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. 2018 Feb:62:18-27.
doi: 10.1016/j.dnarep.2018.01.004. Epub 2018 Jan 9.

A quantitative PCR-based assay reveals that nucleotide excision repair plays a predominant role in the removal of DNA-protein crosslinks from plasmids transfected into mammalian cells

Lisa N Chesner  1 , Colin Campbell  2
Affiliations

A quantitative PCR-based assay reveals that nucleotide excision repair plays a predominant role in the removal of DNA-protein crosslinks from plasmids transfected into mammalian cells

Lisa N Chesner et al. DNA Repair (Amst). 2018 Feb.

Abstract

DNA-protein crosslinks (DPCs) are complex DNA lesions that induce mutagenesis and cell death. DPCs are created by common antitumor drugs, reactive oxygen species, and endogenous aldehydes. Since these agents create other types of DNA damage in addition to DPCs, identification of the mechanisms of DPC repair is challenging. In this study, we created plasmid substrates containing site-specific DPC lesions, as well as plasmids harboring lesions that are selectively repaired by the base excision or nucleotide excision repair (NER) pathways. These substrates were transfected into mammalian cells and a quantitative real-time PCR assay employed to study their repair. This assay revealed that DPC lesions were rapidly repaired in wild-type human and Chinese hamster derived cells, as were plasmids harboring an oxoguanine residue (base excision repair substrate) or cholesterol lesion (NER substrate). Interestingly, the DPC substrate was repaired in human cells nearly three times as efficiently as in Chinese hamster cells (>75% vs ∼25% repair at 8 h post-transfection), while there was no significant species-specific difference in the efficiency with which the cholesterol lesion was repaired (∼60% repair). Experiments revealed that both human and hamster cells deficient in NER due to mutations in the xeroderma pigmentosum A or D genes were five to ten-fold less able to repair the cholesterol and DPC lesions than were wild-type control clones, and that both the global genome and transcription-coupled sub-pathways of NER were capable of repairing DPCs. In addition, analyses using this PCR-based assay revealed that a 4 kDa peptide DNA crosslink was repaired nearly twice as efficiently as was a ∼38 kDa DPC, suggesting that proteolytic degradation of crosslinked proteins occurs during DPC repair. These results highlight the utility of this PCR-based assay to study DNA repair and indicate that the NER machinery rapidly and efficiently repairs plasmid DPC lesions in mammalian cells.

Keywords: DNA-protein crosslinks; Human oxoguanine glycosylase 1; Nucleotide excision repair; qPCR.

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Conflict of interest statement

Conflict of Interest: The authors have no conflicts of interest to disclose.

