跨损伤DNA合成无错旁路途径的作用机制

Translesion DNA synthesis (TLS) plays a critical role in the rescue of the stalled replication fork and the maintenance of the genome stability, which can be divided into error-prone and error-free pathways. It has been shown that the replicant DNA polymerase could be replaced by the TLS polymerase when the replication fork meets DNA lesion. The specialized DNA polymerases, TLS polymerases, are the central enzymes, which could direct synthesize against the DNA lesion site due to its unique structural features. To perform the error-free translesion DNA synthesis, TLS polymerases are required to be recruited to the lesion site at right time following inserting the correct nucleotide opposite the DNA lesion. On the other hand, benzo[a]pyrene is one of the most common environmental pollutants, which can metabolically convert normal DNA to BPDE lesions in human bodies. The BPDE lesion is extreme toxic, which further cause cancer if could not be repaired correctly. Recently, it has been shown that the DNA polymerase κ is the only TLS polymerase bypassing the BPDE lesion DNA in human bodies. However, the detailed mechanism is still unclear. Here, we will investigate the intriguing mechanisms of the error-free translesion DNA synthesis mediated by human DNA polymerase kappa, using molecular biology, biochemical and genetic means. Specifically, we will: 1) In-depth study of the mechanism of the maintenance of genome stability by TLS, especially the role of human DNA polymerase κ in the bypass of the BPDE lesion DNA repair. This will clarify the mechanism of the genome mutagenesis induced by the misincorporation against BPDE lesion DNA. 2) Reveal the mechanism of the human DNA polymerase κ recruitment by mono-ubiquitinated PCNA protein. This will tell us the mechanism of the TLS regulation in vivo. Together, the results of this project will add valuable information about the mechanisms of the error-free translesion DNA synthesis pathway. Moreover, considering the deficiency of TLS pathway has been linked to cancers, these results could also have implication in the clinical medicine.

跨损伤DNA合成（TLS）依赖一类特殊的DNA聚合酶，能够在DNA复制期拯救由DNA损伤导致的复制叉停滞，分为无错旁路和易错旁路两种途径。TLS的无错旁路途径需要TLS聚合酶被及时的招募至DNA损伤位点并在其对侧掺入正确的核苷酸。苯并（a）芘是一种常见的环境污染物，其代谢产物BPDE加合DNA具有极强的细胞毒性。最新的实验证据指出DNA聚合酶κ作为唯一的TLS聚合酶介导了BPDE加合DNA的TLS无错旁路途径，但具体的分子机制尚不清楚。为此，我们将开展以下几个方面的研究：1、研究人源DNA聚合酶κ对于BPDE位点的TLS过程，进而揭示由BPDE位点错误插入造成基因突变的分子机制。2、探索人源DNA聚合酶κ与泛素和PCNA蛋白的识别模式，明确其招募调控机制。该研究成果将阐明人源DNA聚合酶κ介导的TLS无错旁路途径的作用机制，同时鉴于其与癌症的发生直接相关，在临床医学具有潜在的应用价值。

DNA聚合酶κ具有高效的跨损伤合成活性，其能够在BPDE加合物对面掺入核苷酸并进行延伸，介导了TLS无错旁路途径。在这过程之中，PCNA的单泛素化修饰对于其在正确的时间和正确的地点行使正确的反应至关重要。经过4年的研究，本项目研究完成并达到了原申请书的项目计划和任务书的要求和指标。我们的研究得出以下结论：（1）通过优化表达系统，纯化获得了实验所需的DNA聚合酶κ，PCNA等蛋白。（2）通过体外延伸和动力学实验，明确了DNA聚合酶κ跨损伤合成的动力学基础。（3）解析获得了DNA聚合酶κ-DNA-dCMPNPP三元复合体的晶体结构，并通过生化等实验揭示了其工作机制。（4）明确了泛素修饰PCNA激活DNA聚合酶κ跨损伤合成的机制。此外，我们还研究了翻译后修饰调控PCNA及其互作蛋白相互作用的机制。这些研究成果阐明了DNA聚合酶κ介导的跨损伤DNA合成无错旁路途径的作用机制，为后续的研究提供了新的思路。