DNA polymerase IV mediates efficient and quick recovery of replication forks stalled at N<sup>2</sup>-dG adducts

Ikeda, Mio; Furukohri, Asako; Philippin, Gaëlle; Loechler, Edward L.; Akiyama, Masahiro; Katayama, Tsutomu; Fuchs, Robert P. P.; Maki, Hisaji

doi:10.1093/nar/gku547

Cited by 35 publications

(26 citation statements)

References 48 publications

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“…Weakening the gate still dramatically increased Pol V-mediated TLS in cells ( Figure 5C), suggesting that the interaction between Pol III and the β2 clamp likely influences polymerase switching independent of whether TLS occurs at the fork or in a gap ( Figure 6B). Biochemical studies presented here and in prior work, demonstrate that Pol IV can efficiently carry out TLS at the fork for at least a subset of lesions (Gabbai et al, 2014;Ikeda et al, 2014). Cellular evidence for Pol IV-mediated TLS at the fork remains limited but the role of dinB in MMS-induced mutagenesis provides circumstantial evidence.…”

Section: Repriming Is a Failsafe Mechanism For Rescuing Stalled Replisupporting

confidence: 56%

A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes

Chang

Naiman

Thrall

et al. 2019

Preprint

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DNA lesions stall the replisome and proper resolution of these obstructions is critical for genome stability.Replisomes can directly replicate past a lesion by error-prone translesion synthesis. Alternatively, replisomes can reprime DNA synthesis downstream of the lesion, creating a single-stranded DNA gap that is repaired primarily in an error-free, homology-directed manner. Here we demonstrate how structural changes within the bacterial replisome determine the resolution pathway of lesion-stalled replisomes. This pathway selection is controlled by a dynamic interaction between the proofreading subunit of the replicative polymerase and the processivity clamp, which sets a kinetic barrier to restrict access of TLS polymerases to the primer/template junction. Failure of TLS polymerases to overcome this barrier leads to repriming, which competes kinetically with TLS. Our results demonstrate that independent of its exonuclease activity, and the concomitant increase in TLS polymerase levels, higher fractions of stalled replisomes are resolved through TLS (Fuchs, 2016;.

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Section: Repriming Is a Failsafe Mechanism For Rescuing Stalled Replisupporting

confidence: 56%

A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes

Chang

Naiman

Thrall

et al. 2019

Preprint

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“…This appeared to be inconsistent with the 4.9-h mean lifetime (k off ϭ 5.7 ϫ 10 Ϫ5 ) observed for ␣-DnaX dissociation (10) and is the primary subject of this investigation. In addition to a putative exchange of Pol III into the replication fork, exchange of other polymerases, particularly Pol IV, has been well documented (11)(12)(13)(14)(15)(16). Two different mechanisms have been proposed for Pol IV exchange.…”

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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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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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“…Pol IV is relatively abundant (250 molecules per cell) even in non-SOS-induced cells (9). These high levels suggest that Pol IV may gain access to replicating DNA, and indeed, Pol IV can displace the replicase DNA polymerase III from the sliding clamp in vitro (10,11). Pol IV can also elongate from misaligned primer/template termini (12).…”

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confidence: 99%

Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

et al. 2015

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Escherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure of E. coli to DNAdamaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ϳ60% of the foci consisted of three Pol IV molecules, while ϳ40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that the in vitro interaction between Rep and Pol IV reported previously also occurs in vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ϳ70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecA in vivo and is recruited to sites of DSBs to aid in the restoration of DNA replication. IMPORTANCEDNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstrate in vivo localization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings provide in vivo evidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.

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DNA polymerase IV mediates efficient and quick recovery of replication forks stalled at N²-dG adducts

Cited by 35 publications

References 48 publications

A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes

A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes

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

Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

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DNA polymerase IV mediates efficient and quick recovery of replication forks stalled at N2-dG adducts

Cited by 35 publications

References 48 publications

A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes

A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes

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

Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

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DNA polymerase IV mediates efficient and quick recovery of replication forks stalled at N²-dG adducts