Yeast chromatin structure and function

描述（由申请人提供）：我们的总体目标是确定染色体结构和染色质重塑酶如何影响基因组稳定性。特别是，我们感兴趣的是这些因素如何调节DNA双链断裂（DSB）的同源重组（HR）的修复，以及它们如何控制复制叉的进展和稳定性。这些途径中的任一种的缺陷直接影响细胞存活和基因组完整性的维持，导致突变、基因易位、总染色体重排或细胞致死。在过去的预算期间，开发了生化测定法来剖析染色质底物上HR的早期步骤，并重建了抑制重组并要求ATP依赖性染色质重塑的异染色质样结构。此外，Ino80.com染色质重塑酶被证明是体内复制叉稳定性的关键调节因子。我们的总体策略是继续利用生物化学和分子遗传学方法的强大组合来解剖DSB修复过程中和复制过程中染色质结构的动态，使用芽殖酵母作为实验系统。本提案中描述的实验涉及四个目标。第一个目的是研究染色质重塑酶在DSB加工中的作用。Ino80.com这个目标使用遗传方法来剖析Ino 80如何被招募到DSB以及它如何有助于处理。生化研究也将重建DSB处理在体外核小体基板。目的2描述了生物化学研究，研究在同源重组的早期步骤中，在最初的联合分子形成过程中发生的染色质结构的变化。目标3中描述的研究将使用体内和体外方法来研究Ino80.com和Htz 1组蛋白变体之间的功能相互作用。目的4描述了一种新的组合的单分子，分析超离心，组蛋白-组蛋白和组蛋白-DNA交联的方法来剖析爵士异染色质的结构特征。

DESCRIPTION (provided by applicant): Our overall objective is to determine how chromosome structure and chromatin remodeling enzymes influence genome stability. In particular, we are interested in how these factors regulate the repair of DNA double strand breaks (DSBs) by homologous recombination (HR) and how they control the progression and stability of replication forks. Defects in either of these pathways directly impact cell survival and maintenance of genome integrity, leading to mutations, gene translocations, gross chromosomal rearrangements, or cellular lethality. During the past budget period, biochemical assays were developed to dissect the early steps of HR on chromatin substrates, and heterochromatin-like structures were reconstituted that repress recombination and impose a requirement for ATP-dependent chromatin remodeling. In addition, the conserved Ino80.com chromatin remodeling enzyme was shown to be a key regulator of replication fork stability in vivo. Our general strategy is to continue to exploit a powerful combination of biochemical and molecular genetic approaches to dissect the dynamics of chromatin structure during the repair of DSBs and during the replication process, using budding yeast as the experimental system. Experiments described in this proposal address four aims. The first aim investigates the role of the Ino80.com chromatin remodeling enzyme in DSB processing. This aim uses genetic approaches to dissect how Ino80 is recruited to a DSB and how it contributes to processing. Biochemical studies are also described which will reconstitute DSB processing in vitro on nucleosomal substrates. Aim 2 describes biochemical studies that investigate changes in chromatin structure that occur during formation of the initial joint molecule during early steps of homologous recombination. Studies described in Aim 3 will use in vivo and in vitro methods to investigate functional interactions between Ino80.com and the Htz1 histone variant. Aim 4 describes a novel combination of single molecule, analytical ultracentrifugation, histone-histone and histone-DNA crosslinking methods to dissect the structural features of Sir heterochromatin.