Interaction of the UvrABC endonuclease with DNA containing a psoralen monoadduct or cross-link. Differential effects of superhelical density and comparison of preincision complexes

• Published in: The Journal of biological chemistry Vol. 266; no. 36; pp. 24748 - 24756
• Main Authors: MUNN, M. M, RUPP, W. D
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 25-12-1991
• Subjects: Autoradiography; Base Sequence; Biological and medical sciences; Cross-Linking Reagents; DNA - chemistry; DNA - drug effects; DNA - metabolism; DNA Fingerprinting; DNA, Superhelical - drug effects; Electrophoresis, Polyacrylamide Gel; Endodeoxyribonucleases - metabolism; Escherichia coli; Escherichia coli Proteins; Fundamental and applied biological sciences. Psychology; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Mutagenesis. Repair; Nucleic Acid Conformation; Nucleic Acid Heteroduplexes; Trioxsalen - analogs & derivatives; Trioxsalen - metabolism; Trioxsalen - pharmacology; Enzyme; DNA; Escherichia coli; Footprinting technique; Bacteria; Molecular interaction; Endonuclease; Psoralen derivatives; Enterobacteriaceae; Conformation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)54293-9

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.