Unraveling Bloom Helicase role downstream of Nucleotide Excision Repair for ssDNA formation and DNA Damage Checkpoint activation

Cells are persistently exposed to ultraviolet (UV) radiation, which creates bulky DNA lesions that distort the double helix. Efficient recognition and repair of these lesions are essential to prevent the accumulation of genotoxic damage. We previously showed that EXO1, a 5′–3′ exonuclease, is recruited to a subset of UV-induced DNA damage sites, where its activity generates extended single-stranded DNA (ssDNA) gaps and promotes robust activation of the DNA damage checkpoint downstream of nucleotide excision repair (NER). Following NER-mediated lesion recognition and excision, we observed the recruitment of Y family translesion synthesis (TLS) polymerases at local UV damage sites. This recruitment is strictly EXO1-dependent, indicating that TLS polymerases act downstream of NER, specifically at closely spaced opposing lesions (COLs). Such lesions, occurring on both DNA strands, impede canonical repair synthesis and require additional processing to ensure repair completion. Tight coordination of these events is critical to prevent the formation of unscheduled double-strand breaks (uDSBs), particularly in non-proliferating cells. NER incision by the XPG and XPF–ERCC1 endonucleases generates repair intermediates containing a 3′ hydroxyl (3′OH) and a 5′ phosphate (5′P). While repair synthesis initiates from the 3′OH, it can stall when a second lesion is present on the template strand. In parallel, the exposed 5′P end can be resected by EXO1, enlarging the gap and leaving the 3′OH unextended, thereby contributing to further ssDNA accumulation. We further identified the 3′–5′ helicase BLM as a late-recruited factor at UV damage sites. BLM accumulation coincides with replication protein A (RPA) loading, consistent with extended ssDNA formation. Notably, BLM recruitment is independent of EXO1, suggesting that BLM acts on exposed 3′OH ends or associated ssDNA structures to stabilize or further process these repair intermediates.