Interaction of the UvrABC endonuclease with DNA containing a psoralen monoadduct or cross-link. Differential effects of superhelical density and comparison of preincision complexes.
The effect of negative supercoiling on UvrABC incision of covalently closed duplex DNA circles containing either a furan-side monoadduct or a cross-link of 4'-hydroxymethyl-4,5',8-trimethylpsoralen at a unique site was examined. The rate of UvrABC incision of these DNA substrates was measured as a function of superhelical density, sigma, for values of sigma between 0 and -0.050. The monoadducted DNA substrate was incised at close to the maximum rate at all superhelical densities, with only a slight stimulation of activity between sigma = 0 and -0.035. In contrast, efficient UvrABC incision of the cross-linked DNA substrate required the DNA to be underwound, and activity showed a linear dependence on superhelical density up to sigma = -0.035. DNase I protection studies show that in the presence of both UvrA and UvrB a protein complex binds to the site of a psoralen monoadduct or cross-link in linear DNA. This UvrA-UvrB-dependent complex binds with similar affinity to both the monoadducted and the cross-linked DNA helices. However, differences in the DNase I footprint on these two DNA substrates indicate that the interaction of this protein complex is different at these two lesions. The addition of UvrC to linear DNA molecules that are saturated at the site of the lesion with the UvrA-UvrB-dependent complex resulted in efficient nicking of the monoadducted DNA, but not the cross-linked DNA. Thus, the properties of a DNA lesion site that lead to UvrAB recognition and binding are not necessarily sufficient to allow incision when all three Uvr subunits are present. We propose that after recognition and binding of a lesion site by the UvrAB complex and prior to incision, the damaged DNA helix undergoes a conformational change such as unwinding or melting that is induced by the lesion-bound Uvr complex.
Munn
M M
MM
Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut 06510.
Rupp
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WD
D001345:Autoradiography; D001483:Base Sequence; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D016172:DNA Fingerprinting; D004278:DNA, Superhelical / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes; D014307:Trioxsalen / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology
D003432:Cross-Linking Reagents; D004278:DNA, Superhelical; D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; C016507:hydroxymethyltrioxsalen; D004247:DNA; D004706:Endodeoxyribonucleases; C036385:endodeoxyribonuclease uvrABC; D014307:Trioxsalen