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. 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. Recently, we have demonstrated that HOP2-MND1 induces changes in the conformation of the eukaryotic recombinase RAD51 that profoundly alter the basic properties of RAD51. HOP2-MND1 enhances the interaction of RAD51 with nucleotide cofactors and modifies its DNA-binding specificity in a manner that stimulates DNA strand exchange. It enables RAD51 DNA strand exchange in the absence of divalent metal ions required for ATP binding .The magnitude of the changes induced in RAD51 defines HOP2-MND1 as a 'molecular trigger' of RAD51 DNA strand exchange. 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. One example is the use of RNAi knockdowns of RAD51 in mouse testicular cells reveals its role in meiotic homologous recombination in mammals. In most eukaryotes, the recombinases RAD51 and DMC1 play an essential role in the repair of double strand breaks through meiotic homologous recombination (HR). In mammals, knocking-out DMC1 results in an arrest of meiosis at early prophase I, without completion of HR. However, the function of RAD51 during meiotic HR in mammals remains unclear, due to the embryonic lethality of the RAD51 knockout mouse. Here we present our functional studies of RAD51 during mouse spermatogenesis using siRNA to knockdown RAD51 both in vivo by injecting siRNAs into mouse seminiferous tubules and in an in vitro spermatogenesis system with cultured spermatocytes. Our results reveals that RAD51 is indispensable for mouse meiotic HR. Also, we demonstrated by examining the meiotic phenotype of Mnd1 knockouot mice generated by us, that the in vivo DSB repair catalyzed solely by HOP2 supports homologous chromosome pairing and synapsis. In addition, we showed that in vitro HOP2 promotes the co-aggregation of ssDNA with duplex DNA, binds to ssDNA leading to unstacking of bases, and promotes the formation of a three-strand synaptic intermediate. This work not only confirmed that HOP2 is a bona fide recombinase in vitro but it also showed that it could function as one in vivo (Pezza et al. (2014) Nucleic Acids Res. 42, 2346).

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10
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2016
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U.S. National Inst Diabetes/Digst/Kidney
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Dai, Jieqiong; Voloshin, Oleg; Potapova, Svetlana et al. (2017) Meiotic Knockdown and Complementation Reveals Essential Role of RAD51 in Mouse Spermatogenesis. Cell Rep 18:1383-1394
Bugreev, Dmitry V; Huang, Fei; Mazina, Olga M et al. (2014) HOP2-MND1 modulates RAD51 binding to nucleotides and DNA. Nat Commun 5:4198
Pezza, Roberto J; Voloshin, Oleg N; Volodin, Alexander A et al. (2014) The dual role of HOP2 in mammalian meiotic homologous recombination. Nucleic Acids Res 42:2346-57
Broering, Tyler J; Alavattam, Kris G; Sadreyev, Ruslan I et al. (2014) BRCA1 establishes DNA damage signaling and pericentric heterochromatin of the X chromosome in male meiosis. J Cell Biol 205:663-75
Moktan, Hem; Guiraldelli, Michel F; Eyster, Craig A et al. (2014) Solution structure and DNA-binding properties of the winged helix domain of the meiotic recombination HOP2 protein. J Biol Chem 289:14682-91
Margolin, Gennady; Khil, Pavel P; Kim, Joongbaek et al. (2014) Integrated transcriptome analysis of mouse spermatogenesis. BMC Genomics 15:39
Zhao, Weixing; Saro, Dorina; Hammel, Michal et al. (2014) Mechanistic insights into the role of Hop2-Mnd1 in meiotic homologous DNA pairing. Nucleic Acids Res 42:906-17
Naumova, Anna K; Fayer, Shawn; Leung, Jacky et al. (2013) Dynamics of response to asynapsis and meiotic silencing in spermatocytes from Robertsonian translocation carriers. PLoS One 8:e75970
Boateng, Kingsley A; Bellani, Marina A; Gregoretti, Ivan V et al. (2013) Homologous pairing preceding SPO11-mediated double-strand breaks in mice. Dev Cell 24:196-205
Fukuda, Tomoyuki; Pratto, Florencia; Schimenti, John C et al. (2012) Phosphorylation of chromosome core components may serve as axis marks for the status of chromosomal events during mammalian meiosis. PLoS Genet 8:e1002485

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