Cell cycle regulation of polymerase theta mediated end-joining underlies its sensitivity to loss of BLM-DNA2

DNA double-strand breaks (DSBs) are predominantly resolved by the error-prone non-homologous end-joining (NHEJ) or high-fidelity homologous recombination (HR) DNA repair pathways. NHEJ involves minimal end-processing and drives direct ligation of the broken ends, while HR first requires a DSB resection step that degrades 5’ strands to generate 3’ ssDNA tails necessary for homology search. When HR fails, an alternative end-joining pathway involving polymerase theta can serve as a backup repair mechanism by annealing and ligating microhomologies present in ssDNA tails on either side of a resected DSB. Resection is the early step that commits a DSB to repair by HR, and is therefore considered a prerequisite for TMEJ as well. It has been proposed that the exonuclease EXO1 and the helicase/nuclease complex of BLM-DNA2 have redundant roles in resection, and therefore may both contribute to TMEJ. Here, we use a sensitive deep-sequencing approach to identify DSB repair outcomes at an endogenous locus in response to genetic and chemical perturbation of the resection machinery. While we observe that both EXO1 and BLM-DNA2 contribute to resection, our findings highlight a predominant role for BLM-DNA2 in facilitating TMEJ. However, careful cell cycle analysis revealed that BLM-DNA primarily contributes to TMEJ by ensuring normal progression to the G2/M transition, required for TMEJ to become activated. We propose that rather than direct, mechanistic roles in resection, the critical functions of BLM and DNA2 in genome replication largely account for their genetic contribution to efficient TMEJ.