Repair of DNA containing interstrand crosslinks in Escherichia coli: sequential excision and recombination

Abstract

The repair of DNA containing interstrand crosslinks induced by psoralen-plus-light in E. coli cells has been investigated. During a 30-minute incubation after psoralen-plus-light treatment, crosslinks were excised and the cellular DNA was cut into discrete pieces. The molecular weight of these pieces corresponds to about twice the single-strand distance between crosslinks, as measured by sedimentation velocity in alkaline sucrose. During further incubation, these DNA fragments were covalently joined into high molecular weight DNA. This joining did not occur in cells carrying a mutation at recA; in these strains the DNA was further degraded to smaller polynucleotides and acid-soluble material. The possibility that repair of crosslinked DNA involves strand exchanges between homologous duplexes was investigated. Cells were grown in (13)C,(15)N-containing medium for several generations, then switched to medium of normal density that also contained [(3)H]thymidine for about 0.5 generation. After the crosslinking treatment, the cells were incubated in medium of normal density in order for repair to occur. The DNA was extracted and centrifuged in alkaline CsCl density gradients, where the light and heavy strands were separated. Molecules of intermediate density that contained (3)H accumulated during repair in wild-type cells, but not in control cells or treated recA(-) cells. After molecular weight reduction of the intermediate-density DNA, the (3)H could be separated from the heavy strands, demonstrating that covalent joining between heavy and light strands of homologous duplexes accompanies repair. A mechanism involving sequential excision and genetic recombination is proposed for the repair of DNA containing interstrand crosslinks.