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.
Li
L
L
Departments of Experimental Radiation Oncology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Peterson
C A
CA
Lu
X
X
Wei
P
P
Legerski
R J
RJ
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