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Specific Aim 1中，有人提出，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在损伤的相反方向进行核苷酸整合，Pol-kappa从插入的核苷酸延伸。相比之下，Pol-eta只能通过中度扭曲的病变进行复制。这些想法将使用丙烯加合物进行测试，丙烯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中含量很高。在DNA中，鸟嘌呤的活性N2与α、β -不饱和醛或烯醛（包括丙烯醛和巴豆醛）发生反应形成丙烯加合物，烯醛和烯醛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，反式-4-羟基-2-壬烯醛是ω -6多不饱和脂肪酸的独特氧化产物。在Specific Aim 2中，有人提出Pol-kappa可以通过结合下一个正确的核苷酸或使用特定的引物-模板错位机制产生-1缺失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引物末端的熟练程度将通过稳态动力学分析来检验。在Specific Aim 3中，研究人员提出，由于Pol-kappa和Pol-zeta具有从错配引物末端延伸的能力，它们可以在未受损和紫外线受损的人类细胞中起到诱变作用。这一想法将通过对裂变酵母s.p ombe进行遗传研究来验证，以检验Pol-kappa和Pol-zeta对自发诱变和紫外线诱变的相对贡献。在Specific Aim 4中，将研究从复制聚合酶（Pol-delta）到翻译合成聚合酶在病变部位发生转换的方式，并描述PCNA作为这一过程中关键中介的作用。我们对人类细胞克服DNA复制障碍的方式的理解应该通过这些研究大大增强。

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.