is proposed that this delay is the result of a second checkpoint associated with the pathway leading to crossovers. This DSB-independent checkpoint requires the Drosophila homolog of PCH2, an AAA-adenosine triphosphatase (Joyce, 2009a).

In Saccharomyces cerevisiae and Caenorhabditis elegans, it has been suggested that a PCH2-dependent checkpoint pathway responds to synapsis defects independent of DSBs (Bhalla, 2005; Wu, 2006). However, some Drosophila mutants with PCH2-dependent delays in pachytene do not have obvious defects in synapsis. Thus, these results point to a defect in the pathway leading to crossover formation as the trigger that activates the checkpoint. Interestingly, the synapsis mutants analyzed in other organisms also have crossover defects, suggesting that there may be a common mechanism related to crossover specification for triggering the checkpoint in all three species (Joyce, 2009a).

As in most other cell types, there is a checkpoint response to unrepaired DSBs in Drosophila female meiosis. The results of this study define a second and distinct DSB-independent checkpoint that operates during pachytene. Mutations in exchange class (e.g., hdm, mei-9, and mus312) and DSB repair genes (e.g., okr, spn-A, spn-B, spn-D, and mei-41) cause a delay in the timing of at least two events: the chromatin-remodeling response to DSBs (phosphorylation of HIS2AV) and, through a process that is DSB independent, the selection of a single oocyte. Both of the delay phenotypes that were observed can be explained if their timing is linked to the progression through pachytene. A delay in pachytene has also been proposed to explain why Rad51 foci, DSB response markers, persist into late pachytene in synapsis-defective C. elegans mutants (Joyce, 2009a).

The results show that the proposed pachytene checkpoint depends on the Drosophila pch2 ortholog. In C. elegans and S. cerevisiae, pch2 is required for a DSB-independent checkpoint pathway that responds to synapsis defects (Bhalla, 2005; Wu, 2006). In Drosophila, however, two sets of observations suggest that synapsis defects may not be the trigger of the PCH2-dependent checkpoint. First, immunofluorescent studies using the SC components C(2)M (and C(3)G suggest that exchange mutants (e.g., hdm and mei-9) and DSB repair mutants (e.g., okr and spn-D) are able to form SC. Indeed, complete reconstructions from electron micrographs have shown that mei-9 mutants synapse their chromosomes normally. Second, c(3)G mutations, which abolish synapsis in Drosophila, do not trigger pachytene delays (Joyce, 2009a).

Because the exchange mutants have reduced crossover formation but no detectable synapsis defects, the results point to a defect in the pathway that leads to crossovers as the mechanism that triggers pachytene delays. Interestingly, the synapsis mutants analyzed in C. elegans and S. cerevisiae also have defects in crossover production, suggesting there may be a common mechanism to activate the PCH2-dependent checkpoint in all three species. In fact, a non-null crossover-defective zip1 allele in budding yeast was reported to exhibit normal synapsis by immunofluorescence but still activated the PCH2-dependent checkpoint (Mitra, 2007). In C. elegans and S. cerevisiae, it could be a secondary consequence of the synapsis defects on the crossover pathway that triggers the pch2-dependent checkpoint pathway (Joyce, 2009a).

As with most checkpoints, there are two components to the PCH2-dependent checkpoint. First, activation of the checkpoint signal must depend on a specific substrate in the cell (such as a DSB in the canonical DNA repair checkpoint). Second, there must be a process that turns off the checkpoint signal. The first component of the PCH2-dependent checkpoint depends on the precondition genes, but not on DSB formation. This is based on the observation that mutations in the precondition genes mei-218 and rec suppress the pachytene delay phenotypes while mutations in the DSB formation genes mei-P22 and mei-W68 do not. Precondition genes such as mei-218 and rec may be required for establishing the pattern of crossovers, such as their distribution and frequency. Both MEI-218 and REC have homology to MCM proteins (Blanton, 2005) and recently a hypomorphic allele of the Drosophila mcm5 gene has been found to have a precondition mutant phenotype (Lake, 2007). In addition to their role in DNA replication, MCM proteins affect chromosome structure in as yet poorly defined ways and may interact with checkpoint and recombination proteins (Bailis, 2008). Thus, the function of precondition gene products could include modifying the meiotic chromosome structure (see Zickler, 1999), which in turn interacts with and is required to activate the PCH2-dependent checkpoint signal (Joyce, 2009a).

These data are also consistent with previous models that place mei-218 function upstream of exchange genes in the generation of crossovers (Sekelsky, 1995; Bhagat, 2004). However, mei-218 and rec also suppressed the pachytene delay phenotypes observed in the DSB repair mutants, spn-A and spn-D, which provides evidence for precondition gene products functioning early, during, or prior to the first steps of DSB repair. While it is possible that the effects of precondition mutations on pachytene progression and crossover formation are not related, the simplest model is that precondition genes function early in the repair process, close to the time of DSB formation, to commit a subset of DSBs to the crossover pathway. Such an early time for crossover decision has also been proposed in budding yeast (Bishop, 2004; Fung, 2004) and in C. elegans (Couteau, 2005; Joyce, 2009a and references therein).

