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

Mallik, Sarita; Popodi, Ellen; Hanson, Allen R.; Foster, Patricia L.

doi:10.1128/jb.00101-15

Cited by 23 publications

(35 citation statements)

References 80 publications

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“…While over 100 single‐ and double‐resistant mutants were identified with the rpoB library, only 4 unique mutations were identified with the SPM only template primarily located in the PAM‐proximal RRDR (Appendix Fig S3). The resistance could be an outcome of unintended mutations introduced in the template DNA during PCR, or by error‐prone polymerases expressed during the SOS response to DSBs at the target (Mallik et al , ). Regardless, we observed over an order of magnitude more resistant CFUs representing over 25‐fold more unique variants in our error‐prone library compared to the cells transformed with the SPM only template.…”

Section: Resultsmentioning

confidence: 99%

CRISPR /Cas9 recombineering‐mediated deep mutational scanning of essential genes in Escherichia coli

Choudhury

Fenster

Fankhauser

et al. 2020

Molecular Systems Biology

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Deep mutational scanning can provide significant insights into the function of essential genes in bacteria. Here, we developed a high‐throughput method for mutating essential genes of Escherichia coli in their native genetic context. We used Cas9‐mediated recombineering to introduce a library of mutations, created by error‐prone PCR, within a gene fragment on the genome using a single gRNA pre‐validated for high efficiency. Tracking mutation frequency through deep sequencing revealed biases in the position and the number of the introduced mutations. We overcame these biases by increasing the homology arm length and blocking mismatch repair to achieve a mutation efficiency of 85% for non‐essential genes and 55% for essential genes. These experiments also improved our understanding of poorly characterized recombineering process using dsDNA donors with single nucleotide changes. Finally, we applied our technology to target rpoB, the beta subunit of RNA polymerase, to study resistance against rifampicin. In a single experiment, we validate multiple biochemical and clinical observations made in the previous decades and provide insights into resistance compensation with the study of double mutants.

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Section: Resultsmentioning

confidence: 99%

CRISPR /Cas9 recombineering‐mediated deep mutational scanning of essential genes in Escherichia coli

Choudhury

Fenster

Fankhauser

et al. 2020

Molecular Systems Biology

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“…This interaction might facilitate pol IV to function in strand exchange (33). Second, fluorescently labelled pol IV colocalizes with RecA extensively at sites of induced DSBs when expressed from a low-copy plasmid (27). Similarly, in cells treated with ciprofloxacin, pol IV highly colocalizes with RecA* structures (32).…”

Section: Mainmentioning

confidence: 99%

DNA double-strand breaks induced by reactive oxygen species promote DNA polymerase IV activity inEscherichia coli

Henrikus

Henry

McDonald

et al. 2019

Preprint

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Under many conditions the killing of bacterial cells by antibiotics is potentiated by DNA damage induced by reactive oxygen species (ROS)1–3. A primary cause of ROS-induced cell death is the accumulation of DNA double-strand breaks (DSBs)1,4–6. DNA polymerase IV (pol IV), an error-prone DNA polymerase produced at elevated levels in cells experiencing DNA damage, has been implicated both in ROS-dependent killing and in DSBR7–15. Here, we show using single-molecule fluorescence microscopy that ROS-induced DSBs promote pol IV activity in two ways. First, exposure to the antibiotics ciprofloxacin and trimethoprim triggers an SOS-mediated increase in intracellular pol IV concentrations that is strongly dependent on both ROS and DSBR. Second, in cells that constitutively express pol IV, treatment with an ROS scavenger dramatically reduces the number of pol IV foci formed upon exposure to antibiotics, indicating a role for pol IV in the repair of ROS-induced DSBs.

