DNA replication checkpoints in fission yeast

裂殖酵母中的 DNA 复制检查点

• 批准号: 6574867
• 负责人: GENNARO D'URSO
• 金额: $ 32.15万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6574867
• 关键词: DNA damage; DNA repair; DNA replication; DNA replication origin; Saccharomyces cerevisiae; Schizosaccharomyces pombe; cell cycle; gene expression; genetic screening; genetic transcription; microarray technology; molecular cloning; mutant; polymerase chain reaction; protein structure function; tissue /cell culture; two dimensional gel electrophoresis; yeasts

DESCRIPTION (provided by applicant)Understanding the mechanisms that result in increased genetic instability is of great interest to those studying the development of cancer. Inhibition of DNA replication can lead to increases in the frequency of gross chromosomal rearrangements (GCRs), that can arise from DNA damage associated with stalled or collapsed replication forks. In the yeast, Schizosaccharomyces pombe, cells activate specific checkpoint pathways in response to DNA damage, and these checkpoints are essential to allow time for repair. We have discovered a novel checkpoint phenotype associated with mutants in DNA replication initiation proteins called rid (for replication initiation defective). When grown under semi-permissive conditions, rid mutants are delayed in the cell cycle but fail to activate the inter-S phase checkpoint. However, these cells still require the checkpoint kinase Chk1 for viability, suggesting an alternative checkpoint is activated. We propose to investigate the nature of the damage generated in rid mutants and to identify checkpoint/repair proteins required for rid viability. To do this, we will employ both genetic screens and gene microarray analysis. We will also determine whether mutation rates, including the incidence of GCRS, increase in the rid or checkpoint mutants. The results of the proposed experiments should provide a much better understanding of checkpoint pathways that respond to defects in the initiation of DNA replication. Considering the conserved aspects of many of these processes, our experimental findings are likely to be applicable to higher eukaryotic cells, including human. Moreover, our results will likely identify new drug targets for cancer therapy, and reveal potentially less mutagenic strategies to block cell cycle progression in cancer cells.

描述(由申请人提供)了解导致遗传不稳定性增加的机制对研究癌症发展的人非常感兴趣。抑制DNA复制可导致总染色体重排(GCRs)频率的增加，这可能是由于复制叉子停滞或坍塌导致的DNA损伤。在酵母中，细胞激活特定的检查点通路以响应DNA损伤，这些检查点对于有时间进行修复是必不可少的。我们在DNA复制启动蛋白中发现了一种与突变相关的新的检查点表型，称为RID(复制启动缺陷)。当在半允许的条件下生长时，RID突变体在细胞周期中被延迟，但无法激活S间期检查点。然而，这些细胞仍然需要检查点激酶Chk1才能存活，这表明另一个检查点被激活。我们建议调查RID突变体所产生的损伤的性质，并鉴定RID活性所需的检查点/修复蛋白。为此，我们将同时使用基因筛查和基因微阵列分析。我们还将确定突变率，包括GCR的发生率，是否会增加RID或检查点突变。拟议的实验结果应该可以更好地理解检查点路径，这些路径对DNA复制启动中的缺陷做出反应。考虑到其中许多过程的保守性，我们的实验结果很可能适用于包括人类在内的高等真核细胞。此外，我们的结果可能会确定癌症治疗的新药物靶点，并揭示潜在的较少突变的策略来阻止癌细胞的细胞周期进展。