The actual incision determines the efficiency of repair of cisplatin-damaged DNA by the Escherichia coli UvrABC endonuclease.
The UvrABC endonuclease from Escherichia coli repairs a broad spectrum of DNA lesions with variable efficiencies. The effectiveness of repair is influenced by the nature of the lesion, the local DNA sequence, and/or the topology of the DNA. To get a better understanding of the aspects of this multistep repair reaction that determine the effectiveness of repair, we compared the incision efficiencies of linear DNA fragments containing either a site-specific cis-[Pt(NH3)2(d(GpG)-N7(1),-N7(2)]] or a cis- Pt(NH3)2[d(GpCpG)-N7(1),-N7(3)]] adduct. Overall the DNA with the cis-PtGG adduct was incised about 3.5 times more efficiently than the cis-Pt.GCG-containing DNA. The rate of UvrB-DNA preincision complex formation for both lesions was similar and high in relation to the incision. DNase I footprints, however, showed that the local structure of the two preincision complexes is different. An assay was developed to measure the binding of UvrC to the preincision complexes and it was found that the binding rate of UvrC to the more slowly incised cis-Pt.GCG preincision complex was higher than to the cis-Pt.GG preincision complex. This most likely reflects a qualitative difference in preincision complex structures. For both lesions the binding of UvrC to the preincision complex was fast compared to the kinetics of actual incision. Apparently, direct incision of cisplatin damage requires an additional conformational change after the binding of UvrC.
Visse
R
R
Department of Biochemistry, Gorlaeus Laboratories, Leiden University, The Netherlands.
van Gool
A J
AJ
Moolenaar
G F
GF
de Ruijter
M
M
van de Putte
P
P
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D002945:Cisplatin / Q000494:pharmacology*; D004247:DNA / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D004249:DNA Damage; D004265:DNA Helicases; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008969:Molecular Sequence Data
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C058093:UvrB protein, E coli; D004247:DNA; D004706:Endodeoxyribonucleases; C066567:UvrC protein, E coli; C036385:endodeoxyribonuclease uvrABC; C068631:UvrA protein, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D002945:Cisplatin