Role of human DNA polymerase kappa in replicative bypass of DNA lesions

描述（由申请人提供）：翻译合成（TLS） DNA聚合酶（pol）通过DNA损伤促进复制。人类拥有4个属于y家族的TLS pol， pol ？,吗?,吗?启示录1，还有另一个波尔，波尔？它属于b族。这些TLS pol采用高度专门化的机制，通过DNA损伤进行复制。这些呢，阿宝？擅长从庞大的N2-dG小沟槽病变的对面延伸。波尔吗?也可以对其他类型的DNA病变进行TLS；然而，在这种情况下，病变旁路能力并不局限于Pol？，因为其他TLS poll也可以在其旁路中发挥作用。为了阐明Pol？在人类细胞病变搭桥中，我们将使用生化、遗传和结构相结合的方法。在Aim 1中，Pol？(i) n型卡扣，允许Pol？（ii）其活性位点对模板-引物连接处小凹槽的开放性，将通过确定突变对N2-dG小凹槽加合物和其他类型DNA损伤的延伸反应的影响来分析。在目标2中，波尔？将使用新设计的基于sv40的质粒系统来分析通过不同类型的DNA损伤促进人类细胞复制的机制。待研究的DNA损伤包括由细胞DNA氧化损伤引起的8-氧鸟嘌呤（8- oxog）和胸腺嘧啶乙二醇（TG）；开环的N2-(3-羟基丙基-2'-脱氧鸟苷[(r)- 3hopdg]和N2- dg与醛或自由基攻击膜内脂质产生的烯醛反应产生的庞大的反式4-羟基-2-非烯醛-脱氧鸟苷（HNE-dG）加合物；多环苯并[a]芘7,8-二醇9,10-环氧化物（BPDE） N2-dG加合物是由于暴露于环境污染物和致癌物而产生的。为了更深入地了解波尔？实际上对这些DNA损伤进行TLS，在Aim 3中，我们将确定Pol？与8-oxoG， TG和顺式同步TT二聚体以及(r)-3HOPdG， HNE和BPDE的N2-dG小槽加合物。精通Pol？从N2-dG加合物对面插入的C延伸到另一个DNA Pol，例如？或Rev1，将确保通过这种微小的凹槽DNA加合物进行无错误复制。由于人体细胞中通过细胞氧化反应和暴露于化学和环境致癌物形成了大量的N2-dG加合物，Pol？将通过保持低突变率对基因组稳定性产生重大影响，从而减少人类致癌的发生率。拟议的研究与癌症生物学和病因学高度相关，因为它们将揭示人类细胞如何将DNA病变的致突变和致癌潜力降至最低。公共卫生相关性：DNA损伤是由于细胞氧化损伤和暴露于环境污染物和致癌物而在人类细胞中产生的。翻译合成DNA聚合酶通过DNA损伤促进无错误复制，通过保持低突变率帮助维持基因组的稳定性，从而降低癌症的发病率。拟议的研究将检验人类DNA聚合酶的作用。促进无差错病变旁路。

DESCRIPTION (provided by applicant): Translesion synthesis (TLS) DNA polymerases (Pols) promote replication through DNA lesions. Humans possess four TLS Pols that belong to the Y-family, Pols ?, ?, ?, and Rev1, and another Pol, Pol?, that belongs to the B-family. These TLS Pols employ highly specialized mechanisms for replicating through DNA lesions. Of these, Pol? is adept at extending opposite from bulky N2-dG minor groove lesions. Pol? can also carry out TLS opposite other types of DNA lesions; in that case, however, the lesion bypass ability is not limited to Pol?, as other TLS Pols can also function in their bypass. To elucidate the role of Pol? in lesion bypass in human cells, we will use a combined biochemical, genetic, and structural approach. In Aim 1, the role of two unique structural features of Pol?, (i) the N-clasp which allows Pol? to encircle DNA, and (ii) the openness of its active site towards the minor groove at the template-primer junction, will be analyzed by determining the effects of mutations on the extension reaction from N2-dG minor groove adducts and other types of DNA lesions as well. In Aim 2, the role of Pol? in promoting replication through different types of DNA lesions in human cells will be analyzed using a newly devised SV40-based plasmid system. Among the DNA lesions to be studied are 8- oxoguanine (8-oxoG) and thymine glycol (TG) that result from cellular oxidative DNA damage; the ring-opened N2-(3-hydroxyl propyl-2'-deoxygunaosine [(r)-3HOPdG] and the bulky trans-4-hydroxy-2-non-enal- deoxyguanosine (HNE-dG) adducts that result from the reaction of N2-dG with aldehydes or enals generated from free radical attack on lipids in membranes; and the multi-cyclic benzo[a]pyrene 7,8-diol 9,10-epoxide (BPDE) N2-dG adduct that results from exposure to environmental pollutants and carcinogens. To gain a deeper understanding of how Pol? actually performs TLS opposite these DNA lesions, in Aim 3, we will determine crystal structures of Pol? with 8-oxoG, TG, and cis-syn TT dimer, as well as with the N2-dG minor groove adducts of (r)-3HOPdG, HNE, and BPDE. The proficient ability of Pol? for extending from the C inserted opposite the N2-dG adducts by another DNA Pol, such as ? or Rev1, would ensure error-free replication through such minor groove DNA adducts. Since a large variety of N2-dG adducts are formed in human cells from cellular oxidative reactions and from exposure to chemical and environmental carcinogens, Pol? will have a major impact on genome stability by keeping the rate of mutations low, reducing thereby the incidence of carcinogenesis in humans. The proposed studies are highly relevant for cancer biology and etiology as they will reveal how human cells minimize the mutagenic and carcinogenic potential of DNA lesions. PUBLIC HEALTH RELEVANCE: DNA lesions are generated in human cells from cellular oxidative damage and from exposure to environmental pollutants and carcinogens. By promoting error-free replication through DNA lesions, translesion synthesis DNA polymerases help to maintain genomic stability by keeping the rate of mutations low, and thereby reducing the incidence of cancers. The proposed studies will examine the role of human DNA polymerase ? in promoting error-free lesion bypass.