Although it was not clear that Mnd1 knockout mice would be substantially different from Hop2 mice it has now become clear that these mice are a treasure trove of genetic information about mammalian meiosis. Unlike the Hop2 mice, the Mnd1 mice have a significant proportion of spermatocyte nuclei that show homologously paired chromosome synapsis,repair all their double-strand breaks progress up to late pachytene but do not show any crossovers. These results strongly suggest that, as has been proposed for budding yeast, there are two main pathways (DSBR, Double-Strand Break Repair and SDSA, Strand Displacement and Strand-Annealing) for the repair of double-strand breaks in mice (DSBR leads to mainly crossovers and SDSA results in non-crossovers exclusively) and that Hop2 can act on both pathways. This mouse might also provide insights into how chromosome interactions are channeled primarily between homologs versus sisters, a fundamental requirement leading to the crossovers that ensure the proper segregation of chromosomes. Recently, we have confirmed that Hop2 is only expressed in those Mnd1 knockout spermatocytes that synapse their chromosomes completely. This finding strongly suggests that Hop2 is responsible for this synapsis and since these spermatocytes that show complete synapsis have proceeded to a stage late in pachytene when crossovers would have normally appeared, this finding also indicates that the repair has occurred via the SDSA. We have also shown that although the involvement of Hop2 in the SDSA pathway has been revealed in the Mnd1 knockout background, Hop2 is most likely involved in this pathway in the wild-type mouse as there is 2 to 3 times as much Hop2 protein as there is Mnd1 protein in wild-type spermatocytes, that is, there is an excess of Hop2 beyond that required to form the Hop2/Mnd1 heterodimer that functions to stimulate the RecA-like recombinases, Rad51 and Dmc1. Recently, we have established that there is early DSB- and homologous recombination independent homologous pairing of chromosomes in mammalian meiosis. The pairing and alignment of homologous chromosomes in meiosis is arguably the premiere genetic event. The prevailing view, for which we have provided some biochemical support (Yancey-Wrona and Camerini-Otero (1995) Current Biology 5, 1149), is that in most organisms from yeast to man, this pairing and alignment results from a genome-wide search for homology triggered by the introduction of double-strand breaks (DSBs) by SPO11 and mediated by the homologous recombination (HR) biochemical machinery. We now have shown that a significant level of pairing in replicating mouse spermatogenic cells entering meiosis (meiotic S-phase) precedes SPO11 cleavage of chromosomal DNA. These data, obtained from fluorescent in situ hybridization in either structurally preserved nuclei or tissue sections, constitute the first report of such early pairing in mammals. Using a mutant mouse lacking the catalytic activity of SPO11, we have shown that early chromosome pairing requires SPO11, but is independent of its ability to make DSBs. This finding is consistent with previous observations in budding yeast (Cha et al. (2000) Genes and Development 14: 493). Furthermore, an examination of this pairing in mutant mice, deficient for several HR proteins confirmed that it is unlikely that HR is required in this process. Intriguingly, this early pairing requires SUN1, a protein involved in telomere attachment to the nuclear membrane (Ding X. et al. (2007) Dev Cell 12:863;Chi Y. et al. (2009) Development 136:965) and essential for gametogenesis. Furthermore, we find that the DSB-independent pairing at telomeres is stable while that at interstitial (non-telomeric) sites is transient. We have proposed that the reversibility and transience of the interstitial pairing along the length of the chromosomes may be required to allow the homologous recombination machinery to mediate the strand invasion that is the hallmark of the more precise and intimate alignment of the chromosomal DNA at the nucleotide level. That is, we posit that in meiosis, homologous recombination triggered by a DSB is not in fact, responsible for a genome-wide homology search but rather, proofreads the initial pairing to mediate the final stages of proper chromosomal synapsis. Finally, we are now investigating possible mechanisms for this early homologous pairing. In this regard, in a collaboration with the laboratory of Yoshi Watanabe at the University of Tokyo, we investigated the role of the cohesin complex in Pre-DSB pairing. Analysis of pairing prior to SPO11 mediated cleavage in mice lacking the meiotic cohesin complex shows that it also plays a significant role in this early pairing similar to its role in the late pairing (synapsis) later in prophase Most recently, we have been investigating approaches to dissect the pathways involved in early meiosis that do not rely on the use of gene knock-outs or other transgenic mice.

Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2013
Total Cost
$659,424
Indirect Cost
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State
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