Resolving Holliday Junctions with Escherichia coli UvrD Helicase

S., Carter, Annamarie; Tahmaseb, Kambiz; Compton, Sarah A.; Matson, Steven W.

doi:10.1074/jbc.m111.314047

Cited by 11 publications

(12 citation statements)

References 50 publications

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“…We deleted or mutated a number of key genes whose products were involved in homologous recombination and the SOS response that we had previously examined for involvement in OFL persistence within stationary-phase WT cultures (ΔrecA, lexA3, ΔrecB, ΔrecF, ΔrecN, and ΔruvA) (15). In addition, to cover a wider breadth of DNA repair pathways, we also examined the effects of deleting uvrD, which encodes a helicase involved in methyl-directed mismatch repair, nucleotide excision repair, and implicated in replication fork restart (33); sulA, which inhibits cell division during the SOS response (34); and mutM, nfo, and ung, which are involved in base excision repair (35).…”

Section: Resultsmentioning

confidence: 99%

Timing of DNA damage responses impacts persistence to fluoroquinolones

Mok

Brynildsen

2018

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

120

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Bacterial persisters are subpopulations of phenotypic variants in isogenic cultures that can survive lethal doses of antibiotics. Their tolerances are often attributed to reduced activities of antibiotic targets, which limit corruption and damage in persisters compared with bacteria that die from treatment. However, that model does not hold for nongrowing populations treated with ofloxacin, a fluoroquinolone, where antibiotic-induced damage is comparable between cells that live and those that die. To understand how those persisters achieve this feat, we employed a genetic system that uses orthogonal control of MazF and MazE, a toxin and its cognate antitoxin, to generate model persisters that are uniformly tolerant to ofloxacin. Despite this complete tolerance, MazF model persisters required the same DNA repair machinery (RecA, RecB, and SOS induction) to survive ofloxacin treatment as their nongrowing, WT counterparts and exhibited similar indicators of DNA damage from treatment. Further investigation revealed that, following treatment, the timing of DNA repair was critical to MazF persister survival because, when repair was delayed until after growth and DNA synthesis resumed, survival was compromised. In addition, we found that, with nongrowing, WT planktonic and biofilm populations, stalling the resumption of growth and DNA synthesis after the conclusion of fluoroquinolone treatment with a prevalent type of stress at infection sites (nutrient limitation) led to near complete survival. These findings illustrate that the timing of events, such as DNA repair, following fluoroquinolone treatment is important to persister survival and provide further evidence that knowledge of the postantibiotic recovery period is critical to understanding persistence phenotypes.

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Section: Resultsmentioning

confidence: 99%

Timing of DNA damage responses impacts persistence to fluoroquinolones

Mok

Brynildsen

2018

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

120

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“…The higher level of activity observed on these DNA molecules relative to linear plasmid may be due to the higher concentration of dsDNA ends or possibly an affinity for junction DNA as suggested previously. 69…”

Section: Resultsmentioning

confidence: 99%

Rep and UvrD Antagonize One Another at Stalled Replication Forks and This Is Exacerbated by SSB

et al. 2019

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The Rep and UvrD DNA helicases are proposed to act at stalled DNA replication forks to facilitate replication restart when RNA polymerase stalls forks. To clarify the role of these DNA helicases in fork rescue, we used a coupled spectrophotometric ATPase assay to determine how they act on model fork substrates. For both enzymes, activity is low on regressed fork structures, suggesting that they act prior to the regression step that generates a Holliday junction. In fact, the preferred cofactors for both enzymes are forks with a gap in the nascent leading strand, consistent with the 3′–5′ direction of translocation. Surprisingly, for Rep, this specificity is altered in the presence of stoichiometric amounts of a single-strand DNA-binding protein (SSB) relative to a fork with a gap in the nascent lagging strand. Even though Rep and UvrD are similar in structure, elevated concentrations of SSB inhibit Rep, but they have little to no effect on UvrD. Furthermore, Rep and UvrD antagonize one another at a fork. This is surprising given that these helicases have been shown to form a heterodimer and are proposed to act together to rescue an RNA polymerase-stalled fork. Consequently, the results herein indicate that although Rep and UvrD can act on similar fork substrates, they cannot function on the same fork simultaneously.

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“…The Holliday junction processing RuvAB [ 32 ], UvrD, and HolA were also downregulated proteins of the 3R genes. UvrD resolves Holliday junctions and is part of the mismatch and nucleotide excision repair pathways [ 33 , 34 ]. HolA catalyzes DNA replication as a component of the Polymerase III holoenzyme [ 35 ].…”

Section: Discussionmentioning

confidence: 99%

Comparative proteomic analysis of Neisseria meningitidis wildtype and dprA null mutant strains links DNA processing to pilus biogenesis

et al. 2017

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Background: DNA processing chain A (DprA) is a DNA binding protein which is ubiquitous in bacteria, and is required for DNA transformation to various extents among bacterial species. However, the interaction of DprA with competence and recombination proteins is poorly understood. Therefore, the proteomes of whole Neisseria meningitidis (Nm) wildtype and dprA mutant cells were compared. Such a comparative proteomic analysis increases our understanding of the interactions of DprA with other Nm components and may elucidate its potential role beyond DNA processing in transformation. Results: Using label-free quantitative proteomics, a total of 1057 unique Nm proteins were identified, out of which 100 were quantified as differentially abundant (P ≤ 0.05 and fold change ≥ |2|) in the dprA null mutant. Proteins involved in homologous recombination (RecA, UvrD and HolA), pilus biogenesis (PilG, PilT1, PilT2, PilM, PilO, PilQ, PilF and PilE), cell division, including core energy metabolism, and response to oxidative stress were downregulated in the Nm dprA null mutant. The mass spectrometry data are available via ProteomeXchange with identifier PXD006121. Immunoblotting and co-immunoprecipitation were employed to validate the association of DprA with PilG. The analysis revealed reduced amounts of PilG in the dprA null mutant and reduced amounts of DprA in the Nm pilG null mutant. Moreover, a number of pilus biogenesis proteins were shown to interact with DprA and /or PilG. Conclusions: DprA interacts with proteins essential for Nm DNA recombination in transformation, pilus biogenesis, and other functions associated with the inner membrane. Inverse downregulation of Nm DprA and PilG expression in the corresponding mutants indicates a link between DNA processing and pilus biogenesis.

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Resolving Holliday Junctions with Escherichia coli UvrD Helicase

Cited by 11 publications

References 50 publications

Timing of DNA damage responses impacts persistence to fluoroquinolones

Timing of DNA damage responses impacts persistence to fluoroquinolones

Rep and UvrD Antagonize One Another at Stalled Replication Forks and This Is Exacerbated by SSB

Comparative proteomic analysis of Neisseria meningitidis wildtype and dprA null mutant strains links DNA processing to pilus biogenesis

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