The second component, which turns off the checkpoint signal, depends on a previously undescribed DSB-independent function of the DSB repair and exchange genes. If the initial activation of the checkpoint involves precondition gene-dependent changes in chromosome structure, then the DSB repair and exchange genes may function to reverse these changes or block how they interact with the checkpoint. Importantly, a defect in any one of the DSB repair or exchange proteins can trigger the checkpoint. One possibility is that all the proteins required for meiotic recombination preassemble for a 'dry run' prior to the actual repair of DSBs. The absence of a functional DSB repair or exchange protein would result in a reduction or impairment of DSB repair complexes capable of generating crossovers and modifying the activity of the PCH2 checkpoint. In support of this hypothesis, exchange gene products are known to form a complex (see Joyce, 2009b). Whether the exchange and DSB repair proteins form one or multiple complexes has yet to be determined. Preassembling repair complexes before programmed DSB formation occurs could suppress alternative repair pathways as well as provide a mechanism to ensure the proper number of crossovers (Joyce, 2009a).

Another implication of the delay phenotypes is that the exchange and DSB repair genes are required in early pachytene before the phosphorylation of HIS2AV. A function at this time is not surprising for the DSB repair proteins, which are presumably recruited shortly after the break is formed. It is surprisingly early, however, for the exchange genes, considering they are thought to function in the resolution step, relatively late in the repair process. However, if all the proteins required for crossover repair preassemble as proposed above, relatively early mutant phenotypes could be the result (Joyce, 2009a).

Although Drosophila pch2 single mutants had no significant change in crossover distribution or frequency, the hdm; pch2 double mutant had fewer crossovers than the hdm single mutant, establishing a functional link between the checkpoint and generating crossovers. The activated PCH2-dependent checkpoint may promote crossover formation in some situations, such as when crossover formation is compromised. Additional crossovers may form due to an extended 'window of opportunity' to generate crossovers or the activation of additional crossover-promoting gene products (Joyce, 2009a).

Despite the sequence conservation of PCH2 in many organisms, a conservation of function is not clear. For example, the PCH2-dependent checkpoint causes a pachytene delay in flies and budding yeast (San-Segundo, 1999) but apoptosis in nematodes (Bhalla, 2005). Most surprising, the mouse PCH2 homolog is required to complete recombination events but may not have a checkpoint function (Li, 2007). One way to reconcile these differences may be that PCH2 has a role in regulating the timing of important transitions during pachytene. PCH2 may be constitutively active in early pachytene until turned off by activities of proteins involved in crossover formation. This is supported by the observation that mutations in budding yeast pch2 cause delays in the progression of both the crossover and noncrossover pathways but do not affect the final frequency of these events (Borner, 2008). In the future, it will be important to identify what the PCH2-dependent checkpoint responds to. In Drosophila, for example, it will be interesting to know if the proposed interaction of the precondition or repair gene products with the PCH2-dependent checkpoint is restricted to DSB sites or more generally dispersed along the chromosome (Joyce, 2009a).

Pch2 links chromosome axis remodeling at future crossover sites and crossover distribution during yeast meiosis

Segregation of homologous chromosomes during meiosis I depends on appropriately positioned crossovers/chiasmata. Crossover assurance ensures at least one crossover per homolog pair, while interference reduces double crossovers. This study investigated the interplay between chromosome axis morphogenesis and non-random crossover placement. Chromosome axes are structurally modified at future crossover sites as indicated by correspondence between crossover designation marker Zip3 and domains enriched for axis ensemble Hop1/Red1. This association is first detected at the zygotene stage, persists until double Holliday junction resolution, and is controlled by the conserved AAA+ ATPase Pch2. Pch2 further mediates crossover interference, although it is dispensable for crossover formation at normal levels. Thus, interference appears to be superimposed on underlying mechanisms of crossover formation. When recombination-initiating DSBs are reduced, Pch2 is also required for viable spore formation, consistent with further functions in chiasma formation. pch2γ mutant defects in crossover interference and spore viability at reduced DSB levels are oppositely modulated by temperature, suggesting contributions of two separable pathways to crossover control. Roles of Pch2 in controlling both chromosome axis morphogenesis and crossover placement suggest linkage between these processes. Pch2 is proposed to reorganize chromosome axes into a tiling array of long-range crossover control modules, resulting in chiasma formation at minimum levels and with maximum spacing (Joshi, 2009. Full text of article).

