Regulation of meiotic recombination in mice

描述(申请人提供)：本申请的长期目标是阐明哺乳动物减数分裂的新分子网络。减数分裂是生殖细胞特有的过程，包括同源染色体的配对、联会、重组和分离。减数分裂引起的基因异常是导致人类出生缺陷和不孕的主要原因。经过广泛重组的减数分裂染色质的结构和功能特性无疑是减数分裂的中心主题。尽管对减数分裂的研究进展很快，但染色质组织和减数分裂过程(如突触和重组)之间的复杂相互作用仍然很大程度上是未知的。特别是哺乳动物减数分裂的研究进展远远落后于其他模式生物的减数分裂研究，这是由于几个关键障碍：成本高，持续时间长，以及许多远距离物种的减数分裂特异蛋白质缺乏序列保守。为了克服这些障碍，我们开发了一种创新的蛋白质组学方法，在小鼠中系统地鉴定了大量未鉴定的哺乳动物减数分裂染色质相关蛋白。目前的应用是研究MEIOB，一种在我们的蛋白质组筛选中发现的新的减数分裂特异蛋白，在调节小鼠的减数分裂重组和染色体突触中的作用。MEIOB是普遍表达的RPA1的序列类似物。我们发现MEIOB与单链DNA(SsDNA)结合，并显示出3‘到5’ssDNA特异的核酸外切酶活性。MEIOB在减数分裂染色体上形成明显的焦点。在睾丸中，MEIOB与RPA和SPATA22形成复合体。这些蛋白质共定位在减数分裂染色体上的焦点上。此外，无论性别，Meiob缺失突变小鼠都表现出减数分裂重组和染色体突触失败，导致不育。我们的结果有力地支持了MEIOB在减数分裂重组中的独特作用--第二步末端捕获。我们的具体目标是：1)检测MEIOB及其相关蛋白对单链DNA的促进作用；2)确定MEIOB单链DNA结合活性对减数分裂重组的要求；3)阐明广泛表达的单链DNA结合蛋白RPA1在减数分裂重组中的作用。总之，我们的研究将揭示两个相关的单链DNA结合蛋白MEIOB和RPA1调控哺乳动物减数分裂重组的分子机制，并将为人类不孕不育和出生缺陷的病因学提供见解。

DESCRIPTION (provided by applicant): The long-term objective of the current application is to elucidate novel molecular networks underlying mammalian meiosis. Meiosis, a process unique to germ cells, involves pairing, synapsis, recombination, and segregation of homologous chromosomes. Genetic abnormalities resulting from meiosis are a leading cause of birth defects and infertility in humans. The structural and functional properties of meiotic chromatin, which undergoes extensive reorganization, are undoubtedly the central theme of meiosis. Despite rapid progress in understanding meiosis, the complex interplay between chromatin organization and meiotic processes (such as synapsis and recombination) remains largely unknown. In particular, the progress in understanding mammalian meiosis has lagged far behind meiotic studies in other model organisms, due to several critical barriers: high cost, long duration, and the lack of sequence conservation of many meiosis-specific proteins across distant species. To overcome these roadblocks, we have developed an innovative proteomics approach to systematically identify a large number of uncharacterized mammalian meiotic chromatin-associated proteins in mice. The current application is to investigate the role of MEIOB, a novel meiosis-specific protein identified in our proteomics screen, in regulating meiotic recombination and chromosomal synapsis in mice. MEIOB is a sequence paralogue of the ubiquitously expressing RPA1. We find that MEIOB binds to single-stranded DNA (ssDNA) and exhibits 3' to 5' ssDNA-specific exonuclease activity. MEIOB forms distinct foci on meiotic chromosomes. In testes, MEIOB forms a complex with RPA and SPATA22. These proteins colocalize in foci on meiotic chromosomes. Furthermore, Meiob-null mutant mice of both sexes exhibit failures in meiotic recombination and chromosomal synapsis, resulting in sterility. Our results strongly support that MEIOB functions at a distinct step -second end capture - in meiotic recombination. Our specific aims are: 1) to examine promotion of single-stranded DNA annealing by MEIOB and its associated proteins; 2) to determine the requirement of MEIOB ssDNA-binding activity for meiotic recombination in vivo; 3) to elucidate the role of RPA1, a ubiquitously expressed ssDNA-binding protein, in meiotic recombination. Together, our studies will uncover the molecular mechanisms underlying the regulation of meiotic recombination in mammals by two related ssDNA-binding proteins MEIOB and RPA1, and will provide insights into the etiology of infertility and birth defects in humans.