Spatiotemporal characterization of single-stranded DNA Intermediates after UV Irradiation: I: Post-replication gaps...

When E. coli cells are UV irradiated, replisome encounters with some DNA lesions lead to lesion skipping and formation of ssDNA gaps. These gaps are protected by SSB and repaired through the RecFOR recombinational DNA repair pathway. However, many questions about this pathway remain unanswered. Here, we used a fluorescent SSB fusion that supports normal growth in the absence of WT SSB under most conditions to directly visualize the real-time formation and resolution of ssDNA intermediates in cells lacking factors (RecB, RecJ, RecF, and RecO), that facilitate recombinational DNA repair pathways under slow growth conditions. Upon DNA damage, SSB-bound features of various sizes increased within these cells. In WT cells, ssDNA gaps appeared and were resolved at a steady state level that persisted for hours. Formation of most ssDNA gaps was not dependent on RecB function. Large increases in ssDNA gaps were observed in cells lacking RecFORJ functions, particularly in the absence of RecO. These findings indicate that: (a) When hundreds of UV lesions are introduced into the genome, at least some lesions remain unaddressed by nucleotide excision repair (NER) for several hours under slow growth conditions. (b) Replisome encounters with DNA lesions rapidly generate ssDNA gaps. (c) A relatively small portion of the ssDNA foci appearing in WT cells may reflect breaks processed by the RecBCD system. (d) Most prominent SSB features reflect post-replication gaps repaired by RecFORJ. Lack of RecFORJ functions leads to accumulation of unresolved gaps over time. (e) RecF is not required for post-replication gap formation. Overall, the results provide direct visualization of complex UV-induced changes in DNA metabolism caused by replisome encounters with UV-generated pyrimidine dimers. Combined with a decades-long literature of related results and proposals, a unified view of how E. coli responds to UV irradiation can be put forward.