Initiation of the UvrABC nuclease cleavage reaction. Efficiency of incision is not correlated with UvrA binding affinity.
The incision steps of Escherichia coli nucleotide excision repair are mediated by the UvrABC nuclease complex. We have previously shown that the UvrABC nuclease specifically incises apyrimidinic (AP) sites less efficiently than o-benzylhydroxylamine-modified apyrimidinic (BA) sites. To investigate these differences, quantitative DNase I footprinting titration studies were performed. The UvrA binding isotherms were similar for both the AP site (Kd = 6 x 10(-9) M) and the bulkier BA lesion (Kd = 14 x 10(-9) M), despite the fact that the extent of incision differs for these two lesions. It was also found that the relative binding affinity of the preincision UvrA2B complex to the AP and BA substrates differs significantly with estimated apparent equilibrium dissociation constants (Kd) of 4 x 10(-9) M and 80 x 10(-9) to 120 x 10(-9) M, respectively. These results indicate that incision efficiency does not correlate to UvrA binding affinity, but is a direct result of interactions between the UvrA2B complex and the site of the DNA damage. It is also shown that high UvrA concentrations are inhibitory to the UvrABC nuclease reaction.
Snowden
A
A
Department of Pathology, University of Vermont, Burlington 05405.
Van Houten
B
B
D001483:Base Sequence; D004249:DNA Damage; D004260:DNA Repair; D003850:Deoxyribonuclease I; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009710:Nucleotide Mapping; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis
D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D004706:Endodeoxyribonucleases; D003850:Deoxyribonuclease I; C036385:endodeoxyribonuclease uvrABC