A comparison of the rates of reaction and function of UVRB in UVRABC- and UVRAB-mediated anthramycin-N2-guanine-DNA repair.
The repair of anthramycin-DNA adducts by the UVR proteins in Escherichia coli follows two pathways: the adducts may be incised by the combined actions of UVRA, UVRB, and UVRC, or alternatively, the anthramycin may be removed by UVRA and UVRB in the absence of UVRC and with no DNA strand incision. To assess the competition between these two competing pathways, the rate of UVRABC-mediated excision repair of anthramycin-N2-guanine DNA adducts and the rate of UVRAB-mediated removal of the adduct were measured with single end-labeled DNAs under identical reaction conditions. UVR protein concentrations of 15 nM UVRA, 100 nM UVRB, and 10 nM UVRC protein were chosen to mimic in vivo concentrations. With these UVR protein concentrations and anthramycin-DNA concentrations of 1-2 nM the incision reaction and the release reactions are described by first-order kinetics. The rate of the UVRABC reaction, measured as the increase in incised fragments, was six to seven times faster than the rate of the UVRAB reaction, measured as the decrease in incised fragments. The UVRABC incision rate on anthramycin-modified linear DNA was four to five times the incision rate measured on the same DNA irradiated with ultraviolet light. We also investigated the role of the ATPase function of UVRB in UVRAB-mediated anthramycin removal. We found that a UVRB analogue with alanine at arginine 51, which retains near wild type ATPase activity, supported removal of anthramycin in the presence of UVRA, whereas a UVRB analogue with alanine at lysine 45, which abolishes the ATPase activity, did not. UVRB*, a specific proteolytic cleavage product of UVRB which retains the ATPase activity, did support removal of anthramycin in the presence of UVRA.
Nazimiec
M
M
University of Texas System M. D. Anderson Cancer Center, Smithville 78957.
Grossman
L
L
Tang
M S
MS
D000876:Anthramycin / Q000378:metabolism* / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism*; D018736:DNA Adducts; D004260:DNA Repair; D004269:DNA, Bacterial / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D004338:Drug Combinations; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000528:radiation effects; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D006147:Guanine; D007700:Kinetics; D010957:Plasmids; D013997:Time Factors
D018736:DNA Adducts; D004269:DNA, Bacterial; D004338:Drug Combinations; D029968:Escherichia coli Proteins; C020204:anthramycin-DNA; D000876:Anthramycin; D006147:Guanine; D004247:DNA; D004706:Endodeoxyribonucleases; C036385:endodeoxyribonuclease uvrABC