Control of DNA Replication and Cell Division

描述(由申请人提供)： 本研究的总体目标是以裂殖酵母为模式生物，研究真核细胞对复制扰动的反应，以维持基因组的稳定性。当面临染色体扰动时，真核生物激活一组复杂的信号转导反应通路，称为检查点，以调节细胞周期转换，促进DNA修复，在某些情况下诱导细胞凋亡。这些反应的失败会导致基因组不稳定，从而促进肿瘤的发展。此外，细胞还进化出其他过程来处理基因组的扰动，以重新开始复制。细胞可以使用大量的转移性DNA聚合酶来进行无错误或易错误的合成，这有可能产生点突变和单碱基移码。细胞还可以使用重组/修复来重新启动复制，这有可能导致细胞中的缺失突变。复制基因的突变通常导致以缺失突变和点突变为特征的突变子表型。关于检查点如何响应复制突变引起的复制扰动，我们知之甚少。我们最近的研究表明，检查点基因的突变显著影响复制突变体中的突变子表型。这些发现使我们提出了假设：为了响应基因组的扰动，检查点钳将转录聚合酶招募到染色质上进行诱变合成，从而产生点突变，并招募重组/修复因子(S)来促进缺失突变。相反，CDS1激酶调节重组/修复因子，以防止缺失突变。这些过程结合在一起将有助于从停滞的复制中恢复以重新启动DNA复制。 为检验这一假设而提出的具体目标是： (1)。为了重新启动停滞的复制分叉，检查点钳位和/或检查点钳位加载器如何招募转位聚合酶进行突变合成？ (2)。检查点效应蛋白激酶CDS1如何在防止缺失突变发生中发挥作用，以及检查点钳制/钳制加载器是否促进细胞中的缺失突变，以防止复制叉崩溃并重新启动停滞的复制叉？

DESCRIPTION (provided by applicant): The overall goal of this research program is to investigate how eukaryotic cells respond to replication perturbations in order to maintain the genomic stability by using fission yeast as the model organism. When faced with chromosomal perturbations, eukaryotic organisms activate a complex set of signal transduction response pathways called checkpoints to regulate cell-cycle transition, to facilitate DNA repair or in some cases to induce apoptosis. Failure of these responses results in genomic instability, which can promote tumor development. In addition, cells have evolved other processes to deal with the genomic perturbations to restart replication. Cells can use a large repertoire of translesional DNA polymerases to perform either error-free or error-prone synthesis, which has the potential to generate point mutations and single base frameshifts. Cells can also use recombination/repair to restart replication, which has the potential of inducing deletion mutations in cells. Mutations in replication genes often induce a mutator phenotype characterized by deletion mutations and point mutations. Little is known about how checkpoints respond to the replication perturbations caused by replication mutants. Our recent studies have shown that mutations in checkpoint genes significantly affect the mutator phenotype in the replication mutants. These findings have led us to hypothesize: In response to genomic perturbation, the checkpoint-clamp recruits translesional polymerases onto chromatin for mutagenic synthesis which generates point mutations and recruits recombination/repair factor(s) to promote deletion mutations. In contrast, Cds1 kinase regulates the recombination/repair factors to prevent deletion mutations. Together these processes will facilitate recovery from a stalled replication to restart DNA replication. Specific Aims proposed to test this hypothesis are: (1). How do the checkpoint clamp and/or checkpoint-clamp-loader recruit translesional polymerases for mutagenic synthesis in order to restart a stalled replication fork? (2). How does the checkpoint effector kinase Cds1 function in preventing deletion mutations to occur, and does the checkpoint clamp/clamp-loader promote deletion mutations in cells to prevent replication fork from collapse and to restart a stalled replication fork?