Interstrand cross-links induce DNA synthesis in damaged and undamaged plasmids in mammalian cell extracts.
Mammalian cell extracts have been shown to carry out damage-specific DNA repair synthesis induced by a variety of lesions, including those created by UV and cisplatin. Here, we show that a single psoralen interstrand cross-link induces DNA synthesis in both the damaged plasmid and a second homologous unmodified plasmid coincubated in the extract. The presence of the second plasmid strongly stimulates repair synthesis in the cross-linked plasmid. Heterologous DNAs also stimulate repair synthesis to variable extents. Psoralen monoadducts and double-strand breaks do not induce repair synthesis in the unmodified plasmid, indicating that such incorporation is specific to interstrand cross-links. This induced repair synthesis is consistent with previous evidence indicating a recombinational mode of repair for interstrand cross-links. DNA synthesis is compromised in extracts from mutants (deficient in ERCC1, XPF, XRCC2, and XRCC3) which are all sensitive to DNA cross-linking agents but is normal in extracts from mutants (XP-A, XP-C, and XP-G) which are much less sensitive. Extracts from Fanconi anemia cells exhibit an intermediate to wild-type level of activity dependent upon the complementation group. The DNA synthesis deficit in ERCC1- and XPF-deficient extracts is restored by addition of purified ERCC1-XPF heterodimer. This system provides a biochemical assay for investigating mechanisms of interstrand cross-link repair and should also facilitate the identification and functional characterization of cellular proteins involved in repair of these lesions.
Departments of Experimental Radiation Oncology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
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D000818:Animals; D002460:Cell Line; D002474:Cell-Free System; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004249:DNA Damage; D004260:DNA Repair; D004274:DNA, Recombinant / Q000096:biosynthesis* / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D004720:Endonucleases; D005199:Fanconi Anemia / Q000235:genetics / Q000473:pathology; D005363:Ficusin / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D008322:Mammals / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D010957:Plasmids / Q000187:drug effects / Q000235:genetics*; D011506:Proteins / Q000235:genetics / Q000502:physiology*; D051135:Rad51 Recombinase; D011995:Recombination, Genetic; D014020:Tissue Extracts; D014983:Xeroderma Pigmentosum / Q000235:genetics / Q000473:pathology
D003432:Cross-Linking Reagents; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D011506:Proteins; D014020:Tissue Extracts; C095888:xeroderma pigmentosum group F protein; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; C495154:Rad51 protein, mouse; C071447:ERCC1 protein, human; D004720:Endonucleases; C499360:Ercc1 protein, mouse; D005363:Ficusin