Preparation of psoralen-cross-linked R-loops and generation of large deletions by their repair in vivo.
A technique for site-selective introduction of psoralens into DNA has been developed. Irradiation of Escherichia coli 16 S rRNA with aminomethyltrimethyl psoralen (AMT) at 390 nm leads to the incorporation of AMT monoadducts in double-stranded regions of the RNA, but no cross-links. The AMT-containing RNA hybridizes efficiently to a supercoiled plasmid, pCS3, a derivative of pBR313 containing an insert of part of the E. coli rrnC cistron including the 3' 60% of the 16 S rDNA. When the resulting hybrids are re-irradiated at 360 nm, psoralen cross-links form between the rRNA and the rDNA, resulting in covalent R-loops. These were partially purified and used to transform E. coli after various nuclease treatments, employing an ampicillin selection to make cell survival dependent on successful repair of the psoralen-containing region. S1 nuclease-treated AMT-containing covalent R-looped plasmids show efficient transformation, and 9 mutants with large deletions were isolated by random screening of 100 colonies. These fell into 3 specific classes, each of which appears to start some place within the AMT-cross-linked rDNA. The structure of one mutant has been analyzed by DNA sequencing which shows a deletion which extends approximately 6800 nucleotides from nucleotide 868 of 16 S rDNA to nucleotide 315 on the tetracycline gene. Some hints of symmetry exist around the point of the deletion, but the pattern is not a striking one. This technique should be useful in making covalent D-loops as well as R-loops. It offers a number of potential applications for site-specific DNA modification and studies of DNA repair.
D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli; D011564:Furocoumarins / Q000494:pharmacology*; D008854:Microscopy, Electron; D009690:Nucleic Acid Conformation / Q000187:drug effects* / Q000528:radiation effects; D010957:Plasmids; D012335:RNA, Ribosomal / Q000378:metabolism; D014307:Trioxsalen / Q000031:analogs & derivatives / Q000494:pharmacology; D014466:Ultraviolet Rays
D004269:DNA, Bacterial; D011564:Furocoumarins; D012335:RNA, Ribosomal; D004247:DNA; C018822:aminomethyltrioxsalen; D014307:Trioxsalen