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

Moore, Jessica M.; Correa, Raúl; Rosenberg, Susan M.; Hastings, P.J.

doi:10.1371/journal.pgen.1006733

Cited by 27 publications

(37 citation statements)

References 112 publications

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“…The high degree of colocalisation we observed between pol IV and the RecA* probe when treating with ciprofloxacin, together with the binding of pol IV to RecA*-like structures in vitro and in vivo , adds to a growing body of evidence supporting the participation of pol IV in homologous recombination (Ponder et al ., 2005; Lovett, 2006; Williams et al ., 2010; Shee et al ., 2011; Rosenberg et al ., 2012; Shee et al ., 2012b; Shee et al ., 2012a; Pomerantz et al ., 2013a; Pomerantz et al ., 2013b; Moore et al ., 2017). In ciprofloxacin-treated cells, pol IV colocalises with the RecA* probe (this study) far more frequently than it colocalises with the replisome marker τ (Henrikus et al ., 2018b).…”

Section: Discussionmentioning

confidence: 99%

“…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%

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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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Section: Discussionmentioning

confidence: 99%

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

View full text Add to dashboard Cite

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“…These observations suggest that pol IV promotes the formation of DBSs due to the BER-mediated removal of closely spaced 8-oxo-dGTPs incorporated by pol IV (16). Other studies indicate that pol IV has a role in the repair of DSBs (20,21,30,31,22–29): First, pol IV physically interacts with the RecA recombinase and RecA nucleoprotein filaments (RecA*); a key player in DSB repair (DSBR) (26, 32). This interaction might facilitate pol IV to function in strand exchange (33).…”

Section: Mainmentioning

confidence: 99%

“…Similarly, in cells treated with ciprofloxacin, pol IV highly colocalizes with RecA* structures (32). Third, genetic studies reveal that the gene encoding pol IV, dinB , is required for both induced and spontaneous error-prone DSBR (20–25). Fourth, intermediates of DSBR known as recombination D-loops are efficiently utilized as substrates by pol IV in vitro (28, 34).…”

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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“…However, even in fully repair-proficient cell strains the accuracy of the DNA repair system is fundamentally limited by the stochastic nature of the molecular interactions involved (1, 2): For example, proteins that signal or repair DNA damage perform a random target search and therefore have a finite chance of overlooking lesions (3–7). The repair process itself can also be error-prone and cause mutations, loss, or rearrangements of genetic material (8–12). Traditionally, research has focused on genetic defects and such “intrinsic errors” in DNA repair – i.e.…”

Section: Introductionmentioning

confidence: 99%

Gene expression noise randomizes the adaptive response to DNA alkylation damage in E. coli

Uphoff

2019

Preprint

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5 6 DNA damage caused by alkylating chemicals induces an adaptive response in Escherichia coli 7 cells that increases their tolerance to further damage. Signalling of the response occurs through 8 methylation of the Ada protein which acts as a damage sensor and induces its own gene expression 9 through a positive feedback loop. However, random fluctuations in the abundance of Ada 10 jeopardize the reliability of the induction signal. I developed a quantitative model to test how gene 11 expression noise and feedback amplification affect the fidelity of the adaptive response. A 12 remarkably simple model accurately reproduced experimental observations from single-cell 13 measurements of gene expression dynamics in a microfluidic device. Stochastic simulations 14 showed that delays in the adaptive response are a direct consequence of the very low number of 15 Ada molecules present to signal DNA damage. For cells that have zero copies of Ada, response 16 activation becomes a memoryless process that is dictated by an exponential waiting time 17 distribution between basal Ada expression events. Experiments also confirmed the model 18 prediction that the strength of the adaptive response drops with increasing growth rate of cells. 19 20

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

Cited by 27 publications

References 112 publications

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

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

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

Gene expression noise randomizes the adaptive response to DNA alkylation damage in E. coli

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