Single Stranded DNA: The Genome's Achilles Heel

单链 DNA：基因组的致命弱点

基本信息

批准号：
7882988
负责人：
WENYI FENG
金额：
$ 7.32万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-20 至 2010-08-15
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7882988
关键词：
Biological Assay Cells Chromosome Breakage Chromosome Fragile Sites Complex Coupled DNA DNA Damage DNA Sequence Rearrangement DNA biosynthesis DNA mapping Defect Deoxyribonucleotides Detection Development Diagnostic Disease Ensure Eukaryota Event Excision Exposure to Generations Genes Genetic Genetic Processes Genetic Screening Genome Genomic Instability Genomics Goals Heel Human Human Genome Incubated Inherited Karyotype determination procedure Label Lead Location Malignant Neoplasms Maps Methods Mitosis Molecular Mutagens Mutation Organism Partner in relationship Pharmaceutical Preparations Phosphotransferases Principal Investigator Process Production Pulsed-Field Gel Electrophoresis Recovery Replication Origin Replication-Associated Process Reporting Research Research Personnel Role Saccharomyces cerevisiae Screening procedure Secure Single-Stranded DNA Site Southern Blotting Suppressor Genes Survivors Techniques Testing Work Yeasts base comparative genomic hybridization density human disease hydroxyurea member mutant prevent programs tool transmission process

项目摘要

DESCRIPTION (provided by applicant): Chromosomal DNA replication is a highly choreographed process that must take place in an orderly and timely fashion to secure the accurate transmission of genetic information. My general objectives are to understand the mechanisms by which defects in chromosomal DNA replication give rise to genomic instability and disease development. This research plan is centered around exploring the hypothesis that single-stranded DNA (ssDNA) production upon replication fork stalling and destabilization in replication checkpoint-deficient mutants is a detrimental event that prevents complete synthesis of the genome, causes chromosomal breakage and genomic instability. I base this hypothesis on the following observations: (1) Saccharomyces cerevisiae replication checkpoint-deficient rad53 mutant cells contain stalled replication forks and accumulate large regions of ssDNA upon treatment with the genotoxic agent hydroxyurea (HU); (2) the ability of rad53 cells to recover from transient exposure to HU decreases as the duration of exposure increases; (3) viability of these mutant cells after transient exposure to HU cannot be rescued by simply delaying mitosis; (4) rad53 cells, upon removal of HU, are able to synthesize much of their genomic DNA, but certain regions of the genome are under-replicated. In order to test this hypothesis, I devised three specific aims to: (1) Investigate the consequence of replication fork instability using rad53 cells in HU through analyzing replication dynamics, ssDNA production, and chromosomal integrity by microarray-based density transfer, microarray-based genomic ssDNA labeling, and pulse field gel electrophoresis, respectively. (2) Elucidate the mechanism and the consequence of replication fork instability by conducting a genetic screen to isolate multi-copy suppressors of rad53 lethality after transient exposure to HU. (3) Adapt a genomic ssDNA mapping technique that I previously developed in yeast to (a) map origins of replication in human cells; (b) identify chromosomal fragile sites and map potential sites of genomic rearrangement that may lead to disease development in human cells. This comprehensive research plan will enable us to gain broader understanding of how defects in eukaryotic DNA replication give rise to genomic instability and human diseases such as cancer. In the long term, I hope that this research will aid in promoting the development of diagnostic and treatment tools for human diseases.

描述（由申请人提供）：染色体DNA复制是一个精心编排的过程，必须以有序，及时的方式进行，以确保遗传信息的准确传播。我的一般目标是了解染色体DNA复制中缺陷的机制会导致基因组不稳定性和疾病发育。该研究计划集中在探索以下假设：复制叉子后单链DNA（ssDNA）产生复制和不稳定的复制检查点缺陷点突变体是一个有害事件，可防止基因组的完全融合，导致染色体破裂和基因组不稳定。我基于以下观察结果为基础：（1）酿酒酵母复制检查点缺陷rad53突变细胞包含停滞的复制叉，并在用遗传毒性剂羟基脲（HU）处理后积累了大型ssDNA区域；（2）随着暴露持续时间的增加，Rad53细胞从短暂暴露中恢复为HU的能力会降低；（3）这些突变细胞在短暂暴露于HU后的生存能力不能简单地延迟有丝分裂来挽救；（4）RAD53细胞在去除HU后能够合成其大部分基因组DNA，但基因组的某些区域的重复不足。为了检验这一假设，我设计了三个具体目标：（1）通过分析基于微阵列的密度转移，基于微阵列的基因组ssDNA标记和脉冲场凝胶电泳，研究了使用RAD53细胞在HU中使用RAD53细胞的复制叉不稳定性的结果。（2）通过进行遗传筛查来分离瞬时暴露于HU后的Rad53致死性的多拷贝抑制器，阐明了复制叉不稳定性的机制和后果。（3）调整我以前在酵母中开发的基因组ssDNA映射技术，以（a）人类细胞中复制的映射起源；（b）鉴定染色体脆弱的位点，并绘制可能导致人类细胞疾病发展的基因组重排的潜在位点。这项全面的研究计划将使我们能够更广泛地了解真核DNA复制中的缺陷如何产生基因组不稳定性和癌症等人类疾病。从长远来看，我希望这项研究将有助于促进针对人类疾病的诊断和治疗工具的开发。