Compartmentalization of the replication fork by single-stranded DNA binding protein regulates translesion synthesis

Abstract

DNA replication is mediated by the coordinated actions of multiple enzymes within replisomes. Processivity clamps tether many of these enzymes to DNA, allowing access to the primer/template junction. Many clamp-interacting proteins (CLIPs) are involved in genome maintenance pathways including translesion synthesis (TLS). Despite their abundance, DNA replication in bacteria is not perturbed by these CLIPs. Here we show that while the TLS polymerase Pol IV is largely excluded from moving replisomes, the remodeling of ssDNA binding protein (SSB) upon replisome stalling enriches Pol IV at replication forks. This enrichment is indispensable for Pol IV-mediated TLS on both the leading and lagging strands as it enables Pol IV-processivity clamp binding by overcoming the gatekeeping role of the Pol III epsilon subunit. As we have demonstrated for the Pol IV-SSB interaction, we propose that the binding of CLIPs to the processivity clamp must be preceded by interactions with factors that serve as localization markers for their site of action. Figure 4. The Pol IV-SSB interaction enriches Pol IV near lesion-stalled replisomes in cells. A. Schematic diagrams of PAmCherry fusions of Pol IV and RecQ WH -Pol IV LF variants. 303 VWP 305 , rim interacting residues; 346 QLVLGL 351 , the CBM of Pol IV; (G4S)4, a flexible linker. B. (Top panels) Representative fluorescence micrographs of an SSB-mYpet focus (left) and a single photoactivated Pol IV-PAmCherry molecule (right) with overlays showing the cell outlines. (Bottom panels) Corresponding brightfield micrographs with overlays showing SSB foci (red circle, left) or all detected Pol IV tracks (right) (Scale bars: 1 µM).C. Distributions of the mean distance between each static Pol IV track and the nearest SSB focus for Pol IV WT and mutants in cells treated with 100 mM MMS. D. Radial distribution functions g(r) for Pol IV WT and mutants in cells treated with 100 mM MMS. Also shown are random g(r) functions for each data set (dotted lines).

Coordination between nucleotide excision repair and specialized polymerase DnaE2 action enables DNA damage survival in non-replicating bacteria

“…While PolIII activity could directly contribute to gap filling (Isogawa et al, 2018; Sedgwick & Bridges, 1974; Soubry et al, 2019), it is also likely that it is the loading of the β-clamp that is essential for DnaE2 activity (Bunting et al, 2003; Chang et al, 2019; Fujii & Fuchs, 2004; Jérôme Wagner et al, 2009). Additionally, recent studies have highlighted a role for SSB as well in enriching the local pool of PolIV at a lesion, thus enabling polymerase switching (Chang et al, 2020). It would be interesting now to ask how additional components (such as ImuB and other accessory components to DnaE2) contribute to the loading of the ‘specialized replisome’ outside the realms of active replication and whether the properties of the ssDNA gaps generated may vary under different damaging conditions (UV vs MMC).…”

“…Since these error-prone polymerases can synthesize DNA and their activity is mediated by interaction with the β-clamp of the replisome (Bunting et al, 2003;Chang et al, 2019;Fujii & Fuchs, 2004;Thrall et al, 2017;Jérôme Wagner et al, 2009;Warner et al, 2010), action of these polymerases has mostly been studied in the context of replicating cells, as a mechanism that facilitates continued DNA synthesis by acting at or behind the replication fork (Chang et al, 2019(Chang et al, , 2020Indiani et al, 2005;Jeiranian et al, 2013;Marians, 2018). In addition to replicationassociated lesion tolerance, some studies have proposed the possibility of error-prone synthesis in a manner that is replication-independent (Janel-Bintz et al, 2017;Kozmin & Jinks-Robertson, 2013).…”

Coordination between nucleotide excision repair and specialized polymerase DnaE2 action enables DNA damage survival in non-replicating bacteria

“…While PolIII activity could directly contribute to gap-filling ( Isogawa et al, 2018 ; Sedgwick and Bridges, 1974 ; Soubry et al, 2019 ), it is also likely that it is the loading of the β-clamp that is essential for DnaE2 activity ( Bunting et al, 2003 ; Chang et al, 2019 ; Fujii and Fuchs, 2004 ; Wagner et al, 2009 ). Additionally, recent studies have highlighted a role for SSB as well in enriching the local pool of PolIV at a lesion, thus enabling polymerase switching ( Chang et al, 2020 ). The lack of a significant percentage of cells with multiple replisome foci under damage would suggest that only a limited number of long ssDNA gaps are generated per cell or that some repair/replisome component involved in gap processing or gap-filling is limiting.…”

“…Long ssDNA gaps generated by NER serve two purposes: a. Activation of the SOS response for specialized polymerase expression; it is likely that in case of Caulobacter, RecA is essential only for turning on the SOS regulon as DnaE2-mediated synthesis has been previously shown to function independent of RecA (Alves et al, 2017;Galhardo, 2005), unlike E. coli PolV (Goodman, 2014;Nohmi et al, 1988 (Isogawa et al, 2018;Sedgwick & Bridges, 1974;Soubry et al, 2019), it is also likely that it is the loading of the clamp that is essential for DnaE2 activity (Bunting et al, 2003;Chang et al, 2019;Fujii & Fuchs, 2004;Jérôme Wagner et al, 2009). Additionally, recent studies have highlighted a role for SSB as well in enriching the local pool of PolIV at a lesion, thus enabling polymerase switching (Chang et al, 2020). The lack of a significant percentage of cells with multiple replisome foci under damage would suggest that only a limited number of long ssDNA gaps are generated per cell or that some repair/ replisome component involved in gap processing or gap-filling is limiting.…”

“…Since error-prone polymerases can synthesize DNA and their activity is mediated by interaction with the β-clamp of the replisome ( Bunting et al, 2003 ; Chang et al, 2019 ; Fujii and Fuchs, 2004 ; Thrall et al, 2017 ; Wagner et al, 2009 ; Warner et al, 2010 ), action of these polymerases has mostly been studied in the context of replicating cells, as a mechanism that facilitates continued DNA synthesis by acting at or behind the replication fork ( Chang et al, 2019 ; Chang et al, 2020 ; Indiani et al, 2005 ; Jeiranian et al, 2013 ; Marians, 2018 ). In addition to replication-associated lesion tolerance, some studies have proposed the possibility of error-prone synthesis in a manner that is replication-independent ( Janel-Bintz et al, 2017 ; Kozmin and Jinks-Robertson, 2013 ).…”