Distinct effects of the two major types of UV-induced DNA lesions on DNA damage responses
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The most prevalent human carcinogen is sunlight-associated UV, a physiologic dose of which generates thousands of DNA lesions per cell, mostly of two types: cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs). To cope with these lesions that are DNA- distorting and mutagenic, cells trigger ATR-mediated checkpoint signaling to regulate diverse processes including cell cycle arrest, DNA replication, DNA repair, and apoptosis. These ATR- mediated processes determine the mutagenic outcomes of UV-damaged cells. Thus, elucidating the fundamental mechanism by which cells sense UV lesions and activate DNA damage response pathways would be important for our understanding and control of UV carcinogenesis. Although it is known that UV generates distinct types of lesions and triggers DNA damage responses, it remains unclear whether and how these two structurally distinct types of UV lesions are different in their abilities to activate ATR. We hypothesized that there are striking differences in the mechanisms and impact of these two lesion types on DNA replication and DNA damage responses. To determine the individual contributions of CPD and 6-4PP to ATR activation (Aim 1), we generated cells with a single type of lesion (using photolyases that selectively repair either CPD or 6-4PP) and evaluated lesion-specific ATR activation by multiparameter flow cytometry. Strikingly, we found that the ATR-Chk1 pathway is potently activated by 6-4PP, but not by CPD lesions. To investigate the mechanism by which only 6-4PP activates ATR (Aim 2), we examined the effect of each lesion type on replication progression. Using microfluidic-assisted replication track analysis (maRTA), we found that 6-4PPs, but not CPDs, markedly impede DNA replication across the genome. Moreover, we demonstrated that only 6-4PPs preferentially become surrounded by single-stranded DNA in S phase, indicating selective and prolonged replication blockage at 6-4PPs. Taken together, 6-4PPs, although 8- fold fewer in number than CPDs, are critical for replication blockage and activation of the UV- induced ATR-Chk1 pathway. This study identifying the respective roles of CPD and 6-4PP lesions that occur at dipyrimidine sites provides a molecular basis for their remarkably distinct effects on DNA damage responses and insight into the mechanisms of UV carcinogenesis.
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