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“…A series of live-cell studies indicate that pol IV operates in the repair of double-strand breaks (DSBs) (Ponder et al ., 2005; Foster, 2007; Shee et al ., 2011; Rosenberg et al ., 2012; Shee et al ., 2012b; Mallik et al ., 2015; Moore et al ., 2017). Reducing DSB formation (by mitigating the destructive effects of reactive oxygen species) or introducing defects in the end-resection of double-strand breaks (Δ recB mutation) greatly reduces the number of pol IV foci formed in cells treated with ciprofloxacin or trimethoprim (Henrikus et al ., 2019b).…”

Section: Introductionmentioning

confidence: 99%

“…This interaction is proposed to provide pol IV with the ability to participate in DNA synthesis during RecA-dependent strand exchange reactions (Tashjian et al ., 2019). In a fluorescence microscopy study (Mallik et al ., 2015), pol IV was shown to colocalise with RecA structures in vivo . However, the RecA-GFP probe that was used to observe RecA localisation does not differentiate between active forms of RecA (i.e.…”

Section: Introductionmentioning

confidence: 99%

Modulation of DNA polymerase IV activity by UmuD and RecA* observed by single-molecule time-lapse microscopy

McGrath

Jergic

et al. 2019

Preprint

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21DNA polymerase IV (pol IV) is expressed at increased levels in Escherichia coli cells 22 suffering high levels of DNA damage. In a recent single-molecule imaging study, we 23 demonstrated that elevating the pol IV concentration is not sufficient to provide access to binding 24 sites on the nucleoid, suggesting that other factors may recruit pol IV to its substrates once the 25 DNA becomes damaged. Here we extend this work, investigating the proteins UmuD and RecA 26 as potential modulators of pol IV activity. UmuD promotes long-lived association of pol IV with 27 the nucleoid, whereas its cleaved form, UmuDʹ, which accumulates in DNA-damaged cells, 28 109 UmuD in cells treated with the DNA damaging antibiotic ciprofloxacin. In contrast, UmuD′ 110 diminishes pol IV binding. We observed that a large proportion of pol IV foci (up to 40%) 111 colocalise with a RecA* marker in ciprofloxacin-treated cells. The recA(E38K) mutation (also 112 6 known as recA730), which constitutively produces RecA*-like activity (Cazaux et al., 1993; 113 Wang et al., 1993; Ennis et al., 1995), promotes the binding activity of pol IV to the nucleoid, 114 even in the absence of DNA damage. We further showed that pol IV physically interacts with 115 RecA(E38K), which forms RecA*-like structures on single-stranded as well as double-stranded 116 DNA, suggesting that pol IV might also associate with these RecA*-like structures in cells. 117These findings provide evidence for regulatory roles for both UmuD and RecA in modulating the 118 binding activity of pol IV in E. coli cells. RecA* structures that likely mark sites of on-going 119 DSB repair appear to serve as major binding sites for pol IV in live cells treated with 120 ciprofloxacin. 121 Results 122Deletion of umuDC increases pol IV- colocalisation 123 In a previous study, we carried out time-lapse measurements on E. coli cells treated with 124 DNA damaging agents (Henrikus et al., 2018b). We found that the colocalisation of pol IV foci 125 with replisome markers started at ~10% prior to treatment, and dropped to < 5% (i.e. baseline 126 levels) at a time-point 90-100 after the onset of treatment. In a separate study, we observed that 127 pol V (UmuC-mKate2) enters the cytosol and forms foci on the nucleoid at this same 90 min 128 time-point (Robinson et al., 2015). This spatial re-distribution of the UmuC-mKate2 marker 129 required cleavage of UmuD to UmuD′. The similar timing of the changes in pol IV and pol V 130 localisation, together with established links between pol IV activity and UmuD/UmuD′ status 131 described above, led us to hypothesise that UmuD cleavage and/or formation of pol V at the 90 132 min time-point alters the colocalisation of pol IV with replisome markers.133To investigate the effect of pol V and/or its precursors (UmuD and UmuC) on the extent 134 of pol IV focus formation and colocalisation with a replisome marker, we constructed two 135 7 strains: i) dinB-YPet dnaX-mKate2 umuDC + (EAW643, (Henrikus et al., 2018b)) and ii) dinB-136 YPet dnaX-mKate2...

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

Cited by 23 publications

References 80 publications

CRISPR /Cas9 recombineering‐mediated deep mutational scanning of essential genes in Escherichia coli

CRISPR /Cas9 recombineering‐mediated deep mutational scanning of essential genes in Escherichia coli

DNA double-strand breaks induced by reactive oxygen species promote DNA polymerase IV activity inEscherichia coli

Modulation of DNA polymerase IV activity by UmuD and RecA* observed by single-molecule time-lapse microscopy

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