Replisome activity slowdown after exposure to ultraviolet light in Escherichia coli

Abstract

The replisome is a multiprotein machine that is responsible for replicating DNA. During active DNA synthesis, the replisome tightly associates with DNA. In contrast, after DNA damage, the replisome may disassemble, exposing DNA to breaks and threatening cell survival. Using live cell imaging, we studied the effect of UV light on the replisome of Escherichia coli. Surprisingly, our results showed an increase in Pol III holoenzyme (Pol III HE) foci post-UV that do not colocalize with the DnaB helicase. Formation of these foci is independent of active replication forks and dependent on the presence of the χ subunit of the clamp loader, suggesting recruitment of Pol III HE at sites of DNA repair. Our results also showed a decrease of DnaB helicase foci per cell after UV, consistent with the disassembly of a fraction of the replisomes. By labeling newly synthesized DNA, we demonstrated that a drop in the rate of synthesis is not explained by replisome disassembly alone. Instead, we show that most replisomes continue synthesizing DNA at a slower rate after UV. We propose that the slowdown in replisome activity is a strategy to prevent clashes with engaged DNA repair proteins and preserve the integrity of the replication fork.DNA replication | replisome | fluorescence microscopy | UV | Escherichia coli

Visualizing mutagenic repair: novel insights into bacterial translesion synthesis

“…Indeed, several lines of evidence are in favor of the idea that a majority of TLS repair occurs post-replication. It was recently observed that, upon UV exposure, there is an increase in the number of fluorescently tagged Pol III foci that localize away from active replisomes (denoted by the localization of helicase) (Soubry, Wang and Reyes-Lamothe 2019 ). Some of these foci could be repair centers performing post-replicative TLS at the lesions that were skipped by active replisomes.…”

“…For example, studies have observed that mitomycin C-mediated lesions lead to dissociation of the replicative polymerase, PolC, in Bacillus subtilis (Li et al . 2019 ), while UV exposure causes delay in replication (Rudolph, Upton and Lloyd 2007 ) and replisome slowdown in E. coli (Soubry, Wang and Reyes-Lamothe 2019 ). This slowdown is attributed to the inability of Pol III to accommodate bulky DNA modifications and synthesize across them (Yang and Gao 2018 ).…”

Interaction with single‐stranded DNA‐binding protein localizes ribonuclease HI to DNA replication forks and facilitates R‐loop removal

“…Consistent with RNase HI‐YPet foci forming at DNA replication forks, the median distance between a spot of RNase HI‐YPet and the nearest mCherry‐β‐clamp spot is 202 nm (Figure 1c, inset). These values are slightly higher than those previously reported for replisomal components with the ε subunit of DNA Pol III and β‐clamp, which have a median distance of 128 nm (Soubry et al ., 2019). Consequently, we further tested for co‐localization between RNase HI‐YPet and β‐clamp‐mCherry by comparing the distribution of distances to a random distribution of spots in cells, represented by the radial distribution function g ( r ).…”

Non-random segregation of sister chromosomes byEscherichia coliMukBEF axial cores

“…AB1157, lacO240 at ori1 (3908) (hyg), tetO240 at ter3 (1644) (gen), ΔleuB::Plac-lacI-mCherryfrt, ΔgalK::Plac-tetR-mCerulean-frt (Reyes-Lamothe et al, 2008) RRL388 AB1157, frt-mCherry-dnaN (Soubry et al, 2019) RRL396 AB1157, frt-Ypet-dnaB, kan-mCherry-dnaN (Soubry et al, 2019) SN192 AB1157, lacO240 at ori1 (3908) (hyg), tetO240 at ter3 (1644) (gen), ΔleuB::Plac-lacI-mCherryfrt, ΔgalK::Plac-tetR-mCerulean-frt, mukB-mYpet-frt (Nolivos et al, 2016) SN302 AB1157, lacO240 at ori1 (3908) (hyg), tetO240 at ter3 (1644) (gen), ΔleuB::Plac-lacI-mCherryfrt, ΔgalK::Plac-tetR-mCerulean-frt, mukB-mYpet-frt, ΔmatP::cat (Nolivos et al, 2016)…”