DNA replication and post replication DNA repair affect the genomic instability and cancer suppression

DNA 复制和复制后 DNA 修复影响基因组不稳定性和癌症抑制

基本信息

批准号：
16590244
负责人：
SUZUKI Motoshi
金额：
$ 2.3万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

项目摘要

In order to study how cells establish fidelity DNA replication during the lagging strand DNA replication, we isolated active mutants in Saccharomyces cerevisiae pol alpha that have a defect in error discrimination. Among them, L868F pol alpha has a spontaneous error frequency of 3 in 100 nucleotides and 570-fold lower replication fidelity than wild type. In vivo, L868F pol alpha confers a mutator phenotype. For this result, we suggest accurate DNA synthesis by pol alpha is important to suppress genomic instability in cells. We also found L868F pol alpha catalyzed relatively efficient bypass of a cis-syn cyclobutane pyrimidine dimer, extending the 3'-T 26000-fold more efficiently than wild type. This type of activity is characteristic to translesion DNA polymerases, which are classified in another family of DNA polymerases. Interestingly, leucine and phenylalanine residues are conserved in replicative and translesion DNA polymerases, respectively. In a translesion DNA polymerase eta mutant of S. cerevisiae F34L pol eta showed reduced translesion DNA synthesis activity in vitro. The corresponding mutants in human DNA polymerases also showed the similar biochemical features. These data suggest that the phenylalanine and leucine residues may have played a role in the functional evolution of these enzyme classes.The observed high fidelity of chromosomal replication is achieved not only through mechanisms that enhance correct nucleotide insertion by the DNA polymerase but also through ones that correct errors after they occur. We are under investigation of the downstream pathways that may either suppress or promote genomic instability.

为了研究细胞如何在滞后链 DNA 复制过程中建立保真度 DNA 复制，我们在酿酒酵母 pol α 中分离出了具有错误辨别缺陷的活性突变体。其中，L868F pol alpha 的自发错误频率为 100 个核苷酸中 3 个，复制保真度比野生型低 570 倍。在体内，L868F pol alpha 赋予突变表型。对于这一结果，我们认为 pol α 的准确 DNA 合成对于抑制细胞中基因组的不稳定性非常重要。我们还发现 L868F pol α 催化相对有效地绕过顺式环丁烷嘧啶二聚体，比野生型更有效地延伸 3'-T 26000 倍。这种类型的活性是跨损伤 DNA 聚合酶的特征，其属于另一个 DNA 聚合酶家族。有趣的是，亮氨酸和苯丙氨酸残基分别在复制和跨损伤 DNA 聚合酶中保守。在酿酒酵母 F34L pol eta 的跨损伤 DNA 聚合酶 eta 突变体中，体外显示跨损伤 DNA 合成活性降低。人类DNA聚合酶的相应突变体也表现出相似的生化特征。这些数据表明，苯丙氨酸和亮氨酸残基可能在这些酶类的功能进化中发挥了作用。观察到的染色体复制的高保真度不仅是通过增强 DNA 聚合酶正确核苷酸插入的机制实现的，而且还通过以下机制实现：错误发生后予以纠正。我们正在研究可能抑制或促进基因组不稳定性的下游途径。