Single Stranded DNA: The Genome's Achilles Heel

单链 DNA：基因组的致命弱点

• 批准号: 7299315
• 负责人: WENYI FENG
• 金额: $ 8.66万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-30 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7299315
• 关键词: Biological Assay; Cells; Chromosome Breakage; Chromosome Fragile Sites; Complex; Coupled; DNA; DNA Damage; DNA Sequence Rearrangement; DNA biosynthesis; DNA chemical synthesis; DNA mapping; Defect; Deoxyribonucleotides; Detection; Development; Diagnostic; Disease; Ensure; Eukaryota; Eukaryotic Cell; 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

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(申请人提供)：染色体DNA复制是一个高度精心设计的过程，必须以有序和及时的方式进行，以确保遗传信息的准确传递。我的总体目标是了解染色体DNA复制缺陷导致基因组不稳定和疾病发展的机制。这项研究计划的中心是探索这样一种假说，即复制分叉停滞和复制检查点缺陷突变体的不稳定是一种有害事件，会阻止基因组的完全合成，导致染色体断裂和基因组不稳定。我基于以下观察结果提出了这一假说：(1)酿酒酵母复制检查点缺陷的rad53突变细胞包含停滞的复制叉子，并在用遗传毒剂羟基脲(HU)处理后积累了大面积的单链DNA；(2)rad53细胞从短暂暴露于HU的作用中恢复的能力随着暴露时间的延长而降低；(3)这些突变细胞在短暂暴露于HU后的生存能力不能通过简单地推迟有丝分裂来挽救；(4)RAD53细胞在去除HU后，能够合成它们的大部分基因组DNA，但基因组的某些区域复制不足。 为了验证这一假设，我设计了三个具体目标来： (1)分别采用基因芯片密度转移、基因芯片单链DNA标记和脉冲场凝胶电泳法分析了RAD53细胞复制动力学、单链DNA产量和染色体完整性，探讨了复制叉不稳定性的后果。 (2)通过基因筛查来分离瞬时暴露于HU后的rad53致死性多拷贝抑制基因，以阐明复制叉不稳定性的机制和后果。 (3)采用我之前在酵母中开发的基因组ssDNA图谱技术，以(A)绘制人类细胞中复制的起点；(B)识别染色体脆弱部位，并绘制可能导致人类细胞疾病发展的基因组重排的潜在部位。 这一全面的研究计划将使我们能够更广泛地了解真核DNA复制中的缺陷如何导致基因组不稳定和人类疾病，如癌症。从长远来看，我希望这项研究将有助于推动人类疾病诊断和治疗工具的发展。