A Genetic Selection for dinB Mutants Reveals an Interaction between DNA Polymerase IV and the Replicative Polymerase That Is Required for Translesion Synthesis

Scotland, Michelle K.; Heltzel, Justin M. H.; Kath, James E.; Choi, Jung Hee; Berdis, Anthony J.; Loparo, Joseph J.; Sutton, Mark D.

doi:10.1371/journal.pgen.1005507

Cited by 29 publications

(53 citation statements)

References 83 publications

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“…Interaction of Pol IV with Pol III* may trigger a conformational change so that at least one of the two mutual binding sites within ␤ 2, sequestered by Pol III ␣ and ⑀ during elongation (20), become accessible. Consistent with this possibility, residues within or near the start of ␣ helix 5 in Pol IV, which are well removed from the two distinct ␤ 2 clamp binding motifs, are required to displace Pol III* from ␤ 2 assembled on DNA (17). Pol III* exchange could proceed by a similar mechanism, except the primary interacting motif would have to reside within -containing DnaX complexes.…”

Section: Discussionmentioning

confidence: 93%

“…Two different mechanisms have been proposed for Pol IV exchange. One, called the toolbelt model, predicts that Pol IV is recruited through contact with one of the two canonical clamp binding sites, whereas the other predicts recruitment mediated by a direct contact of Pol IV with both Pol III* and an accessory site on the rim of the ␤ 2 sliding clamp, subsequently displacing Pol III to allow binding by Pol IV (12)(13)(14)(15)(17)(18)(19). If operative, the toolbelt model will require modification in light of a recent elegant cryo-EM structure that shows that both of the canonical binding sites within ␤ 2 are occupied by interacting Pol III ␣ and ⑀ subunits of the Pol III HE (20).…”

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confidence: 99%

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DNA Polymerase III, but Not Polymerase IV, Must Be Bound to a τ-Containing DnaX Complex to Enable Exchange into Replication Forks

Yuan¹,

Dohrmann²,

Sutton³

et al. 2016

Journal of Biological Chemistry

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Examples of dynamic polymerase exchange have been previously characterized in model systems provided by coliphages T4 and T7. Using a dominant negative D403E polymerase (Pol) III ␣ that can form initiation complexes and sequester primer termini but not elongate, we investigated the possibility of exchange at the Escherichia coli replication fork on a rolling circle template. Unlike other systems, addition of polymerase alone did not lead to exchange. Only when D403E Pol III was bound to a -containing DnaX complex did exchange occur. In contrast, addition of Pol IV led to rapid exchange in the absence of bound DnaX complex. Examination of Pol III* with varying composition of or the alternative shorter dnaX translation product ␥ showed that -, 2 -, or 3 -DnaX complexes supported equivalent levels of synthesis, identical Okazaki fragment size, and gaps between fragments, possessed the ability to challenge pre-established replication forks, and displayed equivalent susceptibility to challenge by exogenous D403E Pol III*. These findings reveal that redundant interactions at the replication fork must stabilize complexes containing only one . Previously, it was thought that at least two s in the trimeric DnaX complex were required to couple the leading and lagging strand polymerases at the replication fork. Possible mechanisms of exchange are discussed.As with all cellular replicases, the DNA polymerase (Pol) 2 III holoenzyme (HE) of Escherichia coli is tripartite with a sliding clamp processivity factor (␤ 2 ), a clamp loader (DnaX complex, DnaX 3 ␦␦'), and an associated replicative polymerase (Pol III, ␣⑀) (for a review, see Ref. 2). In E. coli and many other bacteria, DnaX encodes two products: a shorter ␥ protein that has ATPase activity and the ability to support clamp loading on single-stranded DNA and a longer protein that has additional domains that interact with the DnaB 6 replicative helicase and Pol III. Cells that cannot express ␥ exhibit defects in UV viability and Pol IV-mediated mutagenesis (3). A proposal has been made that ␥ may be required to avoid the presence of a third Pol III at the replication fork that might outcompete other polymerases, such as Pol IV, that must enter the replication fork to resolve unrepairable lesions and to perform stress-induced mutagenesis functions (2, 3).An E. coli rolling circle replication system has been developed that exploits a 409-nt flapped circular template that has a 50:1 asymmetric GC content, allowing convenient distinction, quantification, and labeling of leading and lagging strands. The asymmetric GC distribution allows specific slowing of lagging strand synthesis by substitution of dGDPNP for dGTP. The V max for insertion of this analog is lower than the natural nucleotide, and the K m is higher, allowing "dialing in" a desired elongation rate without reducing the nucleotide to a level where its concentration is depleted during the progress of the reaction. Because the lagging strand half of the Pol III HE cycles when a new primer is made, even when the pre...

