Effect of 3' flanking neighbors on kinetics of pairing of dCTP or dTTP opposite O6-methylguanine in a defined primed oligonucleotide when Escherichia coli DNA polymerase I is used.

Abstract

0(-Methylguanine (m6G) was incorporated site-specifically into two 25-base oligonucleotides differing only in the nucleotide on the 3' side of the modified base. Templates were primed with oligonucleotides terminating one or two bases prior to the site at which incorporation kinetics were to be investigated. Escherichia coli DNA polymerase I (Klenow fragment) was used to determine the apparent Km and relative V,,x of incorporation of either dCTP or dTTP opposite m6G or G.These data were used to calculate the relative frequency of incorporation opposite the m6G or the unmodified G. When the sequence was 3'-Cm6G-5', there was a 6-to 7-fold preference for formation of a m6G-T pair compared with m6G-C. The m6G-T frequency, based on Vmix/Km, was at least 50-fold greater than that of a G-T pair at the same site. Changing the sequence to 3'-Tm6G-5' had a marked effect on both Km and V.,, of pairs containing m6G and on the incorporation frequency of T opposite m6G, which was then only slightly favored over m6G-C. When replication was started directly opposite m6G, the kinetics appeared unaffected. These data indicate that the frequency of incorporation of C or T opposite m6G in a DNA template is dependent on the flanking neighbors and that a change of even a single base at the 3' position can have a major effect on mutagenic efficiency. Replication using Drosophila Pol a gave the same values for relative frequencies. Pairing of either C or T with m'G on the primer terminus did not significantly inhibit extension of the next normal base pair, in contrast to terminal mismatches of unmodified bases. It is concluded that, in the absence of repair, m6G can exhibit widely differing mutation frequencies which, in these experiments, can be as high as 85% of the replicated base. This variation in frequency of changed pairing could contribute to the occurrence of mutational "hot spots" after replication of damaged DNA.The likely role of 06-alkylguanines as a major factor in mutagenesis by certain alkylating agents was recognized by Loveless about 20 years ago (1). In an attempt to resolve contradictions in the literature, he suggested that O6-methylguanine (m6G) might pair with thymine (T) during replication and thus cause the G-C -* A-T transitions found as the major genetic change after reaction with certain carcinogenic alkylating agents. Further studies in a number of laboratories established that the occurrence, and in particular the persistence of this alkylated base, often correlated with the biological endpoints of mutagenesis and carcinogenesis (2). In accord with the proposed mechanism of mutagenesis, m6G can pair with T both in vitro (3) and in vivo (4), when presented to a polymerase either in the template or as a precursor to DNA synthesis. This model of mutagenesis occurring by base pairing between m6G and T during replication has long been assumed, but only recently has it begun to be tested rigorously [reviewed by Basu and Essigmann (5)]. When an m6G-containing dodecamer was annealed with a series of anal...

The structural basis for the mutagenicity of O 6 -methyl-guanine lesions

“…The cytotoxic effects of DNA-methylating agents have been exploited in their use as potent anticancer agents. O 6 -methyl-guanine (O6MeG) is mutagenic because polymerases frequently misinsert T opposite O6MeG instead of C, both in vivo (9, 10) and in vitro (11)(12)(13). In this study, we present the crystal structures of complexes of a high-fidelity DNA polymerase with substrates representing several steps of nucleotide insertion opposite O6MeG.…”

“…The relative preference for incorporation of T and C opposite an O6MeG lesion varies somewhat with polymerase and sequence context (13). High-fidelity polymerases such as exonuclease-deficient E. coli polymerase I (Klenow fragment) (13) or bacteriophage T7 DNA polymerase (11) show an Ϸ7-fold preference for misinsertion of T. By comparison, these polymerases usually show a several thousand-fold preference for insertion of a correct base-pairing partner when copying a normal, undamaged DNA.…”

“…Phage T7 polymerase shows 170-and 35-fold reductions in efficiency for nucleotide insertion and extension, respectively, as well as a preference for T misinsertion (11). In all polymerases studied to date, incorporation and extension for C and T⅐O6MeG base pairs is more efficient than for of any of the 12 possible unmodified base-base mismatches (11)(12)(13)15).…”

“…Phage T7 polymerase shows 170-and 35-fold reductions in efficiency for nucleotide insertion and extension, respectively, as well as a preference for T misinsertion (11). In all polymerases studied to date, incorporation and extension for C and T⅐O6MeG base pairs is more efficient than for of any of the 12 possible unmodified base-base mismatches (11)(12)(13)15).High-fidelity DNA polymerases copy their templates rapidly and accurately. Structures of Bacillus stearothermophilus DNA polymerase I large fragment (BF) as it replicates undamaged (16-18) and damaged (19-21) DNA substrates yield a detailed picture of the structural mechanisms that ensure fidelity at each step of the reaction.…”

“…High-fidelity polymerases such as exonuclease-deficient E. coli polymerase I (Klenow fragment) (13) or bacteriophage T7 DNA polymerase (11) show an Ϸ7-fold preference for misinsertion of T. By comparison, these polymerases usually show a several thousand-fold preference for insertion of a correct base-pairing partner when copying a normal, undamaged DNA. O6MeG lesions slow the rate of DNA synthesis at the nucleotide incorporation step opposite the lesion (11)(12)(13) and subsequent extension beyond the O6MeG-pair (11,12,14). These kinetic effects vary with polymerase.…”