DNA replication checkpoints in fission yeast

裂殖酵母中的 DNA 复制检查点

• 批准号: 6947138
• 负责人: GENNARO D'URSO
• 金额: $ 3.45万
• 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/6947138
• 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于那些研究癌症发展的人来说非常感兴趣。 DNA 复制的抑制可能导致染色体总重排 (GCR) 频率的增加，这种重排可能是由于与复制叉停滞或崩溃相关的 DNA 损伤而引起的。在粟酒裂殖酵母中，细胞会激活特定的检查点途径来响应 DNA 损伤，这些检查点对于留出修复时间至关重要。我们发现了一种与 DNA 复制起始蛋白突变体相关的新检查点表型，称为“rid”（复制起始缺陷）。当在半允许条件下生长时，rid 突变体的细胞周期延迟，但无法激活 S 期间检查点。然而，这些细胞仍然需要检查点激酶 Chk1 来维持活力，这表明替代检查点被激活。我们建议研究rid突变体中产生的损伤的性质，并鉴定rid活力所需的检查点/修复蛋白。为此，我们将采用遗传筛选和基因微阵列分析。我们还将确定 RID 或检查点突变体的突变率（包括 GCRS 的发生率）是否增加。所提出的实验结果应该可以更好地理解对 DNA 复制起始缺陷做出反应的检查点途径。考虑到许多这些过程的保守性，我们的实验结果可能适用于高等真核细胞，包括人类。此外，我们的结果可能会确定癌症治疗的新药物靶点，并揭示潜在的较少诱变策略来阻止癌细胞的细胞周期进展。