SummaryEscherichia coli dinB encodes the specialized DNA polymerase DinB (Pol IV), which is induced as part of the SOS stress-response system and functions in translesion synthesis (TLS) to relieve the replicative Pol III that is stalled at DNA lesions. As the number of DinB molecules, even in unstressed cells, is greater than that required to accomplish TLS, it is thought that dinB plays some additional physiological role. Here, we overexpressed dinB under the tightly regulable arabinose promoter and looked for a distinct phenotype. Upon induction of dinB expression, progression of the replication fork was immediately inhibited at random genomic positions, and the colony-forming ability of the cells was reduced. Overexpression of mutated dinB alleles revealed that the structural requirements for these two inhibitory effects and for TLS were distinct. The extent of in vivo inhibition displayed by a mutant DinB matched the extent of its in vitro impedance, at near-physiological concentration, of a moving Pol III. We suggest that DinB targets Pol III, thereby acting as a brake on replication fork progression. Because the brake operates when cells have excess DinB, as they do under stress conditions, it may serve as a checkpoint that modulates replication to safeguard genome stability.

An assay that measures synchronized, processive DNA replication by Escherichia coli DNA polymerase III holoenzyme was used to reveal replacement of pol III by the specialized lesion bypass DNA polymerase IV when the replicative polymerase is stalled. When idled replication is restarted, a rapid burst of pol III-catalyzed synthesis accompanied by ϳ7-kb full-length products is strongly inhibited by the presence of pol IV. The production of slower-forming, shorter length DNA reflects a rapid takeover of DNA synthesis by pol IV. Here we demonstrate that pol IV rapidly (<15 s) obstructs the stable interaction between pol III* and the ␤ clamp (the lifetime of the complex is >5 min), causing the removal of pol III* from template DNA. We propose that the rapid replacement of pol III* on the ␤ clamp with pol IV is mediated by two processes, an interaction between pol IV and the ␤ clamp and a separate interaction between pol IV and pol III*. This newly discovered property of pol IV facilitates a dynamic exchange between the two free polymerases at the primer terminus. Our study suggests a model in which the interaction between pol III* and the ␤ clamp is mediated by pol IV to ensure that DNA replication proceeds with minimal interruption.Successful DNA replication requires a high fidelity DNA polymerase to replicate the entire genome accurately. In Escherichia coli, DNA polymerase III holoenzyme (pol III HE) 2 is a replicative polymerase that catalyzes elongation of DNA chains at a rate of about 1 kb/s with high fidelity. pol III HE is a multisubunit complex that contains two pol III core catalytic subassemblies, each linked to a subunit of the DnaX complex. Polymerase activity resides in the ␣ subunit, the largest subunit of pol III HE. pol III*, a subassembly composed of two pol III cores and one DnaX complex, binds to a dimer of the ␤ subunit (the ␤ clamp) loaded onto the primer/template by the DnaX complex to form a stable structure, pol III HE, which can synthesize DNA processively (over 50 kb per binding event) (1, 2).When a replisome encounters a lesion, pol III is generally believed to stall on DNA at a lesion site on the leading strand, resulting in uncoupling of leading and lagging strand DNA synthesis and arrest of the replication fork (3-5). Two major pathways are thought to play a role in overcoming DNA damage at the replication fork; one is recombinational repair, and the other is the translesion synthesis (TLS). In the TLS pathway, a switch is likely to occur from the stalled replicative polymerase to a specialized polymerase, which replaces the former to bypass the lesion. It is not yet clear, however, how a specialized polymerase gains access to the primer terminus when a stalled replicative polymerase exists at the site or how it acts in coordination with the replicative polymerase (6).In E. coli, three polymerases, pol II, pol IV, and pol V, have been identified as specialized polymerases. pol IV, encoded by the dinB gene, belongs to the Y family and is up-regulated by the SOS response (7). pol IV can r...

Escherichia coli DNA polymerase IV (Pol IV, also known as DinB) is a Y-family DNA polymerase capable of catalyzing translesion DNA synthesis (TLS) on certain DNA lesions, and accumulating data suggest that Pol IV may play an important role in copying various kinds of spontaneous DNA damage including N2-dG adducts and alkylated bases. Pol IV has a unique ability to coexist with Pol III on the same β clamp and to positively dissociate Pol III from β clamp in a concentration-dependent manner. Reconstituting the entire process of TLS in vitro using E. coli replication machinery and Pol IV, we observed that a replication fork stalled at (−)-trans-anti-benzo[a]pyrene-N2-dG lesion on the leading strand was efficiently and quickly recovered via two sequential switches from Pol III to Pol IV and back to Pol III. Our results suggest that TLS by Pol IV smoothes the way for the replication fork with minimal interruption.

Long inverted repeats (LIRs), often found in eukaryotic genomes, are unstable in Escherichia coli where they are recognised by the SbcCD (the bacterial Mre11/Rad50 homolog), an endonuclease/exonuclease capable of cleaving hairpin DNA. It has long been postulated that LIRs form hairpin structures exclusively on the lagging-strand template during DNA replication, and SbcCD cleaves these hairpin-containing lagging-strands to generate DNA double-strand breaks. Using a reconstituted oriC plasmid DNA replication system, we have examined how a replication fork behaves when it meets a LIR on DNA. We have shown that leading-strand synthesis stalls transiently within the upstream half of the LIR. Pausing of lagging-strand synthesis at the LIR was not clearly observed, but the pattern of priming sites for Okazaki fragment synthesis was altered within the downstream half of the LIR. We have found that the LIR on a replicating plasmid was cleaved by SbcCD with almost equal frequency on both the leading-and lagging-strand templates. These data strongly suggest that the LIR is readily converted to a cruciform DNA, before the arrival of the fork, creating SbcCDsensitive hairpin structures on both leading-and lagging-strands. We propose a model for the Author ManuscriptThis article is protected by copyright. All rights reserved replication-dependent extrusion of LIRs to form cruciform structures that transiently impede replication fork movement.

Dbf4 is a regulatory subunit for the Cdc7 protein kinase that is required for the initiation of eukaryotic DNA replication, but the precise roles of Dbf4-Cdc7 remain to be determined. Here we identified a Xenopus homolog of Dbf4 (XDbf4) and characterized XDbf4 and Xenopus Cdc7 (XCdc7) in Xenopus egg extracts. XDbf4 formed a complex with XCdc7 in egg extracts and activated XCdc7 kinase activity in vitro. In contrast with Dbf4 in yeast and mammalian cultured cells, the XDbf4 levels in egg extracts did not change during the cell cycle progression. XDbf4 was a phosphoprotein in interphase extracts, and was apparently hyperphosphorylated in cytostatic factor (CSF)-mediated, metaphase-arrested extracts and in mitotic extracts. However, the hyperphosphorylation of XDbf4 did not seem to affect the level of kinase activation, or chromatin binding of the XDbf4-XCdc7 complex. Upon release from CSF-arrest, XDbf4 was partially dephosphorylated and bound to chromatin. Interestingly, XDbf4 was loaded onto chromatin before XCdc7 during DNA replication in egg extracts. These results suggest that the function of XDbf4-XCdc7 during the early embryonic cell cycle is regulated in a manner distinct from that during the somatic cell cycle.