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. 2001 Feb;21(3):713-20.
doi: 10.1128/MCB.21.3.713-720.2001.

Involvement of nucleotide excision repair in a recombination-independent and error-prone pathway of DNA interstrand cross-link repair

X Wang  1 , C A Peterson, H Zheng, R S Nairn, R J Legerski, L Li
Affiliations

Involvement of nucleotide excision repair in a recombination-independent and error-prone pathway of DNA interstrand cross-link repair

X Wang et al. Mol Cell Biol. 2001 Feb.

Abstract

DNA interstrand cross-links (ICLs) block the strand separation necessary for essential DNA functions such as transcription and replication and, hence, represent an important class of DNA lesion. Since both strands of the double helix are affected in cross-linked DNA, it is likely that conservative recombination using undamaged homologous regions as a donor may be required to repair ICLs in an error-free manner. However, in Escherichia coli and yeast, recombination-independent mechanisms of ICL repair have been identified in addition to recombinational repair pathways. To study the repair mechanisms of interstrand cross-links in mammalian cells, we developed an in vivo reactivation assay to examine the removal of interstrand cross-links in cultured cells. A site-specific psoralen cross-link was placed between the promoter and the coding region to inactivate the expression of green fluorescent protein or luciferase genes from reporter plasmids. By monitoring the reactivation of the reporter gene, we showed that a single defined psoralen cross-link was removed in repair-proficient cells in the absence of undamaged homologous sequences, suggesting the existence of an ICL repair pathway that is independent of homologous recombination. Mutant cell lines deficient in the nucleotide excision repair pathway were examined and found to be highly defective in the recombination-independent repair of ICLs, while mutants deficient in homologous recombination were found to be proficient. Mutation analysis of plasmids recovered from transfected cells showed frequent base substitutions at or near positions opposing a cross-linked thymidine residue. Based on these results, we suggest a distinct pathway for DNA interstrand cross-link repair involving nucleotide excision repair and a putative lesion bypass mechanism.

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Figures

FIG. 1
FIG. 1
Recombination-independent removal of a single defined psoralen ICL in repair-proficient HT-1080 fibroblast cells. (A) Preparation of cross-linked reporter substrates as examined by denaturing PAGE. The left panel shows a purified cross-linked oligo (lane 2) and a non-cross-linked control (lane 3). Lane 1 contains a 40-mer oligo marker. The right panel shows the purity of the cross-linked plasmid substrate. Lane 1, pEGFP-N1 plasmid with an unmodified oligo inserted at the HindIII site; lane 2, pEGFP-N1 with a cross-linked oligo inserted at the HindIII site. Both were digested with BamHI and NheI to release a 54-bp fragment followed by end labeling with T4 kinase and resolution by 15% denaturing PAGE. (B) Parallel plated HT-1080 cells were transfected with equal amounts of unmodified pEGFP-N1 plasmid (HT-1080/EGFP) or cross-linked pEGFP-N1 plasmid (HT-1080/CLT). Images in the left panels show representative fluorescent views, and images in the right panels show the bright-field view. (C) Repair of a single psoralen ICL in a luciferase reporter plasmid in the repair-proficient cell lines HT-1080, RKO, AA8, and V79. The relative efficiencies of ICL repair were calculated as the percentage of luciferase activity from cross-linked reporter to that of the unmodified reporter.
FIG. 2
FIG. 2
Involvement of XPA and ERCC1 in recombination-independent repair of ICLs. The repair of cross-linked luciferase reporter is deficient in XPA and ERCC1 mutants but can be restored to normal levels by the reintroduction of wild-type XPA and ERCC1 genes, respectively. C-XPA, XPA mutant complemented with a XPA cDNA expression vector; E1KO7-5, ERCC1-null mutant derived from CHO ATS-49; E1KO-47 (C-ERCC1), E1KO7-5 mutant transformed by stable integration of a wild-type ERCC1 cDNA expression vector.
FIG. 3
FIG. 3
NER mutants are defective in repair reactivation of cross-linked luciferase substrate. (A) Repair efficiency of hamster NER mutants compared to parental AA8 cells. (B) Repair efficiency of XP mutants compared to repair-proficient HT-1080 cells.
FIG. 4
FIG. 4
Recombination-independent repair of ICLs does not require the function of the XRCC2 and XRCC3 genes. Reactivation of the cross-linked luciferase reporter is normal in irs1 and irs1SF mutant cells compared to parental V79 and AA8 cells, respectively.
FIG. 5
FIG. 5
Mutations derived from recombination-independent repair of psoralen ICLs. The rectangle indicates the position of the BsrGI restriction site, and the cross-linked oligonucleotide sequence is underlined. (A) Sequence of the unmodified oligo inserted in the pEGFP-N1 vector. (B) A-to-G transversion found at the site opposite the psoralen-adducted thymidine residue in the nontranscribed strand (triangle). (C) T-to-G transversion found at the site opposite the psoralen-adducted thymidine in the transcribed strand (large triangle), and deletion of a T residue (small triangle). Note that the cross-linked oligo (underlined) was inserted in the opposite polarity. (D) C-to-A transversion found immediately downstream of the A residue opposite the psoralen-adducted thymidine residue (triangle).
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