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

描述（由申请人提供）：转录合成（TLS）DNA聚合酶（Pos）通过DNA损伤促进复制。人类拥有四个属于Y家族的TLS Pos，Pos？，?, ?, Rev 1和另一个Pol，Pol？，它属于B家族。这些TLS Pol采用高度专业化的机制通过DNA损伤进行复制。其中，Pol？擅长于从大的N2-dG小沟病变的对面延伸。波尔？也可以进行与其他类型的DNA损伤相反的TLS;然而，在这种情况下，损伤旁路能力不限于Pol？，因为其他TLS Pol也可以在其旁路中起作用。为了阐明波尔的作用？在人类细胞病变旁路中，我们将使用生物化学、遗传学和结构学相结合的方法。在目的1中，Pol？的两个独特结构特征的作用，(i)N型卡环允许Pol？以包围DNA，和（ii）在模板-引物连接处其活性位点朝向小沟的开放性，将通过确定突变对来自N2-dG小沟加合物和其他类型的DNA损伤的延伸反应的影响来分析。在目标2中，波尔的作用？通过不同类型的DNA损伤在人类细胞中促进复制的作用将使用新设计的基于SV 40的质粒系统进行分析。待研究的DNA损伤中有8-氧代鸟嘌呤（8-oxoG）和胸腺嘧啶乙二醇（TG），它们是由细胞氧化DNA损伤引起的;开环的N2-（3-羟基丙基-2 '-脱氧鸟苷[（r）-3HOPdG]和由N2-dG与自由基攻击膜中脂质产生的醛或烯醛;以及多环苯并[a]芘7，8-二醇9，10-环氧化物（BPDE）N2-dG加合物，其由暴露于环境污染物和致癌物引起。为了更深入地了解波尔？实际上执行TLS对这些DNA病变，在目的3，我们将确定晶体结构的Pol？与8-oxoG、TG和顺式-syn TT二聚体以及与（r）-3HOPdG、HNE和BPDE的N2-dG小沟加合物反应。波尔的熟练能力？从C插入的N2-dG加合物的对面延伸的另一个DNA Pol，如？或Rev 1，将确保通过这种小沟DNA加合物的无错误复制。由于各种各样的N2-dG加合物形成在人体细胞从细胞氧化反应和暴露于化学和环境致癌物，波尔？将通过保持低突变率对基因组稳定性产生重大影响，从而降低人类致癌的发生率。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最大限度地减少DNA损伤的致突变和致癌潜力。公共卫生相关性：DNA损伤是由细胞氧化损伤和暴露于环境污染物和致癌物在人体细胞中产生的。通过促进通过DNA损伤的无错误复制，跨损伤合成DNA聚合酶有助于通过保持低突变率来维持基因组稳定性，从而降低癌症的发病率。拟议的研究将探讨人类DNA聚合酶的作用？在促进无差错的病变旁路方面。

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.