Regulation of mitotic genome stability in yeast.

 描述(申请人提供)：有效修复自发和诱导的DNA损伤是维持真核基因组有丝分裂稳定性的关键。拟议的实验将使用萌芽酵母作为模型，以(1)检查定义的双链断裂的修复，(2)检查转录如何导致潜在DNA模板的不稳定，以及(3)确定在未分裂细胞中发生的遗传变化的性质和机制。双链断裂(DSB)是一种毒性特别大的DNA损伤，有两种不同的修复途径：同源重组(HR)和非同源末端连接(NHEJ)。NHEJ途径连接经常需要处理的末端，这会导致关节处序列的丢失/获得，并使该过程非常容易出错。相比之下，HR通过从完整的染色体复制信息来修复断裂的分子，因此被认为是一个高保真的过程。尽管如此，HR可以改变启动DSB两侧的序列的连接，导致杂合性丧失，或者可以参与分散的重复序列，以产生染色体重排。虽然不是在酵母中生存所必需的，但HR和NHEJ在哺乳动物中都是必不可少的，缺陷已与大量人类疾病有关，包括神经系统疾病、免疫系统功能障碍、早衰综合征和癌症。拟议的实验将定义DSB修复的分子中间体和遗传机制，重点是末端结构/序列如何影响遗传结果。此外，还将研究与自发与DSB诱导的重组相关的链交换中间体，以解决该领域长期存在的一个问题：DSB与单链间隙对自发HR的相对贡献。我们已经证明，转录破坏了潜在的DNA模板的稳定，导致局部升高的突变，从而改变了整个基因组的突变格局。转录相关突变()在很大程度上源于拓扑异构酶1(Top1)的活性，Top1是一种用来消除转录产生的DNA扭转应力的酶。拟议的实验将集中在Top1依赖的突变以及转录和非转录DNA链之间是否存在损伤和突变积累方面的不对称。虽然对双链断裂修复和的研究传统上是通过指数分裂的细胞来完成的，但在未分裂的细胞中也会出现遗传变化。这种变化与人类有丝分裂后组织中发生的变化特别相关，这种变化可能会导致疾病和肿瘤的形成。我们将研究由和NHEJ引起的突变，它们中的每一个都可以是一个复制独立的过程，在不分裂的酵母细胞中。总之，这些研究将为真核细胞分裂和非分裂时基因组稳定性的调节建立新的范式。

 DESCRIPTION (provided by applicant): Efficient repair of spontaneous and induced DNA damage is critical for maintaining the mitotic stability of eukaryotic genomes. The proposed experiments will use budding yeast as a model to (1) examine the repair of defined double-strand breaks, (2) examine how transcription leads to instability of the underlying DNA template, and (3) define the nature and mechanism of genetic changes that occur in non-dividing cells. Double-strand breaks (DSBs) are an especially toxic DNA lesion and are repaired by two distinct pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). The NHEJ pathway ligates ends that often require processing, which results in loss/gain of sequence at the joint and renders the process highly error-prone. By contrast, HR restores a broken molecule by copying information from an intact chromosome, and thus is considered a high-fidelity process. Even so, HR can alter the linkage of sequences that flank an initiating DSB to result in loss of heterozygosity, or can engage dispersed repeated sequences to generate chromosome rearrangements. Though not required for survival in yeast, both HR and NHEJ are essential in mammals and defects have been linked to a large number of human diseases that include neurological disorders, immune system dysfunction, premature aging syndromes and cancer. Proposed experiments will define molecular intermediates and genetic mechanisms of DSB repair, with a focus on how end structure/sequence affects genetic outcomes. In addition, strand-exchange intermediates associated with spontaneous versus DSB-induced recombination will be examined to resolve a long-standing issue in the field: the relative contribution of DSBs versus single-strand gaps to spontaneous HR. We have shown that transcription destabilizes the underlying DNA template, resulting in locally elevated mutagenesis that alters the mutation landscape across the genome. Transcription-associated mutagenesis (TAM) derives largely from the activity of Topoisomerase 1 (Top1), an enzyme recruited to remove DNA torsional stress created by transcription. Proposed experiments will focus on Top1-dependent mutagenesis and on whether there is an asymmetry between the transcribed and non-transcribed DNA strands in terms of damage and mutation accumulation. Though studies of DSB repair and TAM are traditionally done using exponentially dividing cells, genetic changes also arise in non-dividing cells. Such changes are particularly relevant to alterations that occur in post-mitotic human tissue, which can drive disease and tumor formation. We will examine mutagenesis due to TAM and NHEJ, each of which can be a replication-independent process, in non-dividing yeast cells. Together, these studies will establish new paradigms for the regulation of genome stability in dividing as well as non-dividing eukaryotic cells.