低保真性DNA聚合酶pol kappa在BP-N2-dG损伤位点对侧整合正确核苷酸的机制研究

Translesion DNA synthesis (TLS) is one mode of DNA damage tolerance, which could utilize specialized DNA polymerases, called translesion bypass polymerases, to traverse lesions that are blocking replication. Most TLS DNA polymerases are Y-family polymerases, which can synthesize DNA across and beyond a variety of replication-blocking lesions. Polymerase κ is one of the Y-family polymerases found in humans, and Dpo4 is it's homolog in archaea. The type of bypass can either be accurate (error-free) or mutagenic (error-prone), and the error prone TLS is one of the fundamental mechanisms for genome mutagenesis. Benzopyrene (BP) is a ubiquitous cancer-causing environmental pollutant,both in tobacco smoke and as a product of fossil fuel combustion. Its metabolic form can react readily with guanine to form BPDE-N2-dG (G*) adduct. The bypass of the BP-N2-dG adduct by Polκ is error free, while bypass of the same lesion by Dpo4 is error-prone, and their catalytic mechanisms are not fully understood.Crystal structures of Polκ and Dpo4 show that a larger gap is formed in between the little finger (LF) and the finger domain in Polκ compared to that of Dpo4. More sequence variations are found in the finger domains of the active sites at Polκ and Dpo4. Therefore, we suspect that the different amino acids residues in the finger domain and the size of gap are responsible for the discrepancy of translesion bypass of BP-N2-dG lesion catalyzed by Polκ and Dpo4. To test this hypothesis, we 1) mutate several amino acid residues in the finger/little finger domain to reduce the size of gap, 2)replace some residues in the finger domain at Polκ by corresponding amino acids residues derived from Dpo4, and compare Polκ-wt and mutants' ability in bypass of the BPDE-N2-dG lesion in vitro. To gain insights of Polκ function in vivo, the lenti-GFP、lenti-GFP-Polκ wt/mutants plasmids with packing plasmids pCMVDR and pMD2VSVG were co-transfected into 293T cells to produce lentiviral particles. Those lentiviral particles are then used to infect Polκ -/- mouse embryonic fibroblast (MEF) cells. The stable cell line expressing GFP-Polκ wt and mutants fusion proteins are screened by flow cytometry. The effects of mutated amino acids on the recruitments of GFP-Polκ wt and mutants to the replication foci induced by BPDE and the cell survival assays after exposure to the BPDE will be studied. Finally,the computational studies will be carried out to further our understanding underlying the process. The goal of the research is to elucidate the relationship between these polymerase kappa structural features and the TLS process.

跨损伤DNA合成(TLS)是细胞应答DNA损伤的一种耐受机制。TLS聚合酶可以在损伤的对面掺入核苷酸，使停滞的DNA复制得以延续。 Polκ是TLS聚合酶的一个重要成员，Dpo4是其在古菌中的同源物。苯并芘(BP)作为一种常见的有机环境污染物，可在细胞中形成BP-N2-dG(G*)的损伤。研究发现，Polκ可以在G*对面正确掺入dC，而Dpo4介导易错旁路，易导致基因组的突变。通过比对Polκ和Dpo4的序列、结构，我们发现二者活性中心手指域的关键氨基酸有明显的不同，同时Dpo4中小指与手指域形成的gap比Polκ的更窄小。我们推测这些差异可能导致了Polκ与Dpo4保真性的不同。研究将Polκ手指域关键氨基酸突变并替换为Dpo4中相应序列，通过体外酶动力学、体内细胞存活实验等验证野生型和突变型Polκ跨损伤合成DNA能力的变化，研究结果将加深理解Polκ在跨损伤合成中的结构与功能的关系。

烟草、汽车尾气和燃煤排放所产生的苯并芘 (BP) 是一种常见的环境污染物。BP在体内经过代谢产生强致癌产物BPDE，BPDE与DNA上的鸟嘌呤结合形成的加合物（BPDE-dG）与肺癌发生密切相关。跨损伤 DNA 合成 (TLS)聚合酶可以直接在DNA 加合物对面掺入核苷酸，使由于DNA 损伤的存在而停滞的DNA复制得以延续。TLS 可分为无错旁路和易错旁路两种途径，无错旁路途径主要是在DNA加合物对侧掺入正确的核苷酸；而易错旁路途径在损伤的模板对侧掺入错误的核苷酸，从而引发基因组突变。目前人体内共有大约15种DNA聚合酶，Polκ 是迄今已知的唯一能正确跨越BPDE-dG加合物的聚合酶。Polκ可以在BPDE-dG 对面正确掺入dCTP，并进行有效的延伸，从而保护了细胞免受因BPDE-dG 损伤引发的基因组突变,而缺失Polκ则导致BP诱发的基因组突变率显著升高。阐释Polk是如何正确复制BP-dG 加合物的分子机理是本项目的主要研究内容。.利用 Polk在古菌中的同源物Dpo4 易错跨越BP-dG 加合物的特性，通过系统比较Polk与Dpo4 的结构差异，我们首次发现Polκ活性中心与小指之间gap的结构变化决定了其复制BPDE-dG 加合物的延伸能力和保真性；细胞回转实验证实Polk gap结构在调控跨越BP-dG 加合物效应中的重要作用。同时，缺失N 末端51 个氨基酸的Polk表现出减弱的DNA 合成能力，并完全丧失了在BPDE-dG 损伤模板上的延伸能力，揭示了Polκ N-clasp结构在跨越 BPDE-dG损伤过程中的重要功能,阐明了Polκ正确跨越BPDE-dG催化反应的关键分子机制。随着环境污染的加重，癌症、神经变性病、心血管病等多种人类重大疾病的发病率显著上升。本研究成果对于进一步揭示常见环境污染物导致人类重大疾病发生的分子机制，发展相关疾病预防和治疗手段具有重要意义。