Abstract
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.
Highlights
Psoralens produce DNA interstrand cross-links.Cross-links can be toxic and which are thought to be repaired via a sequential ex- mutagenic in a wide variety of organisms, but they can cision and recombination mechanism in Escherichia be repaired
As in the structure formed by UvrABC incision, the furan side of the cross-link was adducted to the oligonucleotide. (For simplicity, the psoralen derivative used in thiswork, HMT, will on occasion be referred to as psoralen.) While an single-stranded DNA (ssDNA) circle per se is not an intermediate in the repair of cross-linked chromosomal DNA, the cross-linked ssDNA substrate serves as a reasonable approximation
With the cross-linked ssDNA substrate,strand exchange could be initiated in an undamaged region of the DNA and just the strand transfer reaction past the crosslinked oligonucleotide could be analyzed
Summary
MM) was dissolved in 98% formamide plus 10 mM EDTA and incubated at 55 "C. 32P-LabeledM13mpl9FSl phage DNA or the crosslinked ssDNA substrate of Fig. 1,path A, was added to 0.18 mM and. Reactions with the cross-linked dsDNA substrate were as above except that thevolume was pl, and they contained3.8-8 pM crosslinked dsDNA substrate, either unmodified or after pol I treatment, 2-8 p~ labeled linear duplex, 2.6-5.5 p M RecA protein, and 0-0.18 p~ SSB asindicated. Reactions were stopped by incubating at 37 'C (10-30 min) in the presence of EDTA (20-25 mM), sodium dodecyl sulfate (0.2-1%), and proteinase K The reaction was stopped by adding EDTA to 15 mM, sodium dodecyl sulfate to 0.1%, proteinase K to 250 pg/ml, and glycogen to 20 pg/ml and incubating at 37 "C for 10 min. The reactions were stopped by adding EDTA to 20 mM, sodium dodecyl sulfate to 0.2%, and proteinase K to 180 pg/ml and incubating a t 37 "C for 15-30 min. Control reactions with 3H-labeled plasmid DNA containing approximately 1nick per plasmid molecule (made by DNase I treatment and verified by sedimentation through an alkaline sucrose gradient) showed thatthe reactions described above were with saturating amounts of pol I (22.5 units/21 p~ DNA) and created gaps of 290 f 30 or 1200 f 200 bases after 10 or 30 min a t 37 "C, respectively
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