The sequence used is analogous to that studied previously to determine the mutation frequency of O6-methylguanine in vitro and in vivo. Synthesis of a 25-mer oligonucleotide template containing O4-methylthymine (m4T) at a unique site is reported. It is postulated that the mutation frequency at a given site is primarily a function of the structure of the sequence around the target site. All enzymes led to approximately the same frequency of transitions. coli DNA polymerase I (Kf), engineered 3'-5' exonuclease-free Kf (exo-free Kf), polymerase alpha-primase complex from Drosophila melanogaster or calf thymus, and human immunodeficient virus-I reverse transcriptase (HIV-I RT).
The enzymes were the Klenow fragment of E. However, both m4T and m6G prefer to form a type of G.T pairing which would lead to the transitions: G.C-A.T or T.A-C.G. The immediate neighbor bases play an important role in determining the frequency of specific changed basepairing and subsequent elongation of the annealed primer. Preliminary data are also reported for another carcinogen product, N2,3-ethenodeoxyguanosine ( epsilon G). We have used site-specific insertion of these derivatives into oligonucleotides and measured the kinetic constants of various pairings, using both prokaryotic and eukaryotic polymerases for replication. Two alkylation products implicated in initiation of carcinogenesis are O6-alkylguanine (m6G) and O4-alkylthymine (m4T). This result suggests that O4MeT lesions can be recognized in this substrate by eukaryotic DNA MTases but the exact biochemical mechanism of methyltransferase inactivation remains to be determined. Surprisingly, the human and yeast MTases were also inactivated by the O4MeT-containing oligomer albeit at IC50 values of 29.5 and 44 nM, respectively.
Both the human and the yeast DNA MTases were efficiently inactivated upon incubation with the O6MeG-containing oligomer (IC50 values of 1.5 and 1.3 nM, respectively). coli Ada MTase displayed a striking preference for O6MeG (IC50 1.25 nM) as compared to O4MeT (IC50 27.5 nM). coli ogt gene product had a relatively high affinity for the O6MeG substrate (IC50 8.1 nM) but had an even higher affinity for the O4MeT substrate (IC50 3 nM). Using chemically synthesized double-stranded 25-base pair oligodeoxynucleotides containing a single O6MeG or a single O4MeT, the concentration of O6MeG or O4MeT substrate that produced 50% inactivation (IC50) was determined for each of four MTases. The suicidal inactivation mechanism of DNA repair methyltransferases (MTases) was exploited to measure the relative efficiencies with which the Escherichia coli, human, and Saccharomyces cerevisiae DNA MTases repair O6-methylguanine (O6MeG) and O4-methylthymine (O4MeT), two of the DNA lesions produced by mutagenic and carcinogenic alkylating agents.
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