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，提出了为了通过高度扭曲的DNA损伤发生复制，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，Pol-kappa从插入的核苷酸延伸。相比之下，Pol-eta被认为只能通过中度扭曲的病变进行复制。这些想法将使用丙加合物进行测试，丙加合物高度扭曲并影响沃森-克里克碱基配对，并且已被证明在人类DNA中存在高水平。丙烷加合物在DNA中由鸟嘌呤的反应性N2与α，β-不饱和醛或烯醛的反应形成，所述α，β-不饱和醛或烯醛包括丙烯醛和巴豆醛，其是环境中的普遍存在的污染物并且也作为脂质过氧化的终产物在细胞中形成，以及反式-4-羟基-2-壬烯醛，其是ω-6多不饱和脂肪酸的独特氧化产物。在具体目标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缺失的特异性引物-模板错位机制来有效地延伸错配的引物末端。将通过稳态动力学分析检查Pol-kappa通过这两种方法延伸错配引物末端的能力。在具体目标3中，提出由于它们从错配引物末端延伸的熟练能力，Pol-kappa和Pol-zeta都将有助于在人类未受损以及UV受损细胞中的诱变。这一想法将通过在裂变酵母S中进行遗传研究来检验。pombe的突变，以检查Pol-kappa和Pol-zeta对自发和UV诱导的诱变的相对贡献。在具体目标4中，将研究从复制聚合酶Pol-delta转换为跨病变合成聚合酶在病变部位发生的方式，并探讨PCNA在这一过程中作为关键中介的作用。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。