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. 2012:2012:271453.
doi: 10.1155/2012/271453. Epub 2012 Sep 27.

UvrD Participation in Nucleotide Excision Repair Is Required for the Recovery of DNA Synthesis following UV-Induced Damage in Escherichia coli

Kelley N Newton  1 , Charmain T Courcelle, Justin Courcelle
Affiliations

UvrD Participation in Nucleotide Excision Repair Is Required for the Recovery of DNA Synthesis following UV-Induced Damage in Escherichia coli

Kelley N Newton et al. J Nucleic Acids. 2012.

Abstract

UvrD is a DNA helicase that participates in nucleotide excision repair and several replication-associated processes, including methyl-directed mismatch repair and recombination. UvrD is capable of displacing oligonucleotides from synthetic forked DNA structures in vitro and is essential for viability in the absence of Rep, a helicase associated with processing replication forks. These observations have led others to propose that UvrD may promote fork regression and facilitate resetting of the replication fork following arrest. However, the molecular activity of UvrD at replication forks in vivo has not been directly examined. In this study, we characterized the role UvrD has in processing and restoring replication forks following arrest by UV-induced DNA damage. We show that UvrD is required for DNA synthesis to recover. However, in the absence of UvrD, the displacement and partial degradation of the nascent DNA at the arrested fork occur normally. In addition, damage-induced replication intermediates persist and accumulate in uvrD mutants in a manner that is similar to that observed in other nucleotide excision repair mutants. These data indicate that, following arrest by DNA damage, UvrD is not required to catalyze fork regression in vivo and suggest that the failure of uvrD mutants to restore DNA synthesis following UV-induced arrest relates to its role in nucleotide excision repair.

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Figures

Figure 1
Figure 1
Cells lacking UvrD are hypersensitive to irradiation with UV light. Survival of wild-type (■), recF (◆), uvrA (▲), uvrD::kan (⚫), and uvrD::tet (◯) cultures following irradiation with the indicated UV doses. Error bars represent the standard error of the mean.
Figure 2
Figure 2
UvrD is required for the recovery of replication following UV irradiation, but not for replication in the absence of damage. Cells grown in the presence of [14C]-thymine were pulse-labeled for 2 min with [3H]-thymidine at the times indicated following either UV irradiation with 27 J/m2 (open symbols) or mock irradiation (closed symbols). Total DNA accumulation (14C incorporation, circles) and rate of synthesis (3H incorporation/ 2 min, squares) are plotted. Graphs represent the average of at least three independent experiments. Error bars represent the standard error of the mean. The level of [3H] and [14C] in preirradiated DNA ranged between 30,000–50,000 cpm and 3000–6000 cpm for all experiments.
Figure 3
Figure 3
In the absence of UvrD, the nascent DNA at stalled replication forks is degraded in a manner similar to other repair mutants. [14C]-thymine-labeled cultures were pulse-labeled with [3H]-thymidine for 5 s before the cells were collected, resuspended in nonradioactive media, and UV-irradiated with 27 J/m2. The fraction of 14C-labeled genomic DNA (□) and 3H-labeled nascent DNA (■) remaining over time is plotted. Graphs represent the average of three independent experiments. The level of [3H] and [14C] in DNA immediately preceding irradiation ranged between 2500–7000 cpm and 1000–2500 cpm in all experiments. Error bars represent the standard error of the mean.
Figure 4
Figure 4
In the absence of UvrD, blocked replication forks persist leading to the accumulation of higher-order recombination intermediates in a manner similar to uvrA mutants. (a) Diagram of structural intermediates observed in the presence or absence of UV-induced damage. (b) Cells containing the pBR322 plasmid were UV-irradiated with 50 J/m2. At the times indicated, genomic DNA was purified, digested with PvuII, and the structural intermediates were examined by two-dimensional agarose gel analysis. Gels shown are representative of at least two independent experiments. (c) The percentage of UV-induced intermediates relative to nonreplicating plasmids over time is plotted. Percentages were quantified as the ratio of radioactivity in either the cone region or the high-order intermediate region over the amount of radioactivity in the nonreplicating region.
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References

    1. Chan GL, Doetsch PW, Haseltine WA. Cyclobutane pyrimidine dimers and (6–4) photoproducts Block polymerization by DNA polymerase I. Biochemistry. 1985;24(21):5723–5728. - PubMed
    1. Lippke JA, Gordon LK, Brash DE, Haseltine WA. Distribution of UV light-induced damage in a defined sequence of human DNA: detection of alkaline-sensitive lesions at pyrimidine nucleoside-cytidine sequences. Proceedings of the National Academy of Sciences of the United States of America. 1981;78(6):3388–3392. - PMC - PubMed
    1. Chow KH, Courcelle J. RecO acts with RecF and RecR to protect and maintain replication forks blocked by UV-induced DNA damage in Escherichia coli . Journal of Biological Chemistry. 2004;279(5):3492–3496. - PubMed
    1. Courcelle J, Hanawalt PC. RecQ and RecJ process blocked replication forks prior to the resumption of replication in UV-irradiated Escherichia coli . Molecular and General Genetics. 1999;262(3):543–551. - PubMed
    1. Courcelle J, Carswell-Crumpton C, Hanawalt PC. recF and recR are required for the resumption of replication at DNA replication forks in Escherichia coli . Proceedings of the National Academy of Sciences of the United States of America. 1997;94(8):3714–3719. - PMC - PubMed

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