DNA polymerase IV primarily operates outside of DNA replication forks in Escherichia coli

Abstract

In Escherichia coli, damage to the chromosomal DNA induces the SOS response, setting in motion a series of different DNA repair and damage tolerance pathways. DNA polymerase IV (pol IV) is one of three specialised DNA polymerases called into action during the SOS response to help cells tolerate certain types of DNA damage. The canonical view in the field is that pol IV primarily acts at replisomes that have stalled on the damaged DNA template. However, the results of several studies indicate that pol IV also acts on other substrates, including single-stranded DNA gaps left behind replisomes that re-initiate replication downstream of a lesion, stalled transcription complexes and recombination intermediates. In this study, we use single-molecule time-lapse microscopy to directly visualize fluorescently labelled pol IV in live cells. We treat cells with the DNA-damaging antibiotic ciprofloxacin, Methylmethane sulfonate (MMS) or ultraviolet light and measure changes in pol IV concentrations and cellular locations through time. We observe that only 5–10% of foci induced by DNA damage form close to replisomes, suggesting that pol IV predominantly carries out non-replisomal functions. The minority of foci that do form close to replisomes exhibit a broad distribution of colocalisation distances, consistent with a significant proportion of pol IV molecules carrying out postreplicative TLS in gaps behind the replisome. Interestingly, the proportion of pol IV foci that form close to replisomes drops dramatically in the period 90–180 min after treatment, despite pol IV concentrations remaining relatively constant. In an SOS-constitutive mutant that expresses high levels of pol IV, few foci are observed in the absence of damage, indicating that within cells access of pol IV to DNA is dependent on the presence of damage, as opposed to concentration-driven competition for binding sites.

The SOS system: A complex and tightly regulated response to DNA damage

“…Impaired Pol III-ssDNA interactions (e.g., when polymerase encounters a hairpin-stem duplex) influence the susceptibility of Pol III HE to Pol IVmediated polymerase exchange (Le et al, 2017). Using single-molecule time-lapse microscopy to directly visualize fluorescently labeled pol IV in live cells, Henrikus et al (2018a) recently demonstrated that over 90% of foci induced by DNA damage form outside of replisome regions, suggesting that pol IV predominantly carries out non-replisomal functions, consistent with the postreplicative TLS in gaps behind the replisome model. However, Pol IV's access to ssDNA gaps is restricted to the first 100 min after induction of the SOS response.…”

DNA double-strand breaks induced by reactive oxygen species promote DNA polymerase IV activity inEscherichia coli

“…7). In a previous study (39), following ciprofloxacin treatment, cells exhibited a similar increase in DinB-YPet concentration; an increase in intracellular DinB-YPet (pol IV) concentrations was measured from 6 ± 1 nM prior to treatment (standard error of the mean, SE) to 34 ± 3 nM (SE) 180 min after ciprofloxacin addition. Interestingly, in this present study, we showed that inclusion of DMSO led to a significant reduction in the expression level of DinB-YPet in ciprofloxacin-treated cells.…”

“…Following antibiotic addition, we recorded time-lapse movies capturing fluorescence from Escherichia coli cells expressing a functional, YPet fusion of the DinB gene from its native promoter ( SI Appendix , Fig. 1 B, C , Materials and Methods) (39, 47). We then monitored pol IV concentrations by measuring the fluorescence intensity of DinB-YPet within cells in the presence or absence of DMSO (2% v/v) and monitored DNA binding activities by counting the number of pol IV foci per cell.…”

“…For some antibiotics, it has however been shown that the SOS response is mostly triggered following DSB processing by RecBCD (36, 37). Notably, upon induction of the SOS response, the cellular concentration of pol IV increases significantly (38, 39). Despite these observations, it remains unclear if pol IV primarily works in recombination intermediates or in the context of replisomes in cells.…”

DNA lesions proximity modulates damage tolerance pathways inEscherichia coli

“…On the other hand, earlier work has suggested that replication forks are able to skip over the lesion, leaving a gap that will be repaired later on (4). This model of "skipping" over the lesion has been recently corroborated by in vitro works showing that repriming can take place on the leading strand (5, 6), (7), and in vivo work showing that DNA Pol IV acts mostly at gaps left behind the replication fork, rather than at stalled replication forks (8). In both models, whether the replication fork stalls at the damage, or skips over the damage, ssDNA gaps are generated downstream the lesion both in the leading and lagging strand, and need to be filled (repaired) in order to achieve chromosomal replication.…”