Search PubMed for articles about Drosophila Pch2

Bailis, J. M., et al. (2008). Minichromosome maintenance proteins interact with checkpoint and recombination proteins to promote s-phase genome stability. Mol. Cell. Biol. 28: 1724-1738. PubMed ID: 18180284

Bhagat, R., et al. (2004). Studies on crossover specific mutants and the distribution of crossing over in Drosophila females. Cytogenet. Genome Res. 107: 160-171. PubMed ID: 15467361

Bhalla, N. and Dernburg, A. F. (2005). A conserved checkpoint monitors meiotic chromosome synapsis in Caenorhabditis elegans. Science 310: 1683-1686. PubMed ID: 16339446

Bishop, D. K. and Zickler, D. (2004). Early decision: meiotic crossover interference prior to stable strand exchange and synapsis. Cell 117: 9-15. PubMed ID: 15066278

Blanton, H. L., et al. (2005). REC, Drosophila MCM8, drives formation of meiotic crossovers. PLoS Genet. 1: e40. PubMed ID: 16189551

Borner, G. V., Barot, A. and Kleckner, N. (2008). Yeast Pch2 promotes domainal axis organization, timely recombination progression, and arrest of defective recombinosomes during meiosis. Proc. Natl. Acad. Sci. 105: 3327-3332. PubMed ID: 18305165

Couteau, F. and Zetka, M. (2005). HTP-1 coordinates synaptonemal complex assembly with homolog alignment during meiosis in C. elegans. Genes Dev. 19: 2744-2756. PubMed ID: 16291647

Fung, J. C., Rockmill, B., Odell, M. and Roeder, G. S. (2004). Imposition of crossover interference through the nonrandom distribution of synapsis initiation complexes. Cell 116: 795-802. PubMed ID: 15035982

Joshi, N., Barot, A., Jamison, C. and Börner, G. V. (2009). Pch2 links chromosome axis remodeling at future crossover sites and crossover distribution during yeast meiosis. PLoS Genet. 5(7): e1000557. PubMed ID: 19629172

Joyce, E. F. and McKim, K. S. (2009a). Drosophila PCH2 is required for a pachytene checkpoint that monitors double-strand-break-independent events leading to meiotic crossover formation. Genetics 181(1): 39-51. PubMed ID: 18957704

Joyce, E. F., Nikhila Tanneti, S. and McKim, K. S. (2009b). Drosophila Hold'em is required for a subset of meiotic crossovers and interacts with the DNA repair endonuclease complex subunits MEI-9 and ERCC1. Genetics 181: 335-340. PubMed ID: 18957705

Keeney, S. (2001), Mechanism and control of meiotic recombination initiation. Curr. Top. Dev. Biol. 52: 1-53. PubMed ID: 11529427

Lake, C. M., Teeter, K., Page, S. L., Nielsen, R. and Hawley, R. S. (2007). A genetic analysis of the Drosophila mcm5 gene defines a domain specifically required for meiotic recombination. Genetics 176(4): 2151-63. PubMed ID: 17565942

Li, X. and Schimenti, J. C. (2007). Mouse pachytene checkpoint 2 (trip13) is required for completing meiotic recombination but not synapsis. PLoS Genet. 3: e130. PubMed ID: 17696610

McKim, K. S. and Hayashi-Hagihara, A. (1998). mei-W68 in Drosophila melanogaster encodes a Spo11 homolog: evidence that the mechanism for initiating meiotic recombination is conserved. Genes Dev. 12: 2932-2942. PubMed ID: 9744869

Mehrotra, S., Hawley, R. S. and McKim, K. S. (2007). Synapsis, double strand breaks and domains of crossover control in females, pp. 125-152 in Recombination and Meiosis, Crossing-Over and Disjunction, edited by R. EGEL and D. LANKENAU. Springer-Verlag, Berlin.

Mitra, N. and Roeder, G. S. (2007). A novel nonnull ZIP1 allele triggers meiotic arrest with synapsed chromosomes in Saccharomyces cerevisiae. Genetics 176: 773-787. PubMed ID: 17435220

San-Segundo, P. A. and Roeder, G. S. (1999). Pch2 links chromatin silencing to meiotic checkpoint control. Cell 97: 313-324. PubMed ID: 10319812

Sekelsky, J. J., et al. (1995). The Drosophila meiotic recombination gene mei-9 encodes a homologue of the yeast excision repair protein Rad1. Genetics 141: 619-627. PubMed ID: 8647398

Wu, H. Y. and Burgess, S. M. (2006). Two distinct surveillance mechanisms monitor meiotic chromosome metabolism in budding yeast. Curr. Biol. 16: 2473-2479. PubMed ID: 17174924

Zickler, D. and Kleckner, N. (1999). Meiotic chromosomes: integrating structure and function. Annu. Rev. Genet. 33: 603-754. PubMed ID: 10690419

date revised: 30 December 2009

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