O6-alkylguanine-DNA alkyltransferase (AGT) repairs O6-alkylguanine residues at different rates depending on the identity of the alkyl group as well as the sequence context. To elucidate the mechanism(s) underlying the differences in rates, we examined the repair of five alkyl groups in three different sequence contexts. The kinact and Km values were determined by measuring the rates of repair of oligodeoxynucleotide duplexes containing the O6-alkylguanine residues with various concentrations of AGT in excess. The time course of the reactions all followed pseudo-first-order kinetics except for one of the O6-ethylguanine substrates, which could be analyzed in a two-phase exponential equation. The differences in rates of repair between the different alkyl groups and the different sequence contexts are dependent on rates of alkyl transfer and not substrate recognition. The relative rates of reaction are in general benzyl>methyl>ethyl>2-hydroxyethyl>4-(3-pyridyl)-4-oxobutyl, but the absolute rates are dependent on sequence. The kinact values between benzyl and 4-(3-pyridyl)-4-oxobutyl range from 2300 to 350000 depending on sequence. The sequence-dependent variation in kinact varied the most for O6-[4-(3-pyridyl)-4-oxobutyl]guanine, which ranged from 0.022 to 0.000016 s(-1). The results are consistent with a mechanism in which the O6-alkylguanine can bind to AGT in either a reactive or an unreactive orientation, the proportion of which depends on the sequence context.

Interactions between the minor groove of the DNA and DNA polymerases appear to play a major role in the catalysis and fidelity of DNA replication. In particular, Arg 668 of Escherichia coli DNA polymerase I (Klenow fragment) makes a critical contact with the N-3-position of guanine at the primer terminus. We investigated the interaction between Arg 668 and the ring oxygen of the incoming deoxynucleotide triphosphate (dNTP) using a combination of site-specific mutagenesis of the protein and atomic substitution of the DNA and dNTP. Hydrogen bonds from Arg 668 were probed with the site-specific mutant R668A. Hydrogen bonds from the DNA were probed with oligodeoxynucleotides containing either guanine or 3-deazaguanine (3DG) at the primer terminus. Hydrogen bonds from the incoming dNTP were probed with (1R,3R,4R)-1-[3-hydroxy-4-(triphosphorylmethyl)cyclopent-1-yl]uracil (dcUTP), an analog of dUTP in which the ring oxygen of the deoxyribose moiety was replaced by a methylene group. We found that the pre-steady-state parameter k pol was decreased 1,600 to 2,000-fold with each of the single substitutions. When the substitutions were combined, there was no additional decrease (R668A and 3DG), a 5-fold decrease (3DG and dcUTP), and a 50-fold decrease (R668A and dcUTP) in k pol . These results are consistent with a hydrogenbonding fork from Arg 668 to the primer terminus and incoming dNTP. These interactions may play an important role in fidelity as well as catalysis of DNA replication.The high fidelity of DNA replication synthesis is accomplished despite the similarity in energy between correctly and incorrectly paired bases (1, 2). Since inter-strand hydrogen bonds cannot account for this selectivity (3, 4), other mechanisms have been proposed to supply the fidelity (reviewed in Ref. 5). These include solvation (6), base stacking (7, 8), steric exclusion (3, 4, 9), and minor groove binding (10). These mechanisms are not mutually exclusive and may all enhance selection for the correct base pairs.The minor groove has been suggested to be a site by which polymerases can check geometry because the O 2 -position of pyrimidines and the N-3-position of purines occupy similar spatial orientations as well as being hydrogen bond acceptors (10). Crystal structures of DNA polymerases bound to DNA have shown that there are many interactions between the protein and the minor groove of DNA (11-15). Site-directed mutagenesis studies have implicated several amino acid residues as being important for catalysis and fidelity of DNA replication (15)(16)(17)(18)(19)(20). Similarly, the use of purine analogs such as 3-deazaadenine (21, 22), 3-deazaguanine (3DG) 1 (23, 24), 9-methyl-1H-imidazo[4,5-b]pyridine and 4-methylbenzimidazole (25,26), and the pyrimidine analogs difluorotoluene (25), 2-aminopyridine, and 3-methyl-2-pyridone (27) also have implicated the minor groove of the DNA as being crucial.X-ray crystallographic studies of polymerases that have a structure similar to that of KF Ϫ (Taq (13) Bacillus stearothermophilus (12) and T7 (14)...

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Bromoalkynol derivatives of diacetone glucose undergo rearrangements to dihaloenol ethers contained in furo[3,4-b]furan cores when treated with halonium-producing reagents. [reaction: see text]