Cis-and trans-acting determinants of replication timing

复制时间的顺式和反式作用决定因素

• 批准号: 6745631
• 负责人: JOEL A HUBERMAN
• 金额: $ 38.17万
• 依托单位: ROSWELL PARK CANCER INSTITUTE CORP
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6745631
• 关键词: DNA binding protein; DNA footprinting; DNA replication origin; Saccharomyces cerevisiae; Schizosaccharomyces pombe; cell growth regulation; chemical structure function; chromatin; functional /structural genomics; fungal genetics; fungal proteins; gel mobility shift assay; genetic regulation; immunoprecipitation; intermolecular interaction; mass spectrometry; molecular cloning; nucleic acid metabolism; nucleic acid sequence; nucleic acid structure; plasmids; polymerase chain reaction; protein purification; recombinant DNA; site directed mutagenesis

DESCRIPTION (provided by applicant): One of the cellular alterations required for development of cancer is "genomic instability," a term that describes a collection of pathways all leading to increased frequency of gene mutation and altered patterns of gene expression. Among the mechanisms causing genomic instability are chromosome breakage, gene amplification, and changes in levels of gene expression. The latter are frequently associated with changes in gene methylation status. All of these mechanisms have been correlated with changes in replication timing, and it is possible that in some cases changes in replication timing may be the primary cause of these phenomena. So far it has been difficult to evaluate the role of changes in replication timing in cancer development, because so little is known about the mechanisms controlling replication timing. Fortunately previous studies in budding and fission yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe), in our laboratory and others, provide entry points into understanding replication timing. In both yeasts we have identified replication origins containing internal cis-acting sequences that determine late replication timing. Here we propose simple strategies that will permit us to precisely localize those sequences and use them to pull out the corresponding trans-acting proteins. In addition, studies in other laboratories and ours have already identified several proteins likely to play roles in replication timing. We intend to investigate the potential roles of all of these proteins in replication timing by studying the effects on timing of mutations in the corresponding genes and by studying the binary interactions between these proteins. The results of the proposed experiments should permit us to develop detailed models of the mechanisms controlling replication timing in both budding and fission yeasts. The conserved aspects of these models are likely to prove applicable to other eukaryotic cells, including human cells. This information may permit the development of drugs that will enhance normal replication timing stability and thus inhibit or reverse the development of cancer.

描述（由申请人提供）： 癌症发展所需的细胞改变之一是“基因组改变” 不稳定”这个术语描述了一系列导致 基因突变频率增加和基因表达模式改变。 导致基因组不稳定的机制包括染色体断裂、基因 扩增和基因表达水平的变化。后者是 通常与基因甲基化状态的变化相关。所有这些 机制与复制时间的变化相关，并且 在某些情况下，复制时间的变化可能是主要的 这些现象的原因。到目前为止，还很难评估其作用。 癌症发展过程中复制时间的变化，因为复制时间太少 了解控制复制计时的机制。幸运的是 之前对芽殖酵母和裂殖酵母（酿酒酵母和 在我们的实验室和其他实验室中，裂殖酵母 (Schizosaccharomyces pombe) 提供了切入点 了解复制时间。在这两种酵母中我们已经鉴定出 复制起点包含决定的内部顺式作用序列 较晚的复制时间。在这里，我们提出简单的策略，使我们能够 精确定位这些序列并使用它们来提取 相应的反式作用蛋白。此外，在其他方面的研究 实验室和我们已经确定了几种可能发挥作用的蛋白质 复制计时中的作用。我们打算研究的潜在作用 通过研究对时间的影响，研究所有这些蛋白质在复制时间中的作用 相应基因的突变并通过研究二元 这些蛋白质之间的相互作用。拟议实验的结果 应该允许我们开发控制机制的详细模型 出芽酵母和裂殖酵母的复制时间。保守的方面 这些模型可能被证明适用于其他真核细胞， 包括人体细胞。这些信息可能有助于药物的开发 这将增强正常的复制定时稳定性，从而抑制或 逆转癌症的发展。