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Section: Discussionmentioning

confidence: 93%

mentioning

confidence: 99%

DNA Polymerase III, but Not Polymerase IV, Must Be Bound to a τ-Containing DnaX Complex to Enable Exchange into Replication Forks

Yuan¹,

Dohrmann²,

Sutton³

et al. 2016

Journal of Biological Chemistry

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“…A requirement for Pol III pausing to allow a switch to Pol IV has been shown previously [76–78]. A simple example of this idea is illustrated in Fig 7 for the formation of SNAs.…”

Section: Discussionmentioning

confidence: 62%

Persistent damaged bases in DNA allow mutagenic break repair in Escherichia coli

et al. 2017

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Bacteria, yeast and human cancer cells possess mechanisms of mutagenesis upregulated by stress responses. Stress-inducible mutagenesis potentially accelerates adaptation, and may provide important models for mutagenesis that drives cancers, host pathogen interactions, antibiotic resistance and possibly much of evolution generally. In Escherichia coli repair of double-strand breaks (DSBs) becomes mutagenic, using low-fidelity DNA polymerases under the control of the SOS DNA-damage response and RpoS general stress response, which upregulate and allow the action of error-prone DNA polymerases IV (DinB), II and V to make mutations during repair. Pol IV is implied to compete with and replace high-fidelity DNA polymerases at the DSB-repair replisome, causing mutagenesis. We report that up-regulated Pol IV is not sufficient for mutagenic break repair (MBR); damaged bases in the DNA are also required, and that in starvation-stressed cells, these are caused by reactive-oxygen species (ROS). First, MBR is reduced by either ROS-scavenging agents or constitutive activation of oxidative-damage responses, both of which reduce cellular ROS levels. The ROS promote MBR other than by causing DSBs, saturating mismatch repair, oxidizing proteins, or inducing the SOS response or the general stress response. We find that ROS drive MBR through oxidized guanines (8-oxo-dG) in DNA, in that overproduction of a glycosylase that removes 8-oxo-dG from DNA prevents MBR. Further, other damaged DNA bases can substitute for 8-oxo-dG because ROS-scavenged cells resume MBR if either DNA pyrimidine dimers or alkylated bases are induced. We hypothesize that damaged bases in DNA pause the replisome and allow the critical switch from high fidelity to error-prone DNA polymerases in the DSB-repair replisome, thus allowing MBR. The data imply that in addition to the indirect stress-response controlled switch to MBR, a direct cis-acting switch to MBR occurs independently of DNA breakage, caused by ROS oxidation of DNA potentially regulated by ROS regulators.

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“…In addition, most organisms possess specialized Pols capable of replicating imperfect templates. There is increasing evidence that these Pols also associate with the replication machinery to provide it with the capacity to cope with damaged DNA (14). Taken together, these findings demonstrate that the composition of the replication fork is more dynamic than previously thought.…”

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confidence: 70%

How MutS finds a needle in a haystack

Sutton

2015

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

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A Genetic Selection for dinB Mutants Reveals an Interaction between DNA Polymerase IV and the Replicative Polymerase That Is Required for Translesion Synthesis

Cited by 29 publications

References 83 publications

DNA Polymerase III, but Not Polymerase IV, Must Be Bound to a τ-Containing DnaX Complex to Enable Exchange into Replication Forks

DNA Polymerase III, but Not Polymerase IV, Must Be Bound to a τ-Containing DnaX Complex to Enable Exchange into Replication Forks

Persistent damaged bases in DNA allow mutagenic break repair in Escherichia coli

How MutS finds a needle in a haystack

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