Mechanism and Regulation of DNA recombination in Saccharomyces cerevisiae

酿酒酵母DNA重组机制及调控

• 批准号: 9912782
• 负责人: Grzegorz A Ira
• 金额: $ 30.65万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9912782
• 关键词: Address; Affect; Animal Model; Automobile Driving; Award; Bacteria; Biochemical; Biological Assay; Cell Cycle Regulation; Cells; Chemicals; Chromosomal Breaks; Chromosomes; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; DNA Sequence Rearrangement; DNA biosynthesis; DNA replication fork; Disease; Double Strand Break Repair; Eukaryota; Event; Evolution; FLP recombinase; Foundations; Future; Genetic; Genetic Recombination; Genome; Genomic Instability; Genomics; Goals; Grant; Health; Human; Investigation; Location; Maintenance; Malignant Neoplasms; Mediating; Meiosis; Methods; Minor; Mitotic; Molecular; Nature; Okazaki fragments; Pathway interactions; Play; Process; Proteins; Regulation; Replication Origin; Replication-Associated Process; Research; Role; Running; S Phase; SPO11 gene; Saccharomyces cerevisiae; Site; Structure; Testing; Work; Yeast Model System; cancer therapy; chromatin immunoprecipitation; cohesin; endodeoxyribonuclease SceI; endonuclease; experimental study; genetic information; helicase; homologous recombination; human disease; insight; irradiation; nuclease; prevent; recombinational repair; recruit; repaired; stem; tool

Homologous recombination (HR) plays an essential role in maintaining stability of genetic information, and even a minor deficiency in HR leads to severe diseases including cancer. Recombination repairs DNA double- strand breaks (DSBs) that occur spontaneously, or are induced by chemicals or irradiation such as used in cancer therapy. Nearly all we know about recombination processes comes from studies of two-ended double strand breaks (DSBs) induced by endonucleases (e.g. I-SceI, HO). However, it is well established that spontaneous chromosomal breaks are predominantly single-ended DSBs (seDSBs), as they arise during DNA replication when a replication fork runs into a nick. In bacteria that contain a single replication origin per genome, broken forks are repaired by Pri proteins capable of reloading the replisome at any genomic location. However, Pri proteins are not conserved, and the mechanism of broken fork repair in eukaryotes remains undefined. Our long-term goal is to understand the molecular mechanisms and regulation of DSB repair including broken replication fork repair, and to understand how deficiencies in these processes affect genomic instability. The objective of this project is to define the mechanistic features of Broken Fork Repair (BFR), which is the most common, yet poorly understood, type of DSB repair. We propose that eukaryotes repair broken replication forks using a combination of the structure-specific nuclease Mus81/Mms4 and a converging fork initiated at the next active or damage-activated origin. We further propose that this mechanism restricts the usage of highly mutagenic DNA synthesis via the well-characterized Break Induced Replication (BIR) process. The central question is whether eukaryotes are able to reestablish replication forks at the site of fork breakage as demonstrated in bacteria. What are the genetic requirements for broken fork repair and how do they differ from mutagenic BIR? What is the fate of replisome proteins at broken forks? These questions will be addressed in the yeast model organism Saccharomyces cerevisiae, where all replication origins are annotated and Flp recombinase-induced broken fork assays are available. We will define whether functional forks can be reestablished and whether dormant origins are activated in the vicinity of the broken fork using the hydrolytic end sequencing (HydEn-seq) method. The stability of the replisome after fork breakage will be studied using chromatin immunoprecipitation. We will also study the role and regulation of structure-specific nucleases in the repair of broken forks. The most common types of genomic rearrangements that occur during BFR and BIR stem from template switches and half crossovers. We will identify the genetic requirements for these events. At the conclusion of this project we expect to: (i) provide new molecular tools to study BFR, (ii) delineate the major mechanism of BFR, and (iii) uncover mechanisms that prevent mutagenic BIR, which is believed to account for a significant fraction of genomic rearrangements associated with human disease. Our work strives to define conserved pathways for the maintenance of chromosome integrity and has strong relevance to human health.

同源重组（HR）在维持遗传信息的稳定性和 即使是人力资源的少量缺乏也会导致包括癌症在内的严重疾病。重组维修DNA双重 自发发生或通过化学物质或辐射引起的链断裂（DSB） 癌症治疗。我们对重组过程的了解几乎来自对两端双重的研究 核酸内切酶诱导的链断裂（DSB）（例如I-SCEI，HO）。但是，已经确定了 自发性染色体断裂主要是单端DSB（SEDSB），因为它们在DNA期间出现 复制叉会陷入划痕时复制。在包含单个复制起源的细菌中 基因组，断裂的叉子通过能够在任何基因组位置重新加载重新加载的PRI蛋白来修复。 但是，PRI蛋白不是保守的，而真核生物中叉骨修复的机制仍然存在 不明确的。我们的长期目标是了解DSB修复的分子机制和调节 包括破裂的复制叉修复，并了解这些过程中的缺陷如何影响基因组 不稳定。该项目的目的是定义破碎的叉修理（BFR）的机械特征， 这是DSB修复的最常见，但知之甚少的类型。我们建议真核生物修复 使用结构特异性核酸酶MUS81/MMS和收敛的结合使用折断复制叉 在下一个活动或损坏激活的起源处启动的叉子。我们进一步提出，这种机制限制了 通过特征良好的断裂诱导复制（BIR）过程来使用高度诱变的DNA合成。 中心问题是真核生物是否能够重新建立叉子破裂现场的复制叉 如细菌所示。破碎的叉子维修的遗传要求是什么？它们有何不同 来自诱变BIR？破碎的叉子在破碎的蛋白质上的命运是什么？这些问题将是 在酵母模型生物糖酿酒酵母中的解决，所有复制起源都是注释的 和FLP重组酶诱导的破叉测定法。我们将定义功能叉是否可以 重建以及是否使用水解的叉子附近激活休眠的起源 结束测序（Hyden-Seq）方法。将使用叉子断裂后重新分散体的稳定性研究 染色质免疫沉淀。我们还将研究结构特异性核酸酶的作用和调节 修理破碎的叉子。 BFR和BIR期间发生的最常见的基因组重排类型 茎来自模板开关和半跨界。我们将确定这些事件的遗传要求。在 我们期望该项目的结论：（i）提供研究BFR的新分子工具，（ii）描述 BFR的主要机制和（iii）揭示了预防诱变BIR的机制，据信这是 占与人类疾病相关的基因组重排的很大一部分。我们的工作努力 定义维持染色体完整性的保守途径，并与 人类健康。