Regulation of DNA recombination in Saccharomyces cerevisiae

酿酒酵母 DNA 重组的调控

基本信息

批准号：
8667465
负责人：
Grzegorz A Ira
金额：
$ 29.55万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-05-01 至 2017-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8667465
关键词：
Aging Animal Model Biochemical Biological Assay Cell Cycle Regulation Cells Chemicals Chromosomal Breaks Cleaved cell DNA DNA Double Strand Break DNA Repair DNA Repair Pathway DNA Sequence Rearrangement DNA Structure DNA biosynthesis DNA-Directed DNA Polymerase Data Defect Development Disease Double Strand Break Repair Enzymes Eukaryota Event Evolution Gene Conversion Genetic Genetic Recombination Genome Genomic Instability Goals Human Immune Infertility Invaded Isotopes Lead Lesion M cell Malignant Neoplasms Mediating Modeling Molecular Molecular Genetics Mutagenesis Neurodegenerative Disorders Pathway interactions Play Polymerase Process Proteins Rad51 recombinase Radiation therapy Reaction Recruitment Activity Regulation Research Resolvase Role Saccharomyces cerevisiae Sequence Homologs Site Telomerase Telomere Maintenance Testing Work cancer therapy chemotherapy chromatin immunoprecipitation cytotoxic density helicase homologous recombination in vivo irradiation migration novel nuclease prevent reconstitution repaired research study

项目摘要

DNA recombination is an essential process that repairs DNA double-strand breaks (DSBs) and gaps that occur spontaneously, or are induced by chemicals or irradiation. In human, defects in recombination result in immune deficiencies, infertility, neurodegenerative disorders, developmental abnormalities, aging and cancer. DSBs as the most cytotoxic lesions are a fundamental component of the most prevalent cancer treatments, radiotherapy and radiomimetic chemotherapy. Therefore defining the genetic requirements and mechanisms of recombination pathways is of critical importance. A fundamental reaction during repair of broken chromosomes by recombination is DNA synthesis that copies homologous sequences from a template DNA molecule. The goal of this project is to understand the mechanisms and regulation of DNA synthesis during recombination, which remains very poorly understood. Two assays will be utilized to examine repair DNA synthesis that reflect two major recombination pathways with distinct DNA synthesis features. Both pathways play different yet important roles in cells. One is the simple repair of two-ended DSBs by gene conversion where both 3' ends prime DNA synthesis and thus only short leading strands are synthesized. The second assay employs break induced replication (BIR), in which a single DSB end invades a template, followed by extensive leading- and lagging- strand DNA synthesis. BIR is thought to be a mechanism of HR-dependent telomere maintenance in the absence of telomerase found in 10- 15% of all cancers. The specific aims are: (1) To understand the unique and redundant functions of multiple DNA polymerases recruited to DSBs and determine which DNA helicases and other enzymes specifically promote DNA synthesis during homologous recombination. The major focus will be on studying Pif1, the DNA helicase that we propose to be the first eukaryotic nonreplicative helicase that stimulates DNA synthesis during recombination. (2) To understand the mechanism of Break Induced Replication. Using isotope density transfer we will establish the mode of DNA synthesis in BIR and determine the role of DNA helicases and structure specific nucleases in BIR. Together we will provide a comprehensive view of DNA synthesis during homologous recombination.

DNA重组是维修DNA双链断裂（DSB）和自发发生或通过化学物质或辐照引起的间隙。在人类中，缺陷重组导致免疫缺陷，不育，神经退行性疾病，发育异常，衰老和癌症。 DSB作为最多的细胞毒性病变是最普遍的癌症治疗，放射疗法和放射性化疗。因此定义了遗传要求和机制重组途径至关重要。修复破裂期间的基本反应重组染色体是DNA的合成，它复制了来自A的同源序列模板DNA分子。该项目的目的是了解机制和调节重组过程中DNA合成的调节，这仍然非常了解。二测定将用于检查修复DNA合成，反映了两个主要重组具有不同DNA合成特征的途径。这两种途径都扮演着不同但重要的角色在细胞中。一个是通过基因转换对两端DSB的简单修复，这两个末端都主要DNA合成，因此仅合成了短领导链。第二个测定法采用休息诱导的复制（BIR），其中单个DSB结束入侵模板，其次是广泛的前导和滞后DNA合成。 BIR被认为是在没有10-中发现的端粒酶的情况下，HR依赖性端粒维持的机制所有癌症中有15％。具体目的是：（1）了解独特和多余的募集到DSB的多种DNA聚合酶的功能并确定哪些DNA解旋酶和其他酶在同源重组过程中特异性促进了DNA合成。这主要重点将是研究PIF1，这是我们建议成为第一个真核生物的DNA解旋酶在重组过程中刺激DNA合成的非复制解旋酶。（2）理解破裂的机制引起了复制。使用同位素密度转移我们将建立 DNA合成在BIR中的模式，并确定DNA解旋酶和结构的作用 BIR中的特定核酸酶。我们将共同提供DNA合成的全面视图在同源重组期间。