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

人类 DNA 聚合酶 kappa 在 DNA 损伤复制旁路中的作用

• 批准号: 7714688
• 负责人: SATYA PRAKASH
• 金额: $ 47.57万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7714688
• 关键词: 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; 7,8-dihydro-8-oxoguanine; Active Sites; Address; Adverse effects; Affect; Aldehydes; Aromatic Polycyclic Hydrocarbons; Automobiles; Benzo(a)pyrene; Binding; Biochemical; Biochemical Genetics; Bypass; Cancer Biology; Carcinogens; Cells; Chemicals; Complex; DNA; DNA Adducts; DNA biosynthesis; DNA lesion; DNA-Directed DNA Polymerase; Deoxyguanosine; Ensure; Environmental Carcinogens; Environmental Pollutants; Epoxy Compounds; Etiology; Exposure to; Family; Free Radicals; Genetic; Genome Stability; Glycols; Guanine; Human; Hydroxyl Radical; Incidence; Lesion; Lipid Peroxidation; Lipids; Malignant Neoplasms; Mammalian Cell; Mediating; Membrane; Minor Groove; Molecular; Mutation; Nucleotides; Plasmids; Polymerase; Process; Reaction; Role; Solvents; Specificity; Structure; System; Testing; Time; UV induced; adduct; base; benzo(a)pyrene 7,8-diol-9,10-epoxide-N2-deoxyguanosine; carcinogenesis; dimer; human DNA; oxidative DNA damage; oxidative damage; public health relevance; thymine glycol

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.

描述（由申请人提供）：跨损伤合成（TLS）DNA聚合酶（Pols）通过DNA损伤促进复制。人类拥有属于 Y 家族的四个 TLS Pol，Pols ?、?、? 和 Rev1，以及另一个属于 B 家族的 Pol，Pol?。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，波尔?擅长从大的 N2-dG 小沟病变对面延伸。波尔？还可以针对其他类型的DNA损伤进行TLS；然而，在这种情况下，病变旁路能力不仅限于 Pol?，因为其他 TLS Pol 也可以在其旁路中发挥作用。阐明波尔的作用？在人类细胞的病变旁路中，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 扣环。围绕 DNA，以及 (ii) 其活性位点对模板引物连接处小沟的开放性，将通过确定突变对 N2-dG 小沟加合物和其他类型 DNA 延伸反应的影响来进行分析病变也。在《目标 2》中，波尔的角色是？将使用新设计的基于 SV40 的质粒系统来分析通过人类细胞中不同类型的 DNA 损伤促进复制的作用。待研究的 DNA 损伤包括由细胞氧化 DNA 损伤引起的 8-氧代鸟嘌呤 (8-oxoG) 和胸腺嘧啶乙二醇 (TG)；反应产生的开环 N2-(3-羟基丙基-2'-脱氧鸟苷 [(r)-3HOPdG] 和庞大的反式-4-羟基-2-非烯醛-脱氧鸟苷 (HNE-dG) 加合物N2-dG 与膜中脂质的自由基攻击产生的醛或烯醛以及多环苯并[a]芘 7,8-二醇；因暴露于环境污染物和致癌物而产生的 9,10-环氧化物 (BPDE) N2-dG 加合物 为了更深入地了解 Pol? 实际上如何针对这些 DNA 损伤发挥 TLS，在目标 3 中，我们将确定 Pol 的晶体结构。 ? 具有 8-oxoG、TG 和 cis-syn TT 二聚体，以及 (r)-3HOPdG、HNE 的 N2-dG 小沟加合物， Pol？通过另一个DNA Pol（例如？或Rev1）从C插入到N2-dG加合物的熟练能力将确保通过此类小沟DNA加合物的无差错复制。 N2-dG 加合物是在人体细胞中通过细胞氧化反应以及接触化学和环境致癌物而形成的，Pol？通过保持较低的突变率，从而降低人类致癌的发生率，对基因组稳定性产生重大影响。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 损伤的诱变和致癌潜力。公共健康相关性：DNA 损伤是由于细胞氧化损伤以及接触环境污染物和致癌物而在人体细胞中产生的。通过 DNA 损伤促进无差错复制，跨损伤合成 DNA 聚合酶可保持较低的突变率，从而有助于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。