Analysis of UvrABC endonuclease reaction intermediates on cisplatin-damaged DNA using mobility shift gel electrophoresis.
One of the least understood steps in the UvrABC mediated excision repair process is the recognition of lesions in the DNA. The isolation of different reaction intermediates is of vital importance for the unraveling of the mechanism. A mobility shift gel electrophoresis assay is described which visualizes such intermediates. After incubation of a DNA substrate containing a specific cisplatin adduct with UvrA alone or with UvrA and UvrB, UvrA.DNA, UvrAB.DNA and UvrB.DNA complexes were observed which could be identified using specific antibodies. At low UvrA concentrations in the presence of UvrB only the UvrB.DNA complex is observed. Bands corresponding to the UvrAB.DNA complex and also other nonspecific bands are found at relatively high UvrA concentrations. The DNase-I footprint for the UvrAB.- and UvrB.DNA complex are very similar and protect about 20 bases. Both complexes are incised in the presence of UvrC with comparable efficiency. The UvrAB.- and the UvrB.DNA complex were both incised at the 8th phosphodiester bond 5' to a specific cisplatin adduct. In addition the UvrAB.DNA complex could also be incised at the 15th phosphodiesterbond 5' to the damaged site. The results suggest that the UvrB.DNA complex is the natural substrate for UvrC-induced incision.
Visse
R
R
Department of Biochemistry, Gorlaeus Laboratories, Leiden University, The Netherlands.
de Ruijter
M
M
Moolenaar
G F
GF
van de Putte
P
P
D002945:Cisplatin / Q000633:toxicity*; D004247:DNA / Q000187:drug effects* / Q000378:metabolism; D004249:DNA Damage; D016172:DNA Fingerprinting; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D029968:Escherichia coli Proteins; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; C036385:endodeoxyribonuclease uvrABC; D002945:Cisplatin