Translesion DNA synthesis in humans

人类跨损伤 DNA 合成

• 批准号: 6796160
• 负责人: LOUISE PRAKASH
• 金额: $ 35.86万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6796160
• 关键词: DNA damage; DNA directed DNA polymerase; DNA repair; Schizosaccharomyces pombe; adduct; environmental contamination; enzyme mechanism; frameshift mutation; isozymes; molecular site; mutagens; nucleic acid biosynthesis; proliferating cell nuclear antigen

DESCRIPTION (provided by applicant): Our long term objectives are to understand the roles of human DNA polymerases, Pol-eta, Pol-iota, and Pol-kappa, in the replication of damaged DNA. In Specific Aim 1, it is proposed that for replication to occur through highly distorting DNA lesions, two different DNA polymerases are required, wherein Pol-iota carries out nucleotide incorporation opposite the lesion and Pol-kappa extends from the inserted nucleotide. By contrast, Pol-eta is suggested to replicate only through moderately distorting lesions. These ideas will be tested using propano adducts that are highly distorting and affect Watson-Crick base pairing, and which have been shown to be present at high levels in human DNA. Propano adducts are formed in DNA from the reaction of the reactive N2 of guanine with alpha,beta-unsaturated aldehydes or enals, which include acrolein and crotonaldehyde, that are ubiquitious pollutants in the environment and which are also formed in cells as the end product of peroxidation of lipids, and trans-4-hydroxy-2-nonenal, a unique oxidation product of omega-6 polyunsaturated fatty acids. In Specific Aim 2, it is proposed that Pol-kappa can efficiently extend mispaired primer termini either by incorporating the next correct nucleotide or by using a specific primer-template misalignment mechanism that generates -1 deletions. The proficiency of Pol-kappa to extend mispaired primer termini by these two means will be examined by steady-state kinetic analyses. In Specific Aim 3, it is proposed that because of their proficient ability to extend from mispaired primer termini, both Pol-kappa and Pol-zeta would contribute to mutagenesis in undamaged as well as UV damaged cells in humans. This idea will be tested by carrying out genetic studies in the fission yeast S. pombe to examine the relative contributions of Pol-kappa and Pol-zeta to spontaneous and UV induced mutagenesis. In Specific Aim 4, the manner by which the switch from the replicative polymerase, Pol-delta, to a translesion synthesis polymerase occurs at the lesion site will be studied and the role of PCNA as the key intermediary in this process delineated. Our understanding of the manner in which human cells overcome blocks to DNA replication should be greatly enhanced by these studies.

描述（由申请人提供）：我们的长期目标是了解人类 DNA 聚合酶 Pol-eta、Pol-iota 和 Pol-kappa 在受损 DNA 复制中的作用。在具体目标 1 中，提出要通过高度扭曲的 DNA 损伤进行复制，需要两种不同的 DNA 聚合酶，其中 Pol-iota 在损伤对面进行核苷酸掺入，而 Pol-kappa 从插入的核苷酸延伸。相比之下，Pol-eta 被认为只能通过适度扭曲的病变进行复制。这些想法将使用高度扭曲并影响沃森-克里克碱基配对的丙醇加合物进行测试，并且已被证明在人类 DNA 中高水平存在。丙醇加合物是由鸟嘌呤的反应性 N2 与 α,β-不饱和醛或烯醛反应形成的，其中包括丙烯醛和巴豆醛，它们是环境中普遍存在的污染物，也是在细胞中形成的最终产物。脂质过氧化，以及反式 4-羟基-2-壬烯醛（omega-6 多不饱和脂肪酸的独特氧化产物）。在特定目标 2 中，提出 Pol-kappa 可以通过掺入下一个正确的核苷酸或通过使用产生 -1 缺失的特定引物模板错配机制来有效延伸错配的引物末端。 Pol-kappa 通过这两种方式延伸错配引物末端的能力将通过稳态动力学分析进行检验。在具体目标 3 中，有人提出，由于 Pol-kappa 和 Pol-zeta 具有从错误配对的引物末端延伸的熟练能力，因此它们都有助于人类未受损和 UV 受损细胞的诱变。这一想法将通过在裂殖酵母 S. pombe 中进行遗传学研究来检验，以检查 Pol-kappa 和 Pol-zeta 对自发和紫外线诱导诱变的相对贡献。在具体目标 4 中，将研究在病变部位发生从复制聚合酶 Pol-delta 到跨病变合成聚合酶的转换方式，并描述 PCNA 作为此过程中关键中介的作用。这些研究应该会大大增强我们对人类细胞克服 DNA 复制障碍的方式的理解。