Role of recombination-dependent DNA damage response mechanisms in the resolution of benzo[a]pyrene-induced DNA replication stress

众所周知，职业性或与吸烟相关的接触人类致癌物质苯并[a]芘(B[a]P)会诱发肺癌。B[a]P的致癌性主要是通过其反应性代谢物抗B[a]P-7，8-二醇-9，10-环氧化物(BPDE)的DNA加合物来实现的。尽管这些DNA加合物如果在S期之前不修复会阻碍DNA复制分叉进程，这一过程被称为DNA复制应激，但容易出错的跨病变合成(TLS)聚合酶旁路通常允许继续复制。TLS过去的BPDE加合物已被广泛研究，并与苯并[a]磷引起的致突变性有关。然而，BPDE加合物也导致同源重组(HR)和姐妹染色单体交换，这表明重组因子作用于基本上停滞不前或崩溃为DNA双链断裂(DSB)的分叉。DNADSB复制分叉进程受损和复制依赖的形成对苯并[a]磷诱导的毒性的贡献以及重组在解决这种复制应激中的作用还知之甚少。该项目将应用在DNA复制应激基础研究中通常使用的实验方法来阐明BPDE加合物是阻止还是崩溃传入的复制装置，以及重组因子如何在真核生物的这些分叉处发挥作用。三个有趣的候选蛋白质参与受损的叉子重塑和重组，PARP1，RAD51和ZRANB3，将被系统地研究它们在复制叉子上的功能和潜在的相互作用，以响应BPDE处理在哺乳动物细胞。他们的影响，减速，失速，以及重新启动或崩溃的个别叉子和新的来源发射将被检查。对BPDE停滞复制叉处的蛋白质动力学的分析将进一步阐明哪些DNA损伤反应机制和蛋白质对恢复至关重要。最后，将评估PARP1、RAD51和ZRANB3在BPDE诱导的重组中的作用，以阐明HR参与BPDE加合物对DNA损伤反应的生化原因。由于不忠诚的重组可能导致总的染色体不稳定，更好地理解参与解决BPDE诱导的复制应激的重组依赖的和潜在的重组DNA损伤反应机制将最终为了解苯并[a]磷引起的突变和毒性提供新的见解。这可能会发现未来预防或保护的创新战略。

Occupational or smoking-related exposure towards the human carcinogen benzo[a]pyrene (B[a]P) is known to induce lung tumours. This carcinogenicity of B[a]P is significantly mediated by DNA adduct formation of its reactive metabolite anti-B[a]P-7,8-diol-9,10-epoxide (BPDE). Although these DNA adducts impair DNA replication fork progression if remaining unrepaired until S-phase, a process termed DNA replication stress, error-prone bypass by translesion synthesis (TLS) polymerases usually allows continued replication. TLS past BPDE adducts has been extensively studied and is implicated in B[a]P-caused mutagenicity. However, BPDE adducts also lead to homologous recombination (HR) and sister chromatid exchanges, suggesting that recombination factors act at forks that are substantially stalled or collapsed into DNA double-strand breaks (DSBs). The contributions of impaired replication fork progression and replication-dependent formation of DNA DSBs to B[a]P-induced toxicity as well as the role of recombination in resolution of this replication stress are very poorly understood. This project will apply experimental approaches typically used in fundamental research on DNA replication stress to illuminate whether BPDE adducts block or collapse the incoming replication apparatus and how recombination factors act at those forks in eukaryotes. Three interesting candidate proteins involved in remodelling and recombination at impaired forks, PARP1, RAD51, and ZRANB3, will be systematically investigated for their function and potential interplay at replication forks in response to BPDE treatment in mammalian cells. Their impact on slowing, stalling, as well as restart or collapse of individual forks and new origin firing will be examined. Analyses of protein dynamics at BPDE-stalled replication forks will additionally clarify which DNA damage response mechanisms and proteins are crucial for recovery. Finally, the role of PARP1, RAD51, and ZRANB3 in BPDE-induced recombination will be assessed to elucidate biochemical reasons for the engagement of HR in DNA damage response to BPDE adducts. As unfaithful recombination could give rise to gross chromosomal instability, a better understanding of recombination-dependent and potentially recombinogenic DNA damage response mechanisms involved in the resolution of BPDE-induced replication stress will ultimately provide novel insight into B[a]P-caused mutagenicity and toxicity. This might uncover innovative strategies for prevention or protection in future.