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方面起着至关重要的作用， 即使是轻微的HR缺陷也会导致包括癌症在内的严重疾病。DNA双链修复 自发发生的或由化学品或辐射诱导的链断裂（DSB），例如 癌症治疗。几乎所有我们对重组过程的了解都来自于对双端双链的研究。 由核酸内切酶（例如I-SceI、HO）诱导的链断裂（DSB）。然而，众所周知， 自发的染色体断裂主要是单端DSB（seDSB），因为它们在DNA断裂过程中出现。 当复制叉进入缺口时，在含有单个复制起点的细菌中， 在基因组中，断裂的叉由能够在任何基因组位置重新加载复制体的Pri蛋白修复。 然而，Pri蛋白并不保守，真核生物中的断叉修复机制仍然存在 未定义。我们的长期目标是了解DSB修复的分子机制和调控 包括断裂的复制叉修复，并了解这些过程中的缺陷如何影响基因组 不稳定本项目的目标是确定断裂叉修复（BFR）的机械特征， 这是最常见但知之甚少的DSB修复类型。我们认为真核生物修复 使用结构特异性核酸酶Mus 81/Mms 4和聚合酶的组合破坏复制叉。 在下一个活动或损坏激活的起源处启动分叉。我们进一步建议，这一机制限制了 通过充分表征的断裂诱导复制（BIR）过程使用高度致突变的DNA合成。 核心问题是真核生物是否能够在复制叉断裂的部位重新建立复制叉 如细菌中所示。断叉修复的遗传要求是什么，它们有什么不同 诱变BIR吗复制体蛋白在分叉处的命运是什么？这些问题将 在酵母模式生物酿酒酵母中解决，其中所有复制起点都有注释 和Flp重组酶诱导的断叉测定法是可用的。我们将定义函数式分叉是否可以 重新建立和休眠的起源是否被激活附近的断裂叉使用水解 末端测序（HydEn-seq）方法。复制体在叉断裂后的稳定性将使用 染色质免疫沉淀。我们还将研究结构特异性核酸酶在细胞中的作用和调节 修理坏掉的叉子。在BFR和BIR期间发生的最常见的基因组重排类型 源于模板交换和半交叉。我们将确定这些事件的遗传要求。在 本项目的结论，我们期望：（i）提供新的分子工具来研究BFR，（ii）描绘 BFR的主要机制，和（iii）揭示防止诱变BIR的机制，据信 占与人类疾病相关的基因组重排的很大一部分。我们的工作力求 以确定维持染色体完整性的保守途径，并与以下方面有很强的相关性： 人体健康