Genetic Pathways Effecting Crossover Control and Meiotic Recombination in Mammals

描述（由申请人提供）：在人类中，50%的自发流产是由于第一次减数分裂时的非分离错误，而90%的唐氏综合征病例可归因于母体减数分裂i的错误。这些错误的主要原因是减数分裂的重组事件调节不当。互惠重组，或交叉，发生在前期I，是必要的系在一起同源染色体直到第一次减数分裂。重组是由DNA双链断裂（DSB）的形成开始的，然后被加工成交叉（CO）或非交叉（NCO）。CO的频率和位置受到严格调控，以确保每条染色体至少有一个CO，并防止CO间隔太近，这一过程被称为干扰，而从大量dsb中选择CO则受到严格的交叉稳态控制。干扰和交叉稳态都被认为出现在DSB修复过程的早期。在包括哺乳动物在内的许多生物中，已经描述了两种CO途径。I类途径由DNA错配修复（MMR）家族的减数分裂成分（MSH4-MSH5和MLH1-MLH3异源二聚体）调控，II类途径由MUS81-EME1调控。PI实验室的研究已经确定，这两种途径之间存在一定程度的整合，这似乎是哺乳动物所特有的，例如，II类途径导致I类交叉中间体的增加，减数分裂染色体上MLH1-MLH3出现的增加就证明了这一点。通过I类通路增加的通量维持了最终的交叉数；大概是因为这些额外的I类事件取代了那些在没有MUS81时不能再发生的II类事件。另一种可能是，由于Mlh3和Mus81的双突变体仍然保留了残留的交叉，因此可能招募了第三种交叉途径来维持最终的交叉计数。无论哪种情况，CO通路之间的整合发生在前期I的后期，这表明了一种监测最终CO输出的新机制，这种机制在时间上与早期的干扰和稳态事件不同。本提案中的研究旨在了解如何实现这两种途径之间的整合。本文提出的初步数据指出了两种可能的介质：BLM解旋酶和新发现的BTBD12内切酶，这是ATM激酶的一个假定靶点。我们的总体假设是，CO途径的选择可能涉及通过两个调节器（BLM和BTBD12）的综合信号传导，每个调节器都为CO处理提供适当的底物和/或根据需要在途径之间转移结构。具体目的是：(1)研究BLM在哺乳动物生殖细胞减数分裂前期I不同阶段的作用；(2)了解BTBD12在减数分裂中的作用以及ATM激酶如何调控这一功能；(3)探索两种CO通路在I前期后期的整合机制，以产生适当的交叉计数。

DESCRIPTION (provided by applicant): In humans, 50% of all spontaneous miscarriages are due to non-disjunction errors at the first meiotic division, while 90% of Down syndrome cases can be attributed to errors in maternal meiosis I. The predominant cause of these errors lies with misregulation of the recombination events that define meiosis. Reciprocal recombination, or crossing over, occurs during prophase I and is essential for tethering homologous chromosomes together until the first meiotic division. Recombination is initiated by the formation of DNA double-strand breaks (DSB) that are then processed to form either crossovers (CO) or noncrossovers (NCO). CO frequency and placement is tightly regulated to ensure at least one CO per chromosome and to prevent closely spaced CO, a process known as interference, while selection of COs from a large pool of DSBs is subject to stringent crossover homeostasis. Both interference and crossover homeostasis are thought to arise early in the DSB repair process. Two CO pathways have been described in a number of organisms, including mammals. The class I pathway is regulated by the meiotic components of the DNA Mismatch repair (MMR) family (MSH4-MSH5 and MLH1-MLH3 heterodimers), while the class II pathway is regulated by MUS81-EME1. Studies in the PI's laboratory have determined that there is a degree of integration between the two pathways that appears to be unique to mammals, such that the Class II pathway leads to an increase in Class I crossover intermediates, as demonstrated by the increase in MLH1-MLH3 appearance on meiotic chromosomes. This increased flux through the Class I pathway maintains the final chiasmata count; presumably because these additional Class I events replace those Class II events that can no longer occur in the absence of MUS81. Alternatively, it is possible that a third crossover pathway is recruited to maintain the final chiasmata tally, since double mutants for both Mlh3 and Mus81 still retain residual chiasmata. In either case, this integration between CO pathways occurs late in prophase I, indicating a novel mechanism for monitoring final CO output that is temporally distinct from the earlier interference and homeostasis events. Studies in this proposal are aimed at understanding how this integration between the two pathways is achieved. Preliminary data presented herein point towards two possible mediators of these events: BLM helicase, and the newly-identified BTBD12 endonuclease, a putative target of the ATM kinase. Our overall hypothesis is that the choice of CO pathway may involve integrated signaling through two regulators, BLM and BTBD12, each of which serve either to provide the appropriate substrate for CO processing and/or to divert structures between pathways, as needed. The specific aims are: (1) to examine the role of BLM at different stages of meiotic prophase I in mammalian germ cells; (2) to understand the meiotic role of BTBD12 and how this function is regulated by ATM kinase; and (3) to explore the mechanisms by which the two CO pathways are integrated in late prophase I to produce the appropriate tally of chiasmata.