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

描述(由申请人提供)：跨损伤合成(TLS)DNA聚合酶(POL)通过DNA损伤促进复制。人类拥有四个属于Y家族的TLS Pol，Pol？，？，？和Rev1，以及属于B家族的另一个Pol，Pol？这些TLS POL使用高度专业化的机制通过DNA损伤进行复制。这些吗，波尔？擅长从体积较大的N2-DG小沟病变向相反方向延伸。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，病变旁路能力并不局限于POL？，因为其他TLS POL也可以在其旁路中发挥作用。来阐明POL的作用？在人类细胞的病变搭桥中，我们将使用生化、遗传和结构相结合的方法。在目标1中，Pol？的两个独特结构特征的作用：(I)允许Pol？的N-卡环。将通过确定突变对N2-DG小沟加合物和其他类型DNA损伤的延伸反应的影响来分析其对DNA的包围，以及(Ii)其活性部位对模板-引物连接处小沟的开放程度。在《目标2》中，波尔的角色是什么？在通过不同类型的DNA促进复制的过程中，将使用新设计的基于SV40的质粒系统来分析人类细胞中的损伤。待研究的DNA损伤包括：8-氧鸟苷(8-oxoguanine，8-oxoG)和胸腺嘧啶二醇(Tg)，它是细胞DNA氧化损伤的结果；开环的N2-(3-羟丙基-2‘-脱氧鸟苷[(R)-3HOPdG])和笨重的反式-4-羟基-2-非烯醛-脱氧鸟苷(HNE-DG)加合物，它是由N2-DG与醛或烯反应产生的，后者是由自由基对膜中脂质的攻击而产生的；以及多环苯并[a]芘7，8-二醇9，10-环氧化物(BPDE)N2-DG加合物，它是暴露在环境污染物和致癌物中产生的。为了更深入地了解波尔是如何做到的？实际上针对这些DNA损伤进行TLS，在目标3中，我们将确定Pol？的晶体结构。与8-oxoG、TG和顺-syn TT二聚体，以及与(R)-3HOPdG、HNE和BPDE的N_2-DG小槽加合物。Pol的熟练程度如何？为了从插入的与N_2-DG加合物相对的C延伸出另一个DNA极，例如？或Rev1，将确保通过这样的小凹槽DNA加合物无错误地复制。由于人体细胞通过细胞氧化反应以及暴露于化学和环境致癌物中形成了大量的N_2-DG加合物，因此Pol？将通过保持较低的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将DNA损伤的突变和致癌潜力降至最低。与公共卫生相关：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。通过促进DNA损伤的无错误复制，跨损伤合成DNA聚合酶通过保持低突变率来帮助维持基因组的稳定性，从而降低癌症的发生率。拟议的研究将检查人类DNA聚合酶的作用？在推广无错误病变搭桥术方面。

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.