Genetic Pathways Effecting Crossover Control and Meiotic Recombination in Mammals

描述(申请人提供)：在人类，50%的自发性流产是由于第一次减数分裂时的不分离错误，而90%的唐氏综合症病例可以归因于母体减数分裂I的错误。这些错误的主要原因是定义减数分裂的重组事件的错误调节。互换重组或交换发生在前期I，对于将同源染色体系在一起直到第一次减数分裂是必不可少的。重组是由DNA双链断裂(DSB)的形成启动的，然后DNA双链断裂被加工成交叉(CO)或非交叉(NCO)。CO的频率和位置受到严格的调控，以确保每条染色体至少有一个CO，并防止紧密间隔的CO，这一过程被称为干扰，而从大量DSB中选择CO受到严格的交叉稳态的影响。干扰和交叉稳态都被认为是在DSB修复过程的早期出现的。已经在包括哺乳动物在内的许多生物体中描述了两条CO途径。I类途径由DNA错配修复(MMR)家族的减数分裂组分(MSH4-MSH5和MLH1-MLH3异源二聚体)调控，而II类途径由MUS81-EME1调控。PI实验室的研究已经确定，这两条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，因此，II类途径导致I类交叉中间产物的增加，这从减数分裂染色体上MLH1-MLH3出现的增加可见一斑。通过I类通路的这种增加的流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没有MUS81的情况下不能再发生的II类事件。或者，由于M1h3和MUS81的双突变体仍然保留残留的交叉，因此可能会招募第三个交叉途径来维持最终的交叉计数。在任何一种情况下，CO通路之间的这种整合都发生在前期I的晚期，这表明了一种新的机制来监测最终的CO输出，这种机制在时间上与早期的干扰和动态平衡事件不同。这项建议中的研究旨在了解这两条途径之间的这种整合是如何实现的。这里提供的初步数据指向了这些事件的两个可能的介体：BLM解旋酶和新发现的BTBD12内切酶，它可能是ATM激酶的靶标。我们的总体假设是，CO途径的选择可能涉及通过两个调节子BLM和BTBD12的整合信号，每个调节子都为CO加工提供合适的底物和/或根据需要在途径之间转移结构。其具体目的是：(1)研究BLM在哺乳动物生殖细胞减数分裂前期I的不同阶段中的作用；(2)了解BTBD12的减数分裂作用以及ATM激酶如何调节这一功能；以及(3)探索这两条CO通路在前期I晚期整合的机制，以产生适当的交叉计数。 公共卫生相关性：同源重组(HR)是一种细胞可以有效修复由各种病理过程引起的DNA损伤的机制。HR也是在减数分裂(产生配子的特殊细胞分裂：卵子和精子)过程中确保母本和父本染色体准确分离的主要机制。如果在这种接触中没有有效的人力资源，可能会出现自发的染色体异常，这可能会导致严重的人类疾病，如唐氏综合症，在人类群体中异常普遍。我们的实验室试图了解在配子发生过程中调节HR的分子事件，从而评估哪些途径可能导致人类减数分裂中的错误。

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. PUBLIC HEALTH RELEVANCE: Homologous recombination (HR) is a mechanism by which cells can efficiently repair DNA damage arising from various pathological processes. HR is also the major mechanism that ensures accurate segregation of maternal and paternal chromosomes during meiosis (the specialized cell division that gives rise to the gametes: eggs and sperm). Without efficient HR in this contact, spontaneous chromosome abnormalities can arise, which can result in serious human diseases such as Down syndrome, are exceptionally prevalent in the human population. Our laboratory seeks to understand the molecular events that regulate HR during gametogenesis and thereby to evaluate which pathways may contribute to the errors seen in human meiosis.