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中的错误。这些错误的主要原因在于错误的重组事件，定义减数分裂。相互重组，或交换，发生在前期I，是必要的拴同源染色体在一起，直到第一次减数分裂。通过形成DNA双链断裂（DSB）来启动双链化，然后将其加工以形成交叉（CO）或非交叉（NCO）。CO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，并防止紧密间隔的CO，这是一种称为干扰的过程，而从大量DSB中选择CO则需要严格的交叉稳态。干扰和交叉稳态都被认为在DSB修复过程的早期出现。在包括哺乳动物在内的许多生物体中已经描述了两种CO途径。I类途径由DNA错配修复（MMR）家族的减数分裂组分（MSH 4-MSH 5和MLH 1-MLH 3异二聚体）调节，而II类途径由MUS 81-EME 1调节。PI实验室的研究已经确定，两种途径之间存在一定程度的整合，这似乎是哺乳动物独有的，因此II类途径导致I类交叉中间体增加，如减数分裂染色体上MLH 1-MLH 3外观增加所证明的。通过I类途径的这种增加的通量维持了最终的交叉计数;可能是因为这些额外的I类事件取代了那些在不存在MUS 81的情况下不再发生的II类事件。或者，可能的是，募集第三交换途径以维持最终的交叉计数，因为Mlh 3和Mus 81的双突变体仍然保留残留的交叉。在任何一种情况下，CO途径之间的这种整合发生在后期前期I，表明一种新的机制，用于监测最终的CO输出，这是时间上不同于早期的干扰和稳态事件。本提案中的研究旨在了解这两种途径之间的整合是如何实现的。本文提供的初步数据指向这些事件的两种可能的介质：BLM解旋酶和新鉴定的BTBD 12核酸内切酶，ATM激酶的推定靶点。我们的总体假设是，CO途径的选择可能涉及通过两个监管机构，BLM和BTBD 12，其中每一个服务，以提供适当的基板CO加工和/或转移结构之间的途径，根据需要整合信号。具体目标是：（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BTBD 12的减数分裂作用以及ATM激酶如何调节该功能;（3）探索两条CO途径在后期前期I整合以产生适当交叉的机制。

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.