Interaction between Escherichia coli DNA polymerase IV and single-stranded DNA-binding protein is required for DNA synthesis on SSB-coated DNA

Furukohri, Asako; Nishikawa, Yoshito; Akiyama, Masahiro; Maki, Hisaji

doi:10.1093/nar/gks264

Cited by 28 publications

(21 citation statements)

References 35 publications

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“…5) would thus not effectively compete with SSB (1.7-3.4 μM; Bobst et al, 1985) in terms of ssDNA binding. Moreover, although DinB also interacts with SSB (Furukohri et al, 2012), we have not been able to detect replacement of SSB on SSB-coated ssDNA by DinB (AF, MTA and HM, unpublished results). Thus, it is highly unlikely that DinB makes ssDNA inaccessible to RecA by masking it to suppress SOS induction.…”

Section: Discussionmentioning

confidence: 82%

<i>Escherichia coli</i> DinB inhibits replication fork progression without significantly inducing the SOS response

et al. 2012

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The SOS response is readily triggered by replication fork stalling caused by DNA damage or a dysfunctional replicative apparatus in Escherichia coli cells. E. coli dinB encodes DinB DNA polymerase and its expression is upregulated during the SOS response. DinB catalyzes translesion DNA synthesis in place of a replicative DNA polymerase III that is stalled at a DNA lesion. We showed previously that DNA replication was suppressed without exogenous DNA damage in cells overproducing DinB. In this report, we confirm that this was due to a dosedependent inhibition of ongoing replication forks by DinB. Interestingly, the DinB-overproducing cells did not significantly induce the SOS response even though DNA replication was perturbed. RecA protein is activated by forming a nucleoprotein filament with single-stranded DNA, which leads to the onset of the SOS response. In the DinB-overproducing cells, RecA was not activated to induce the SOS response. However, the SOS response was observed after heat-inducible activation in strain recA441 (encoding a temperature-sensitive RecA) and after replication blockage in strain dnaE486 (encoding a temperature-sensitive catalytic subunit of the replicative DNA polymerase III) at a non-permissive temperature when DinB was overproduced in these cells. Furthermore, since catalytically inactive DinB could avoid the SOS response to a DinB-promoted fork block, it is unlikely that overproduced DinB takes control of primer extension and thus limits single-stranded DNA. These observations suggest that DinB possesses a feature that suppresses DNA replication but does not abolish the cell's capacity to induce the SOS response. We conclude that DinB impedes replication fork progression in a way that does not activate RecA, in contrast to obstructive DNA lesions and dysfunctional replication machinery.

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Section: Discussionmentioning

confidence: 82%

<i>Escherichia coli</i> DinB inhibits replication fork progression without significantly inducing the SOS response

et al. 2012

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“…One may speculate that SSB on the lagging strand directly contacts the polymerase on the leading strand and inhibits its action. In fact, direct contact of SSB to Pol II and to Pol IV has been documented (35,50). RecA may overcome SSB inhibition by simply occupying the lagging strand adjacent to the leading polymerase and prevent SSB binding (Fig.…”

Section: Discussionmentioning

confidence: 99%

RecA acts as a switch to regulate polymerase occupancy in a moving replication fork

Indiani

Patel

Goodman

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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This report discovers a role of Escherichia coli RecA, the cellular recombinase, in directing the action of several DNA polymerases at the replication fork. Bulk chromosome replication is performed by DNA polymerase (Pol) III. However, E. coli contains translesion synthesis (TLS) Pols II, IV, and V that also function with the helicase, primase, and sliding clamp in the replisome. Surprisingly, we find that RecA specifically activates replisomes that contain TLS Pols. In sharp contrast, RecA severely inhibits the Pol III replisome. Given the opposite effects of RecA on Pol III and TLS replisomes, we propose that RecA acts as a switch to regulate the occupancy of polymerases within a moving replisome. Previous studies have shown that all three Pols function at replication forks with DnaB helicase, primase, the β sliding clamp, and the clamp loader (2). Bulk chromosome replication is performed by Pol III, and the mechanism that regulates the access of TLS Pols to the replisome is largely unexplored.Heavy DNA damage in E. coli induces the SOS response, which slows down chromosomal replication (3) and expresses over 40 proteins that aid cell survival and enable genome replication to continue (4, 5). Induction of the SOS response requires formation of ssDNA by replication forks to which the cellular recombinase, RecA, binds. The binding of RecA to ssDNA facilitates RecAmediated self-cleavage of the LexA repressor, thereby activating the SOS response genes (6). Among these are TLS Pol II (polB) and TLS Pol IV (dinB), which are rapidly (<5 min) up-regulated during the SOS response (7-10). TLS Pol V (UmuD′ 2 C) is induced late (≥45 min) and requires RecA bound to ssDNA (RecA*) for synthetic activity (8,11,12). Pol IV and Pol V belong to the Yfamily Pols (1), whereas Pol II, even though involved in TLS and mutagenesis, is a B-family polymerase (13).During normal chromosome duplication, the intracellular levels of Pols II and IV are not sufficient to take over Pol III-based replisomes (2). Upon DNA damage, the induced levels of TLS Pols take over the cellular replicase (2, 14, 15). Our previous studies have shown that Pols II and IV replace Pol III within the replisome without causing fork collapse, as they preserve the DNAbound helicase and sliding clamp to form TLS Pol replisomes (2). Pol II and Pol IV replisomes progress at significantly slower rates than the intrinsic rate of DnaB helicase, and thus regulate the speed of helicase unwinding. Slower fork progression may give the cell additional time to repair DNA lesions using normal repair processes (e.g., nucleotide excision repair), thereby preventing direct encounters of the replication fork with DNA lesions.It has been suggested that polymerase selection at the replication fork is mainly guided by mass action dictated by the concentration of each polymerase and its affinity for the clamp (1,14,16,17). However, the findings of this report demonstrate that RecA adds an additional level of control, acting as a switch that regulates DNA polymerase access to the replicat...

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“…A subset of CLIPs interacts with ssDNA binding protein (SSB) including all three TLS polymerases (Arad et al, 2008;Furukohri et al, 2012;Molineux and Gefter, 1974). In cells, SSB rapidly associates with ssDNA to protect it from nucleolytic cleavage and chemical damage.…”

Section: Introductionmentioning

confidence: 99%

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

Chang

Thrall

Laureti

et al. 2020

Preprint

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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).

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Interaction between Escherichia coli DNA polymerase IV and single-stranded DNA-binding protein is required for DNA synthesis on SSB-coated DNA

Cited by 28 publications

References 35 publications

<i>Escherichia coli</i> DinB inhibits replication fork progression without significantly inducing the SOS response

<i>Escherichia coli</i> DinB inhibits replication fork progression without significantly inducing the SOS response

RecA acts as a switch to regulate polymerase occupancy in a moving replication fork

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

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