Spatiotemporal characterization of single-stranded DNA intermediates after UV irradiation: II. Rapid growth and effects of recA and recJ

Irradiation of E. coli with UV light results in the formation of post-replication gaps and induction of the SOS response. Here, we investigate the dynamics of single strand gap formation and resolution in cells growing with a 50 min doubling time within specially designed microfluidic chips, making observations on fluorescent gap markers in individual cells over the course of 8 hours. In cells proficient for gap repair, irradiation with UV at 5 J/m 2 triggers an immediate increase in the number and intensity of gap markers. Cells lacking recF , recO , or recA cannot repair gaps via the canonical gap repair pathway and exhibit elevated gap numbers and intensities over many hours. Major conclusions include: (1) Post-replication gaps are a major feature of DNA metabolism after UV irradiation. (2) The long-lived, high-intensity foci observed in recF , recO , or recA mutants are completely dependent on the RecJ nuclease. In the absence of other RecFOR pathway functions, RecJ-mediated enlargement of many gaps continues unabated for extended periods. (3) In the absence of RecA or other RecFOR pathway functions, cells accumulate repair intermediates that are bound by large numbers of SSB molecules. (4) In the absence of RecJ, other pathways, perhaps involving TLS, displace SSB in gaps. Our results also confirm, using a different experimental setup and protocol, that: (a) UV-related repair activities, including nucleotide excision repair of at least some lesions, may continue for multiple cell generations after exposure; and (b) we again see no evidence that RecF facilitates lesion skipping. Overall patterns of gap formation and resolution under rapid growth conditions are consistent with a burst of post-replication gap formation following UV irradiation, postulated in the accompanying report to rapidly trigger the SOS response.