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，细胞已经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，并非所有的DNA损伤都被修复，相反，损伤反而被一组跨损伤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Rev 3能够从错误掺入的核苷酸模板延伸，并且负责损伤诱导的和自发的诱变。对哺乳动物细胞中的酶知之甚少。数据表明，脊椎动物Rev 3的丢失导致基因组不稳定性增加，对DNA损伤的影响更敏感。我们计划研究当野生型和Rev 3-/-细胞用各种DNA损伤剂处理时，Rev 3在损伤耐受性中的贡献。此外，我们将试图确定聚合酶活性是否在损伤旁路中是重要的，以及蛋白质的截短部分是否足以恢复损伤耐受性。最后，数据表明Rev 3 mRNA在不同水平上受到调节。哺乳动物Rev 3蛋白表达模式未知。我们计划产生一种抗体，以研究组织特异性蛋白质表达，并通过免疫印迹分析在遗传毒性应激期间Rev 3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.