Product inhibition and magnesium modulate the dual reaction mode of hOgg1.
8-Oxoguanine (8-oxoG) is a major mutagenic DNA base damage corrected by the base excision repair (BER) pathway, which is initiated by lesion specific DNA glycosylases. The human DNA glycosylase hOgg1 catalyses excision of 8-oxoG followed by strand incision 3' to the abasic site if cytosine is positioned in the complementary strand. Unlike most bifunctional glycosylases, hOgg1 uncouples base removal and strand cleavage. This paper addresses the significance of product inhibition and magnesium for the non-concerted action of hOgg1 activities. The enzymatic activities of hOgg1 were analysed on duplex DNA containing a single 8-oxoG or abasic site opposite cytosine. AP-lyase cleavage of abasic sites was inhibited in the presence of free 8-oxoG, indicating that the product of base excision inhibits the subsequent strand incision step. Assays with DNA containing 8-oxoG showed that free 8-oxoG also inhibited the glycosylase activity. This result suggests that the free 8-oxoG base may retain in the recognition site following N-glycosylic cleavage, implying that product inhibition contribute to uncoupling the activities of hOgg1. Magnesium reduced the efficiency of base excision and strand incision on DNA containing 8-oxoG under single turnover conditions; however, the reduction was more pronounced for the AP-lyase activity. Furthermore, Shiff-base formation between hOgg1 and 8-oxoG containing DNA was abrogated in the presence of magnesium. These results suggest that hOgg1 mainly operates as a monofunctional glycosylase under physiological concentrations of magnesium.
Morland
Ingrid
I
Department of Molecular Biology, Institute of Medical Microbiology, University of Oslo, The National Hospital, N-0027 Oslo, Norway.
Luna
Luisa
L
Gustad
Ellen
E
Seeberg
Erling
E
Bjørås
Magnar
M
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D045647:DNA Glycosylases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D017931:DNA Primers; D004563:Electrochemistry; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D008274:Magnesium / Q000494:pharmacology*; D012545:Schiff Bases / Q000378:metabolism
D017931:DNA Primers; D012545:Schiff Bases; C024829:8-hydroxyguanine; D006147:Guanine; D045647:DNA Glycosylases; C479058:oxoguanine glycosylase 1, human; D008274:Magnesium