Novel repair activities of AlkA (3-methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine, guanine lesions induced by nitric oxide and nitrous acid

Abstract

Nitrosation of guanine in DNA by nitrogen oxides such as nitric oxide (NO) and nitrous acid leads to formation of xanthine (Xan) and oxanine (Oxa), potentially cytotoxic and mutagenic lesions. In the present study, we have examined the repair capacity of DNA N-glycosylases from Escherichia coli for Xan and Oxa. The nicking assay with the defined substrates containing Xan and Oxa revealed that AlkA [in combination with endonuclease (Endo) IV] and Endo VIII recognized Xan in the tested enzymes. The activity (V(max)/K(m)) of AlkA for Xan was 5-fold lower than that for 7-methylguanine, and that of Endo VIII was 50-fold lower than that for thymine glycol. The activity of AlkA and Endo VIII for Xan was further substantiated by the release of [(3)H]Xan from the substrate. The treatment of E.coli with N-methyl-N'-nitro-N-nitrosoguanidine increased the Xan-excising activity in the cell extract from alkA(+) but not alkA(-) strains. The alkA and nei (the Endo VIII gene) double mutant, but not the single mutants, exhibited increased sensitivity to nitrous acid relative to the wild type strain. AlkA and Endo VIII also exhibited excision activity for Oxa, but the activity was much lower than that for Xan.

Oxanine DNA Glycosylase Activity from Mammalian Alkyladenine Glycosylase

“…AlkA, similar to hAAG, is a broad substrate DNA glycosylase that can remove alkylated and deaminated bases. However, the deaminated base DNA glycosylase activities of E. coli AlkA seem low compared with its alkylbase DNA glycosylase activities (18,41). Endonuclease VIII is a DNA glycosylase that removes oxidative base lesions (42,43).…”

“…Endonuclease VIII is a DNA glycosylase that removes oxidative base lesions (42,43). Compared with other lesions, the ODG activities of both AlkA and endo VIII are quite low (18). Enzyme in excess (30-fold) is required to detect cleavage products (18).…”

“…Compared with other lesions, the ODG activities of both AlkA and endo VIII are quite low (18). Enzyme in excess (30-fold) is required to detect cleavage products (18). It is reported (18) that both AlkA and endo VIII preferentially remove oxanine from A/O, C/O, and G/O base pairs.…”

“…Xanthine DNA glycosylase activities of E. coli AlkA and endo VIII proteins have been reported (9,18). E. coli endo V has been shown to have deoxyxanthosine endonuclease activity (19,20).…”

“…coli AlkA and endo VIII contain some glycosylase activity to remove mutagenic oxanine from DNA, but it appears that a large excess of glycosylase proteins is needed to detect limited activities (18). To understand the mutagenicity and potential oxanine DNA glycosylase activities in mammalian systems, we investigated Oxa-containing incorporation by human DNA polymerase ␤.…”