In vitro repair of psoralen-DNA cross-links by RecA, UvrABC, and the 5'-exonuclease of DNA polymerase I.
Psoralens produce DNA interstrand cross-links which are thought to be repaired via a sequential excision and recombination mechanism in Escherichia coli. The first round of incision by UvrABC has been characterized: it results in 11-base oligonucleotide cross-linked to an intact DNA strand (Van Houten, B., Gamper, B., Holbrook, S.R., Hearst, J.E., and Sancar, A. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8077-8081). In the present work, DNA substrates containing 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) cross-links in defined positions are constructed and used to analyze the other steps in repair. It is shown that RecA protein mediates strand transfer past an oligonucleotide cross-linked to a single-stranded DNA circle and that the resulting heteroduplex is a substrate for the UvrABC complex: it excises a double-stranded oligonucleotide which contains the HMT cross-link. It is also found that the first round of UvrABC incision does not lead directly to strand exchange but that an intervening step is needed. That step is carried out in vitro by the 5'-exonuclease activity of DNA polymerase I (pol I) which creates a single-stranded DNA region (a gap) at an incised cross-link such that RecA can initiate strand exchange. Studies using cross-linked oligonucleotides showed that the gap produced by pol I results from the inability of the polymerase to add nucleotides to a 3'-OH end two to three nucleotides away from the furan side of an HMT cross-link. Pol I can, however, extend a 3'-OH end next to the pyrone side of the cross-link. Since UvrABC incises predominantly the furan side of psoralen cross-links in duplex DNA, this discrepancy has important consequences for repair.
Sladek
F M
FM
Department of Molecular Biophysics, Yale University, New Haven, Connecticut 06511.
Munn
M M
MM
Rupp
W D
WD
Howard-Flanders
P
P
D003432:Cross-Linking Reagents; D004247:DNA / Q000187:drug effects*; D004249:DNA Damage; D004256:DNA Polymerase I / Q000502:physiology*; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000502:physiology*; D029968:Escherichia coli Proteins; D005090:Exodeoxyribonucleases / Q000378:metabolism; D011564:Furocoumarins / Q000494:pharmacology*; D011938:Rec A Recombinases / Q000502:physiology*; D011995:Recombination, Genetic
D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D011564:Furocoumarins; D004247:DNA; D011938:Rec A Recombinases; D004256:DNA Polymerase I; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C036385:endodeoxyribonuclease uvrABC