Translesion Synthesis DNA Polymerases and Genome Instability

描述(申请人提供)：自发发生的突变或由外源基因毒物诱导的突变是癌症的根本原因。几乎所有遗传毒物诱导的突变都是由于DNA损伤和专门的跨病变合成(TLS)DNA聚合酶复制受损的DNA造成的，这些聚合酶的准确性低于正常的复制型DNA聚合酶。在复制未受损的DNA时，TLS聚合酶也非常容易出错，因此构成了基因组不稳定的持久来源，需要控制以避免疾病。虽然TLS聚合酶募集到DNA损伤部位的机制相对较好，但限制其参与正常复制的机制却不是很清楚。这项提议试图定义在DNA损伤和生理条件下调节POLζ在体内容易错误合成的程度的全球机制。在具体目标1中，我们将确定依赖检查点的dNTP池提升在塑造POLζ错误签名中的作用。在特定的目标2中，我们将确定POLζ对DNA合成的贡献是如何受到复制动力学和分叉不对称的调节的。酿酒酵母模型系统将用于AIMS 1和2中提出的研究，目的是利用在酵母中获得的数据来进一步促进我们对人类细胞诱变机制的理解。在特定的目标3中，我们将描述人类细胞对复制缺陷的诱变反应的机制。这项拟议的工作将使人们更好地了解在正常细胞中运行的突变过程，以及由环境遗传毒物或治疗干预引起的突变过程。从长远来看，学会操纵这些过程将有助于降低癌症发病率，延缓病情发展，并改善治疗结果。

DESCRIPTION (provided by applicant): Mutations occurring spontaneously or induced by exogenous genotoxicants are a root cause of cancer. Nearly all genotoxicant-induced mutations result from DNA damage and replication of the damaged DNA by specialized translesion synthesis (TLS) DNA polymerases that are less accurate than normal replicative DNA polymerases. TLS polymerases are also highly error-prone when copying undamaged DNA, thus constituting a persistent source of genomic instability that needs to be controlled to avoid disease. While the mechanisms of TLS polymerase recruitment to the sites of DNA damage are understood relatively well, the mechanisms that restrict their participation in the normal replication are much less clear. The PI's laboratory has discovered that the participation of DNA polymerase ζ (Polζ) in the copying of undamaged DNA is promoted by a variety factors that impede the progression of the replication, including defects in the normal replication machinery, fork stalling at natural impediments and treatment with therapeutic replication inhibitors. This proposal seeks to define the global mechanisms that regulate the extent of error-prone synthesis by Polζ in vivo in DNA damaging and physiological conditions. In Specific Aim 1, we will determine the role of checkpoint dependent elevation of dNTP pools in shaping the error signature of Polζ. In Specific Aim 2, we will determine how the contribution of Polζ to DNA synthesis is regulated by the replication dynamics and fork asymmetry. The yeast Saccharomyces cerevisiae model system will be utilized in the studies proposed in Aims 1 and 2, with the goal of using the data obtained in yeast to further advance our understanding of the mechanisms of mutagenesis in human cells. In Specific Aim 3, we will characterize the mechanism of the mutagenic response to replication defects in human cells. The proposed work will lead to a better understanding of the mutagenic processes that operate in normal cells, as well as those induced by environmental genotoxicants or therapeutic interventions. In the long run, learning to manipulate these processes will help reduce cancer incidence, delay progression and improve therapy outcome.