Single Stranded DNA: The Genome's Achilles Heel

描述（由申请人提供）：染色体DNA复制是一个高度编排的过程，必须以有序和及时的方式进行，以确保遗传信息的准确传输。我的总体目标是了解染色体DNA复制缺陷引起基因组不稳定和疾病发展的机制。本研究计划围绕探索以下假设：复制检查点缺陷突变体中复制叉停滞和不稳定时的单链DNA（ssDNA）产生是一种有害事件，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。我的假设基于以下观察：（1）酿酒酵母复制检查点缺陷的rad 53突变细胞含有停滞的复制叉，并在用遗传毒性剂羟基脲（HU）处理后积累大区域的ssDNA;（2）rad 53细胞从短暂暴露于HU中恢复的能力随着暴露持续时间的增加而降低;（3）这些突变细胞在短暂暴露于HU后的生存力不能通过简单地延迟有丝分裂来挽救;（4）rad 53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复制不足。 为了验证这一假设，我设计了三个具体目标： (1)分别通过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分析复制动力学、ssDNA产生和染色体完整性，研究在HU中使用rad 53细胞的复制叉不稳定性的后果。 (2)通过进行遗传筛选以分离短暂暴露于HU后rad 53致死性的多拷贝抑制因子，阐明复制叉不稳定性的机制和后果。 (3)将我以前在酵母中开发的基因组ssDNA作图技术应用于（a）绘制人类细胞中的复制起点;（B）鉴定染色体脆性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复制缺陷如何引起基因组不稳定性和人类疾病，如癌症。从长远来看，我希望这项研究将有助于促进人类疾病诊断和治疗工具的发展。

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.