Formation of DNA repair intermediates and incision by the ATP-dependent UvrB-UvrC endonuclease.
The Escherichia coli UvrB and UvrC proteins play key roles in DNA damage processing and incisions during nucleotide excision repair. To study the DNA structural requirements and protein-DNA intermediates formed during these processes, benzo[a]pyrene diol epoxide-damaged and structure-specific 50-base pair substrates were constructed. DNA fragments containing a preexisting 3' incision were rapidly and efficiently incised 5' to the adduct. Gel mobility shift assays indicated that this substrate supported UvrA dissociation from the UvrB-DNA complex, which led to efficient incision. Experiments with a DNA fragment containing an internal noncomplementary 11-base region surrounding the benzo[a]pyrene diol epoxide adduct indicated that UvrABC nuclease does not require fully duplexed DNA for binding and incision. In the absence of UvrA, UvrB (UvrC) bound to an 11-base noncomplementary region containing a 3' nick (Y substrate), forming a stable protein-DNA complex (Kd approximately 5-10 nM). Formation of this complex was absolutely dependent upon UvrC. Addition to this complex of ATP, but not adenosine 5'-(beta,gamma-iminotriphosphate) or adenosine 5'-(beta, gamma-methylene)triphosphate, caused incision three or four nucleotides 5' to the double strand-single strand junction. The ATPase activity of native UvrB is activated upon interaction with UvrC and enhanced further by the addition of Y substrate. Incision of this Y structure occurs even without DNA damage. Thus the UvrBC complex is a structure-specific, ATP-dependent endonuclease.
Zou
Y
Y
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555, USA.
Walker
R
R
Bassett
H
H
Geacintov
N E
NE
Van Houten
B
B
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D004265:DNA Helicases; D004260:DNA Repair; D004269:DNA, Bacterial / Q000235:genetics*; D004706:Endodeoxyribonucleases; D004720:Endonucleases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C058093:UvrB protein, E coli; D004706:Endodeoxyribonucleases; D004720:Endonucleases; C066567:UvrC protein, E coli; D004265:DNA Helicases