Figures

Figure 1
Figure 1. Quantification of damaged plasmid via strand-specific primer extension/qPCR (SSPE-qPCR)
Repair of damaged plasmids is quantified using SSPE-qPCR: (1) Plasmid DNA is denatured, (2) a primer extension reaction is performed using primer R, (3) the denaturation/primer extension step is repeated and additional 7 cycles. After 8 cycles, primer L is added and Ct values determined using qPCR. The presence of an abasic site, cholesterol, or DPC (A) will cause Taq polymerase to stall, resulting in no full-length product strands. In contrast, when repair has occurred (B), Taq polymerase will produce full-length product strands containing the binding site for primer L.
Figure 2
Figure 2. An AP site blocks Taq-mediated primer extension
Taq polymerase extension reactions were performed as described in the text on 40-mer oligonucleotides containing either an 8-oxo dG residue (8oxo) or an AP site (AP), see methods section for details, in the absence (-) or presence (+) of complementary primer Z (15-mer, see methods). The products were resolved by electrophoresis, stained with ethidium bromide and subject to scanning densitometry. The relative amount of full-length product (40 nt) was quantitated using image J and plotted on the y axis (arbitrary units).
Figure 3
Figure 3. 8-oxo-dG repair efficiency is similar in wild-type and NER-deficient Chinese hamster clones
An oligonucleotide containing an 8-oxo-dG modification (O, in red) is annealed to single-stranded M13 and a primer extension reaction performed as described in the Methods. Covalently closed circular double-stranded DNA is gel-purified, and transfected into wild-type V79 (blue) and NER-deficient V-H1 (red) Chinese hamster cells via lipofection. Low molecular weight DNA was recovered 1, 1.5 and 3-hours post-transfection and treated with OGG1 to convert unrepaired 8-oxo-dG residues to abasic sites. DNA repair assays were then performed as described in the legend to Figure 1. Values depict mean percent repair, ± SEM, N=3.
Figure 4
Figure 4. Cholesterol-DNA adducts are repaired with reduced efficiency in NER-deficient cells
Plasmid DNA containing a cholesterol adduct was transfected into Chinese hamster (blue) and human (orange) cells lines proficient (dark bars) or deficient (light bars) in NER via lipofection and low molecular weight DNA recovered after 8 hours. DNA repair assays were performed as described above. Values depict mean percent repair, ± SEM, N=4, *P < 0.05.
Figure 5
Figure 5. Creation of an OGG1-plasmid DNA crosslink substrate
(A) Plasmid DNA containing an 8-oxo-dG residue (O, in red) was reacted with OGG1 in the presence of sodium cyanoborohydride to create a covalent bond between lysine residue 249 and a deoxyribosyl moiety on the plasmid (see Methods for details, chemical structure depicted in inset). (B) This product was cut with restriction enzymes to generate two fragments: a 2800 bp protein-free DNA fragment and a 4400bp fragment crosslinked to OGG1. (C) The restriction digested material was resolved by agarose gel electrophoresis prior to (lane 2) or following (lane 3) precipitation in the presence of K-SDS (see Methods), and stained with ethidium bromide. The arrow illustrates the selective loss of the OGG1-crosslinked DNA fragment following K-SDS precipitation. Lane 1; molecular weight marker in kilobase pairs.
Figure 6
Figure 6. OGG1-DNA crosslink repair in wild-type cells
Left: Plasmid crosslinked to OGG1 was transfected into V79 cells via lipofection, low molecular weight DNA recovered 3, 5, 7, and 8 hours post-transfection, and repair assays performed as described above. The graph depicts mean percent repair, ± SEM, N=3. Right: Plasmid DNA containing an OGG1 crosslink was transfected into: Chinese hamster ovary (CHO-K1, green), Chinese hamster lung fibroblast (V79, blue), and human (XPDcorr, orange and XPAcorr, purple) cells, low molecular weight DNA recovered 8 hours post-transfection and repair assays performed as described above. Values depict mean percent repair, ± SEM, N=4.
Figure 7
Figure 7. OGG1-DNA crosslinks are repaired more efficiently when present on a template strand than when present on a coding strand
Plasmid pLC119/pLC120 DNA substrates containing an OGG1 crosslink present on the template strand (left), or coding strand (right) of a transcriptional unit (construction described in Methods) were transfected into wild-type (V79, dark bars) and NER-deficient (V-H1, light bars) cells, and repair assays performed as described above. The graph depicts mean percent repair, ± SEM, N=3, *P < 0.05.
Figure 8
Figure 8. A DNA-peptide crosslink is repaired with reduced efficiency in NER-deficient Chinese hamster cells
Plasmid DNA containing a DNA-peptide crosslink (prepared as described in the Methods) was transfected into wild-type (V79, dark bars) or NER deficient (V-H1, light bars) Chinese hamster cells via lipofection, low molecular weight DNA recovered at 3 and 8 hours post-transfection, and repair assays performed as described above. The graph depicts mean percent repair, ± SEM, N=4, *P < 0.05.
Figure 9
Figure 9. Cisplatin-induced DNA-protein crosslinks accumulate at elevated levels in NER-deficient Chinese hamster cells
Wild type (V79, dark bar) and NER-deficient (V-H1, light bar) Chinese hamster cells were exposed to 100 μM cisplatin for one hour in serum-free media, and levels of chromosomal DPCs determined. The graph depicts mean percent of protein-crosslinked DNA (calculated as described in the Methods), ± SEM, N=3, *P < 0.05.
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