Translesion DNA synthesis in humans

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。在特定目标1中，我们提出，为了通过高度扭曲的DNA损伤进行复制，需要两种不同的DNA聚合酶，其中Pol-iota与损伤相对进行核苷酸掺入，Pol-kappa从插入的核苷酸延伸。相比之下，建议仅通过中度扭曲的病变复制POL-ETA。这些想法将使用丙基加合物进行测试，这些加合物高度扭曲并影响沃森-克里克碱基配对，已被证明存在于人类DNA中的高水平。丙烷加合物是由鸟嘌呤的反应性氮气与α，β-不饱和醛或烯醛反应而在DNA中形成的，α，β-不饱和醛或烯醛包括丙烯醛和巴豆醛，它们是环境中普遍存在的污染物，也是细胞中脂质过氧化的最终产物，以及反式-4-羟基-2-壬烯醛，欧米茄-6多不饱和脂肪酸的独特氧化产物。在特定的目标2中，提出了Pol-kappa可以通过加入下一个正确的核苷酸或通过使用产生-1缺失的特定的引物-模板错位机制来有效地延长错配的引物末端。Pol-kappa通过这两种方法延伸错配对引物末端的熟练程度将通过稳态动力学分析来检验。在具体目标3中，有人提出，由于Pol-kappa和Pol-Zeta都具有从错配的引物末端延伸的熟练能力，因此Pol-kappa和Pol-Zeta都将有助于未受损和紫外线损伤的人类细胞的突变。这一想法将通过在裂解酵母S.pombe中进行遗传研究来验证，以检查Pol-kappa和Pol-Zeta在自发和紫外线诱导突变中的相对贡献。在具体目标4中，将研究在病变部位从复制聚合酶POL-Delta向跨损伤合成聚合酶转换的方式，并描述增殖细胞核抗原作为这一过程中的关键中介的作用。通过这些研究，我们对人类细胞克服DNA复制障碍的方式的理解应该会大大增强。

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.