Evidence from in vitro replication that O6-methylguanine can adopt multiple conformations.

Abstract

The effect of O6-methylguanine (m6G) on replication, in a partiafly 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 m'G, replication was virtually complete in -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-O'-methylguanine-methyltransferase, replication was rapid with no blockage. These data suggest that there can be two (or more) conformations of m'G. In these studies the term syn refers to conformers interfering with base-pairing, whereas anti refers to those aflowing such base-pairing. Previous physical studies by others indicate that syn-and ant-conformers of the methyl group relative to the Ni 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 weDl as mutagenic.An important mutagenic lesion formed by methylating agents is 06-methylguanine (m6G). This modified base can pair with thymine (1) and, therefore, generate GC -AT mutations (2,3). This is one of the major mutations observed with methylating agents (for reviews, see refs. 4 and 5). However, m6G is also capable of pairing with cytosine, in which case no mutation would result (1). In addition, there is evidence that m6G is a lethal lesion in eukaryotic and prokaryotic cells (6)(7)(8)(9)(10)(11)(12). These data raise the question of how this simple modification ofguanine can generate such different biological end points.Numerous physical studies have been performed on m6G, and two conformations are reported: (i) The methyl group is syn with respect to the Ni-position of guanine, and it points into the helix, disrupting the hydrogen-bonding potential. (ii) The methyl group is anti with respect to the Ni position ofthe purine, and it points into the major groove. Syn-m6G is the favored conformation in small molecules, as determined by x-ray diffraction (13-15). In addition, syn-m6G was observed when paired with thymine in duplex DNA in solution determined by NMR (16,17). However, anti-m6G was observed when paired with thymine in an x-ray diffraction study (18) of a DNA duplex in crystalline form. Al...

Transcriptional mutagenesis: causes and involvement in tumour development

“…Interestingly, computational modeling with yeast RNAPII showed that bypass of O 6 mG can only occur when the lesion adopts an anti -conformation with the methyl group in the proximal orientation and that accommodation of the misincorporated uracil involves specific hydrogen bonding 64 . As for 8-oxoG, this specific scheme of pairing during transcription is similar to the one used to explain thymine misincorporation during replication 65 .…”

Replication across O6-Methylguanine by Human DNA Polymerase β in Vitro

“…In this respect pol ␤ is similar to KF, T4 and T5 polymerases which also preferentially insert dTMP (17)(18)(19). On the other hand, pol ␤ has an opposing 2-fold (13-fold from a running start) preference for extension from dC-m 6 dG rather than dT-m 6 dG.…”

“…In vitro studies (17)(18)(19), as well as in vivo mutagenesis assays (20,21) have shown that m 6 dG preferentially base pairs with dTMP instead of dCMP, thus giving rise to G to A transition mutations. The importance of these lesions has been repeatedly demonstrated.…”

Bypass of the Major Alkylative DNA Lesion by Human DNA Polymerase η

“…Nonenzymatic methylation by exogenous and endogenous methylating agents (e.g., (S)-adenosylmethionine and N-methyl-N-nitrosourea) [3,4], however, gives rise to a wide variety of genotoxic DNA lesions, including O6-methylguanine and N7-methylguanine [5,6]. O6-methylguanine represents a minor yet highly mutagenic lesion due to its ability to disrupt Watson–Crick base pairing during replication [7,8,9,10]. N7-methyl-2-deoxyguanosine (m7dG) is the predominant alkylative DNA lesion found in cells [11,12].…”