Yeast chromatin structure and function

描述(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通过同源重组(HR)调节DNA双链断裂(DSB)的修复，以及它们如何控制复制分叉的进展和稳定性。这两个途径中的任何一个的缺陷都直接影响细胞的生存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死亡。在过去的预算期间，发展了生化分析来剖析染色质底物上HR的早期步骤，并重组了异染色质样结构，以抑制重组并要求依赖于ATP的染色质重塑。此外，保守的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。我们的总体战略是继续利用生化和分子遗传学的强大组合来剖析DSB修复和复制过程中染色质结构的动态，使用萌芽酵母作为实验系统。这项提案中描述的实验解决了四个目标。第一个目的是研究Ino80.com染色质重塑酶在DSB加工中的作用。这个目标使用遗传方法来剖析INO80是如何被招募到DSB的，以及它是如何促进加工的。还描述了将在核小体底物上重建体外DSB过程的生物化学研究。目的2描述了在同源重组的早期阶段，在初始关节分子形成过程中发生的染色质结构变化的生化研究。AIM 3中描述的研究将使用体内和体外方法来研究Ino80.com和Htz1组蛋白变体之间的功能相互作用。目的4采用单分子、分析超速离心法、组蛋白-组蛋白和组蛋白-DNA交联法研究SIR异染色质的结构特征。 与公共卫生相关：染色体结构在调节细胞分裂过程中DNA的修复和DNA的忠实复制方面发挥着核心作用。因此，这些途径中的缺陷可能会影响细胞的存活和基因组完整性的维持。这里描述的研究将调查调节这些事件的关键酶和结构蛋白的作用。

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. PUBLIC HEALTH RELEVANCE: Chromosome structure plays a central role in regulating the repair of DNA and the faithful copying of DNA during cell division. Consequently, defects in these pathways can impact cell survival and maintenance of genome integrity. The studies described here will investigate the role of key enzymes and structural proteins that regulate these events.