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

已知职业性或与吸烟相关的人类致癌物苯并[a]芘（B[a]P）可诱发肺部肿瘤。B[a]P的致癌性主要由其活性代谢物抗B[a]P-7,8-二醇-9,10-环氧化物（BPDE）的DNA加合物形成介导。虽然这些DNA加合物如果在s期之前未被修复，则会损害DNA复制叉的进展，这一过程被称为DNA复制应激，但翻译合成（TLS）聚合酶容易出错的绕过通常允许继续复制。经过BPDE加合物的TLS已被广泛研究，并与B[a] p引起的致突变性有关。然而，BPDE加合物也会导致同源重组（HR）和姐妹染色单体交换，这表明重组因子在分叉处起作用，而分叉实际上是停滞或坍塌成DNA双链断裂（DSBs）。受损的复制叉进展和依赖复制的DNA dsb形成对B[a] p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，人们知之甚少。本项目将采用DNA复制压力基础研究中常用的实验方法，阐明BPDE加合物是否阻断或破坏进入的复制装置，以及真核生物中重组因子如何在这些分叉处起作用。在哺乳动物细胞中，三个有趣的候选蛋白PARP1， RAD51和ZRANB3参与了受损叉的重塑和重组，将系统地研究它们在BPDE治疗下在复制叉上的功能和潜在的相互作用。它们对单个分叉的减速、失速、重启或崩溃以及新原点发射的影响将被检查。分析bpde停止复制分叉处的蛋白质动力学将进一步阐明哪些DNA损伤反应机制和蛋白质对恢复至关重要。最后，我们将评估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DNA损伤反应的生化原因。由于不忠实的重组可能导致总体染色体不稳定，因此更好地了解重组依赖和潜在的重组性DNA损伤反应机制，涉及解决bpde诱导的复制应激，最终将为B[a] p引起的突变性和毒性提供新的见解。这可能会揭示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.