All four known cyclic adducts formed in DNA by the vinyl chloride metabolite chloroacetaldehyde are released by a human DNA glycosylase.
Proceedings of the National Academy of Sciences of the United States of America 91:3 (1994) 1024-1028
Abstract:
We have previously reported that human cells and tissues contain a 1,N6-ethenoadenine (epsilon A) binding protein, which, through glycosylase activity, releases both 3-methyladenine (m3A) and epsilon A from DNA treated with methylating agents or the vinyl chloride metabolite chloroacetaldehyde, respectively. We now find that both the partially purified human epsilon A-binding protein and cell-free extracts containing the cloned human m3A-DNA glycosylase release all four cyclic etheno adducts--namely epsilon A, 3,N4-ethenocytosine (epsilon C), N2,3-ethenoguanine (N2,3-epsilon G), and 1,N2-ethenoguanine (1,N2-epsilon G). Base release was both time and protein concentration dependent. Both epsilon A and epsilon C were excised at similar rates, while 1,N2-epsilon G and N2,3-epsilon G were released much more slowly under identical conditions. The cleavage of glycosyl bonds of several heterocyclic adducts as well as those of simple methylated adducts by the same human glycosylase appears unusual in enzymology. This raises the question of how such a multiple, divergent activity evolved in humans and what may be its primary substrate.Both O4-methylthymine and O4-ethylthymine preferentially form alkyl T.G pairs that do not block in vitro replication in a defined sequence.
Carcinogenesis 14:9 (1993) 1915-1919
Abstract:
The mutagenic potential of O4-methylthymine (m4T) and O4-ethylthymine (e4T) was determined by a primer extension assay on a 25mer oligonucleotide containing a single site-specifically incorporated modified thymine. The e4T-containing oligonucleotide was prepared by using a new synthetic procedure suitable for large alkyl groups on thymine. The second-order rate constants, K(app)m and V(rel)max, permitted calculation of the frequency of formation and extension of modified base pairs compared to Watson-Crick pairing. With both m4T and e4T, the T.G type pairing was formed at least 10-fold more frequently than the nonmutagenic alkyl T.A pairing. However, there was a small but reproducible preference for m4T.G pairing. In both cases T-->C transitions would result. There was no evidence for formation of alkyl T.C or T.T. These data suggest that reported T-->A transversions by ethylation are not likely to result from O4-alkylthymine. In contrast to insertion, extension beyond alkylthymine under kinetic conditions did not occur with alkyl T.A. but only with the alkyl T.G termini. For this latter T.G type pairing, the larger ethyl group did not hinder extension compared to that of the methyl group, in the sequence studied. Under non-limiting conditions of dNTP concentration and time, complete replication could be demonstrated for both methyl- and ethyl-containing oligonucleotides. We conclude that the increase in size of the alkyl group from methyl to ethyl does not significantly affect the mutagenic potential and type of mutations of O4-alkylthymine in vitro.Evidence from in vitro replication that O6-methylguanine can adopt multiple conformations.
Proceedings of the National Academy of Sciences of the United States of America 90:9 (1993) 3983-3987
Abstract:
The effect of O6-methylguanine (m6G) on replication, in a partially double-stranded defined 25-base oligonucleotide, has been studied under nonlimiting conditions of unmodified dNTPs and over an extended time period, using the Klenow fragment of Escherichia coli DNA polymerase I. The sequence surrounding m6G has flanking cytosines (C-m6G-C), and the initial steady-state kinetics have been reported. When the primer was annealed so that the first base to be replicated was m6G, replication was virtually complete in approximately 5 min, although the reaction appears biphasic. When annealed with a primer where thymine or cytosine is paired opposite template m6G, about half the molecules were replicated in the first 15 sec, and no significant further replication was seen over a 1-hr period. When m6G was dealkylated by DNA-O6-methylguanine-methyltransferase, replication was rapid with no blockage. These data suggest that there can be two (or more) conformations of m6G. In these studies the term syn refers to conformers interfering with base-pairing, whereas anti refers to those allowing such base-pairing. Previous physical studies by others indicate that syn- and anti-conformers of the methyl group relative to the N1 of guanine are possible. Here molecular modeling/computational studies are described, suggesting that syn- and anti-m6G can be of similar energy in DNA, and, therefore, these two conformers may explain the two types of species observed during in vitro replication. An alternative explanation could be the possibility that the different species may manifest differential interactions of m6G with Klenow fragment. These results may provide a rationale for why m6G lesions in vivo have been reported to be lethal as well as mutagenic.Further studies of the mixed acetals of nucleosides.
Biochimie 75:7 (1993) 511-515
Abstract:
We reported in 1988 on a new nucleoside modification reaction: the exocyclic amino groups of (d)adenosine and (d)cytidine react rapidly at ambient temperature with acetaldehyde and alcohols to give stable mixed acetals (N-ethylethoxy-acetal). NH2 + O = CH(CH3) + ROH-->NH-CH(CH3)-O-R + H2O. Here we report in detail on the occurrence of this reaction in very dilute aqueous solution (ie under biological conditions), on its mechanism and kinetics, on the mixed acetal formation with other aldehydes and other nucleic acid components, and on the question of whether these adducts are mutagenic.Both purified human 1,N6-ethenoadenine-binding protein and purified human 3-methyladenine-DNA glycosylase act on 1,N6-ethenoadenine and 3-methyladenine.
Proceedings of the National Academy of Sciences of the United States of America 89:20 (1992) 9386-9390