DNA Polymerase Zeta and Its Role in DNA Damage Tolerance

描述（由申请人提供）：DNA不断受到各种细胞和环境因素的破坏。为了防止DNA突变，细胞进化出多种有效的修复机制来纠正受损的DNA。然而，在复制过程中，并不是所有的DNA损伤都被修复，相反，损伤被一组翻译DNA聚合酶如聚合酶eta和聚合酶zeta （Pol C）所耐受。酵母中的证据表明，Pol δ的催化亚基Rev3能够从错误结合的核苷酸模板延伸，并负责损伤诱导和自发突变。人们对哺乳动物细胞中的这种酶知之甚少。数据表明，脊椎动物Rev3基因的缺失会导致基因组不稳定性增加，并更容易受到DNA损伤的影响。我们计划研究当野生型和Rev3 -/-细胞被各种DNA损伤剂处理时，Rev3对损伤耐受性的贡献。此外，我们将尝试确定聚合酶活性在损伤旁路中是否重要，以及截断的蛋白质部分是否足以恢复损伤耐受性。最后，数据表明Rev3 mRNA在不同水平上受到调控。哺乳动物Rev3蛋白的表达模式尚不清楚。我们计划生成一种抗体，通过免疫印迹分析来研究组织特异性蛋白表达以及Rev3在基因毒性应激期间是否上调。

DESCRIPTION (provided by applicant): DNA is constantly being damaged by a variety of cellular and environmental agents. In order to prevent DNA mutagenesis, cells have evolved a variety of effective repair mechanisms to correct damaged DNA. However, during replication, not all DNA damage is repaired, rather, damage instead is tolerated by a group of translesion DNA polymerases such as Polymerase eta and Polymerase zeta (Pol C). Evidence in yeast indicates that the catalytic subunit of Pol delta, Rev3, is capable of extending from misincorporated nucleotide templates and is responsible for both damage-induced and spontaneous mutagenesis. Very little is known about the enzyme in mammalian cells. Data suggests that loss of vertebrate Rev3 leads to increased genomic instability and greater susceptibility to the effects of DNA damage. We plan to study the contribution of Rev3 in damage tolerance when wild-type and Rev3 -/- cells are treated with various DNA damaging agents. Furthermore, we will attempt to determine if the polymerase activity is important in damage bypass and whether truncated portions of the protein is sufficient to restore damage tolerance. Finally, data indicates that Rev3 mRNA is regulated at various levels. Mammalian Rev3 protein expression patterns are unknown. We plan to generate an antibody in order to study tissue specific protein expression and whether Rev3 is upregulated during periods of genotoxic stress via immunoblot analysis.