Meiotic Chromosome Synapsis and Recombination in Yeast

 描述(申请人提供)：我们研究了减数分裂过程中同源染色体的通讯过程。减数分裂是有性繁殖配子形成的特殊细胞周期。重要的是，减数分裂缺陷是导致人类不孕不育和几种遗传病的原因，特别是唐氏综合症。拟议的研究涉及三个大的领域。I.我们研究了染色体事件如何在没有直接遗传指定位置的情况下以均匀分布的模式发生。具体地说，这种模式表现在减数分裂交叉，最初表现为“交叉干扰”的遗传现象，随后通过细胞学分析延长的中期前期染色体上的交叉相关结构，后来作为部分压缩的双线染色体(“交叉”)上的同源物之间的连接点。我们正在使用成像、蛋白质组学、遗传学、数学模拟和激光纳米外科来解决三个问题：(A)干扰涉及哪些分子？(B)这种模式的生物学影响是什么？(C)通讯是否如我们所建议的那样，通过机械应力的重新分布而发生？我们研究同源染色体配对，这是减数分裂计划的一个中心普遍特征，再次通过三个入口点。(A)我们开发了一种独特的强大系统，用于3D跟踪活的减数分裂酵母细胞中的同源配对Fas焦点，整个过程中都会收集密集的图像 减数分裂时期。该系统准备解决重组依赖和独立配对以及配对期间运动的性质和作用。(B)减数分裂同源配对包括避免和主动消除纠缠(互锁)。通过跟踪Sordaria的染色体路径，我们现在可以回答两个关键问题。(I)互锁决议的机制是什么(通过TopOII或端粒引导的运动)？(Ii)经典花束阶段是调节配对的初始同系接触还是主要参与确保规则的拓扑关系？(C)我们的证据表明，粗糙脉孢菌的重复诱导点突变(RIP)涉及重组非依赖的DNA/DNA配对。作为DSB独立配对的第二种方法，我们正在搜索RIP所需的函数。减数分裂重组发生在与染色体结构轴密切的物理和功能相互作用中，一旦轴之间形成联会复合体(SC)。在Sordaria，我们将扩展最近的发现：(A)开始阐明安装SC时关键的中期-前期转变事件的分子途径；(B)提出一个关于染色单体轴如何在DNA交叉部位变得“交换”的新想法；并揭示与乳腺癌相关的BRCA2对染色体具有不同的作用，而不是归因于它作为RAD51介体的典型作用。最后，为了打开一个全新的了解减数分裂过程的窗口，我们将开发一个鉴定和分析Sordaria减数分裂非编码RNA的系统。

 DESCRIPTION (provided by applicant): We investigate the processes involved in communication along and between homologous chromosomes during meiosis, the specialized cell cycle that underlies gamete formation for sexual reproduction. Importantly, defects in meiosis underlie human infertility and several genetic diseases, notably Downs Syndrome. Proposed research addresses three broad areas. I. We investigate how chromosomal events can occur in an evenly-spaced pattern without direct genetic specification of position. In particular, such patterning is exhibited by meiotic crossovers, manifested originally in the genetic phenomenon of "crossover interference" and subsequently by cytological analyses of crossover-correlated structures along elongated mid-prophase chromosomes and later as points of connections between homologs along partially compacted diplotene chromosomes ("chiasmata"). We are using imaging, proteomics, genetics, mathematical simulations, and laser nanosurgery to address three questions: (A) What molecules are involved in interference? (B) What are the biological implications of this patterning? (C) Does communication occur by redistribution of mechanical stress, as we have proposed? II. We investigate homologous chromosome pairing, a central universal feature of the meiotic program, again by three entry points. (A) We have developed a uniquely powerful system for 3D tracking of homologously-pairing FROS foci in living meiotic yeast cells, with images collected densely over the entire time period of meiosis. This system is poised to address recombination-dependent and -independent pairing and the nature and roles of motion during the pairing period. (B) Meiotic homolog pairing involves both avoidance and active elimination of entanglements (interlocks). By tracking chromosome paths in Sordaria, we can now answer two key questions. (i) What is the mechanism of interlock resolution (by TopoII or by telomere-led movement)? (ii) Does the classical bouquet stage mediate initial homolog contact for pairing or is it involved primarily in ensuring regular topological relationships? (C) Our evidence suggests that repeat-induced point mutation (RIP) in Neurospora crassa involves recombination-independent DNA/DNA pairing. As a second approach to DSB-independent pairing, we are searching for functions required for RIP. III. Meiotic recombination occurs in close physical and functional interaction with chromosome structural axes and, once it forms between axes, the synaptonemal complex (SC). In Sordaria, we will extend recent findings which: (A) begin to elucidate a molecular pathway of events at the critical mid-prophase transition when SC is installed; (B) suggest a new idea for how chromatid axes become "exchanged" at sites of DNA crossovers; and reveal that breast cancer-related BRCA2 has diverse roles for chromosomes beyond those attributable to its canonical role as a Rad51 mediator. Finally, to open an entirely new window onto the meiotic process, we will develop a system for identification and analysis of Sordaria meiotic non-coding RNAs.