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

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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.