Replication past O⁶-Methylguanine by Yeast and Human DNA Polymerase η

Abstract

O6 -Methylguanine (m6G) is formed by the action of alkylating agents such as N-methyl-N-nitro-N-nitrosoguanidine (MNNG) on DNA. m6G is a highly mutagenic and carcinogenic lesion, and it presents a block to synthesis by DNA polymerases. Here, we provide genetic and biochemical evidence for the involvement of yeast and human DNA polymerase (Pol) in the replicative bypass of m6G lesions in DNA. The formation of MNNG-induced mutations is almost abolished in the rad30⌬ pol32⌬ double mutant of yeast, which lacks the RAD30 gene that encodes Pol and the Pol32 subunit of DNA polymerase ␦ (Pol␦). Although Pol␦ can function in the mutagenic bypass of m6G lesions, our biochemical studies indicate that Pol is much more efficient in replicating through m6G than Pol␦. Both Pol and Pol␦ insert a C or a T residue opposite from m6G; Pol, however, is more accurate, as it inserts a C about twice as frequently as Pol␦. Alkylating agents are used in the treatment of malignant tumors, including lymphomas, brain tumors, melanomas, and gastrointestinal carcinomas, and the clinical effectiveness of these agents derives at least in part from their ability to form m6G in DNA. Inactivation of Pol could afford a useful strategy for enhancing the effectiveness of these agents in cancer chemotherapy. O6 -Methylguanine (m6G) is formed in DNA by treatment with alkylating agents such as N-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG). m6G is highly mutagenic, and the mutagenic and carcinogenic potency of alkylating agents closely parallels their ability to form m6G in DNA (31). In yeast as well as higher eukaryotes, m6G specifically induces G ⅐ C to A ⅐ T transition mutations (30,35).Alkylation at the O 6 position of guanine has a profound effect on base pairing properties. Melting studies of DNA duplexes containing m6G have shown that the m6G ⅐ T base pair is energetically less stable than the m6G ⅐ C base pair (10), and nuclear magnetic resonance studies have indicated that the m6G ⅐ T base pair is less hydrogen bonded than the m6G ⅐ C base pair (33, 34). Nevertheless, DNA polymerases incorporate T opposite m6G more often than C, because the m6G ⅐ T mispair retains the Watson-Crick geometry more closely than the m6G ⅐ C base pair. At neutral pH, C is inserted opposite m6G via a wobble configuration, but the phosphodiester links both 3Ј and 5Ј to the C are distorted in this base pair (18,19,24,40,41,46). m6G is a block to synthesis by prokaryotic and eukaryotic DNA polymerases. Extensive steady-state kinetic analyses have indicated that the Escherichia coli Klenow fragment is inhibited by m6G both at the step of insertion of a nucleotide opposite the lesion and at the step of extension from the m6G ⅐ C or m6G ⅐ T base pair (7). Sequenase (T7 DNA polymerase) is partially inhibited by m6G at both these steps (42). Eukaryotic DNA polymerase ␣, required for lagging strand DNA synthesis, is strongly blocked one base before m6G, indicating an inhibition of nucleotide insertion opposite the lesion (42). m6G also blocks DNA polymerase ␤, which is involved i...

Miscoding properties of 1,N6-ethanoadenine, a DNA adduct derived from reaction with the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea

“…Studies using in vitro replication systems have revealed that the three related Y family enzymes, pols η (Rad30A), ι (Rad30B) and κ (DinB1), are capable of bypassing various DNA lesions [e.g. [28][29][30][31][32][33][34][35][36] that are normally blockers of classical DNA polymerases such as pol α and pol β. However, such translesion syntheses can be error-free or error-prone.…”

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

“…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 (11-13) and subsequent extension beyond the O6MeG-pair (11,12,14). These kinetic effects vary with polymerase.…”

“…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.…”

“…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.…”

Bypass of Mutagenic O6-Carboxymethylguanine DNA Adducts by Human Y- and B-Family Polymerases

“…[24][25][26] These O 6 -alkylguanine adducts are relevant in terms of endogenous methylation, tobacco nitrosamine exposure, and the model hepatocarcinogen methylbenzylnitrosamine. In comparing these previously characterized O 6 -alkylG TLS substrates, the larger the alkyl group on the O 6 -position of guanine, the greater the reduction in bypass capacity of the human enzymes.…”