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. 2012;7(12):e52472.
doi: 10.1371/journal.pone.0052472. Epub 2012 Dec 18.

Analysis of CPD Ultraviolet Lesion Bypass in Chicken DT40 Cells: Polymerase η and PCNA Ubiquitylation Play Identical Roles

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Free PMC article

Analysis of CPD Ultraviolet Lesion Bypass in Chicken DT40 Cells: Polymerase η and PCNA Ubiquitylation Play Identical Roles

Agnes Varga et al. PLoS One. .
Free PMC article

Abstract

Translesion synthesis (TLS) provides a mechanism of copying damaged templates during DNA replication. This potentially mutagenic process may operate either at the replication fork or at post-replicative gaps. We used the example of T-T cyclobutane pyrimidine dimer (CPD) bypass to determine the influence of polymerase recruitment via PCNA ubiquitylation versus the REV1 protein on the efficiency and mutagenic outcome of TLS. Using mutant chicken DT40 cell lines we show that, on this numerically most important UV lesion, defects in polymerase η or in PCNA ubiquitylation similarly result in the long-term failure of lesion bypass with persistent strand gaps opposite the lesion, and the elevation of mutations amongst successful TLS events. Our data suggest that PCNA ubiquitylation promotes CPD bypass mainly by recruiting polymerase η, resulting in the majority of CPD lesions bypassed in an error-free manner. In contrast, we find that polymerase ζ is responsible for the majority of CPD-dependent mutations, but has no essential function in the completion of bypass. These findings point to a hierarchy of access of the different TLS polymerases to the lesion, suggesting a temporal order of their recruitment. The similarity of REV1 and REV3 mutant phenotypes confirms that the involvement of polymerase ζ in TLS is largely determined by its recruitment to DNA by REV1. Our data demonstrate the influence of the TLS polymerase recruitment mechanism on the success and accuracy of bypass.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Monitoring lesion bypass in a shuttle plasmid.
(A) A schematic representation of the amplified region of the pQ-CPDs shuttle plasmid. The cloning sites for the lesion-containing oligonucleotide, the lesions on each strand, and the approximate positions of the PCR primers are shown, not to scale. Some of the twelve Dpn I sites are omitted for clarity. (B) Representative sequences obtained from xpa cells, grouped into categories by bypass type (indicated with schematic drawings on the right). (C) The proportion of replicated plasmids recovered from wild type (WT) or xpa mutant cells showing evidence of translesion synthesis (dark grey) or the use of the opposite strand as a repair or bypass template (‘error free’, light grey). (D) Replication efficiency of the pQ-CPDs plasmid in the indicated cell lines, as measured by qPCR (see Materials and Methods). The mean and S.E.M. of 3–8 measurements is shown.
Figure 2
Figure 2. Post-replicative ssDNA regions at CPD lesions detected by PCR.
(A) Representative samples of sequence deletions generated by a PCR reaction using the pure pQ-CPDs plasmid as template. (B) A schematic drawing for the generation of deletions at single-stranded gaps. Replication of a lesion-containing strand may produce a gap in the daughter strand (grey). The gapped strand is not suitable as a PCR template, so the PCR reaction initially must proceed across the lesion (dashed line). (C) Dpn I digested untransfected control pQTc and lesion-containing pQ-CPDs plasmid do not serve as PCR templates. The indicated amounts of plasmid were subjected to Dpn I digests (+) or mock digests, amplified by PCR and analysed on an agarose gel. A positive control sample extracted from xpa cells and subjected to the same treatment is shown in the right hand lane. (D) The percentage of deletions amongst the PCR products of pure plasmids containing CPD lesions on both strands (pQ-CPDs) or only one strand (pQ-1CPD). (E) The percentage of deletions amongst PCR products of plasmids recovered from transfected cells of the indicated genotypes. The mean percentage and S.E.M. of 3–7 experiments are shown. (F) A reduction of deletion-bearing PCR products when the plasmid recovered from xpa pcnaK164R cells is pre-treated with the ssDNA specific mung bean nuclease. The mean and S.E.M. of 3 experiments are shown.
Figure 3
Figure 3. The genetic dependence of successful CPD bypass.
The proportion of replicated plasmids recovered from cells of the indicated genotypes showing evidence of translesion synthesis (dark grey) or the use of the opposite strand as an alternative template (‘template switch’, light grey). Sequences with PCR-generated gaps, bearing evidence of incomplete bypass, are omitted. The level of TLS in the control xpa cell line is shown by a dashed line. 95% binomial confidence intervals are shown.
Figure 4
Figure 4. The mutagenic spectrum of CPD bypass.
(A) The arrangement of lesions in the pQ-CPDs and pQ-CPDo plasmids. (B) The percentage of pooled CPDs and CPDo TLS events that are different from an AA insertion, grouped by mutation type: base change at the lesion (white), an extra A opposite the base 5′ to the lesion (AAA, light grey), one-base deletion (dark grey). (C) The frequency of different mutagenic base insertions opposite the two bases of the thymine dimer. The height of the letters indicates the frequency of occurrence of the corresponding DNA base.
Figure 5
Figure 5. Cell cycle analysis after UV irradiation.
DT40 cells of the indicated genotypes were treated with 1 J/m2 UV radiation, and harvested at the indicated times. The cells were treated with a pulse of BrdU before fixation. Their DNA content was analysed using propidium iodide staining (horizontal, linear axis), and their rate of DNA replication using anti-BrdU-FITC antibody staining (vertical, logarithmic axis). The separate panel on the right indicates the rationale for assigning cell populations to different cell cycle phases, including an ‘arrested S’ population with a DNA content between G1 and G2 levels but no active DNA synthesis. The percentage of cells in each category is shown on the individual panels.

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Grant support

This work was supported by St George's, University of London, and by the Hungarian Academy of Sciences (Momentum grant LP2011-015 to D.S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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