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

Abstract

Replication in vivo across unrepaired O 6 -methylguanine (m 6 dG) lesions by mammalian DNA polymerase ␤ (pol ␤) during short patch repair may contribute to the cytotoxicity and mutagenesis of m 6 dG. We have employed in vitro steady state kinetic analysis to investigate the replication of oligonucleotide templates containing site-specific m 6 dG by human pol ␤. Our results show that m 6 dG is a strong but not absolute block to replication by pol ␤. pol ␤ exhibits mixed kinetic discrimination during overall replication across dG and m 6 dG. pol ␤ preferentially inserts dTMP rather than dCMP opposite m 6 dG. However, pol ␤ extends from the dC-m 6 dG base pair more efficiently than from the dTm 6 dG base pair. This is in strong contrast to other polymerases such as the exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I (exo ؊ KF) that preferentially extends dT-m 6 dG by a factor of 10 over dC-m 6 dG. When both insertion and extension are considered, pol ␤ has a 15-fold overall preference for incorporation of the mutagenic substrate dTTP rather than the nonmutagenic substrate dCTP during replication across m 6 dG. This suggests that pol ␤, in concert with the T:G-specific thymine DNA glycosylase, may be intricately involved in the futile cytotoxic repair induced by m 6 dG. Our results also suggest that replication across m 6 dG by pol ␤ may contribute to m 6 dG-induced G 3 A transition mutations.1 is a nuclear DNA repair polymerase extensively characterized by Wilson (1, 2). It mainly carries out gap filling during short patch base excision repair (3-7). pol ␤ fills short (up to six nucleotides long) DNA gaps (4 -8) and has been found to act in concert with uracil glycosylase (3) and the human G:T-specific thymine glycosylase (9). pol ␤ is also implicated in the repair of DNA damage induced by anticancer agents such as bleomycin, ␥-irradiation (10), cisplatin (11), and alkylating agents (12). Resistance to cisplatin may be partially due to overexpression of pol ␤ (11). Tumors may also develop resistance to radiation therapy and chemotherapy (e.g. cisplatin and alkylating agents) by increased efficiency of DNA repair. pol ␤ has been found to be altered in some human cancers (13,14), suggesting its importance in maintaining genomic stability. However, pol ␤ is the most error prone of all known polymerases tested in vitro (15).Mechanistic studies of pol ␤ are important for understanding its diverse roles in DNA replication and repair under circumstances leading to mutagenesis, cytotoxicity, tumor progression, and the resistance of tumors to anticancer therapy. Furthermore, the small size, single subunit composition, availability of crystal structure, and absence of confounding associated activities (such as 3Ј-5Ј exonuclease) make pol ␤ a good model for the study of DNA polymerase structure and function.O 6 -Methylguanine (m 6 dG) is a mutagenic and cytotoxic DNA adduct that can be formed in vivo by such diverse agents as tobacco smoke, methylnitrosourea, and other S n 1 methylating agents (16)....

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

“…Although Pol␤ can replicate through m6G in DNA, it does so 10,000-fold less efficiently than the replication of undamaged DNA, and Pol␤ inserts primarily a T residue (ϳ95%) opposite m6G (38). Further, our genetic studies in yeast have yielded no evidence that might impute a role for Pol␤ in m6G bypass.…”

“…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 in base excision repair (38). Thus, although the m6G ⅐ T base pair is more Watson-Crick-like in geometry than the m6G ⅐ C pair, DNA polymerases are quite inefficient in incorporating even a T opposite m6G.…”

Kinetics of the Action of Thymine DNA Glycosylase

“…The removal of thymine from SMe G⅐T base pairs suggests that some SMe G⅐T base pairs may be processed by this base excision pathway and thus avoid recognition by the proteins of the postreplicative mismatch repair pathway. Although it has been suggested that, in the closely analogous case of OMe G⅐T base pairs, base excision repair might itself contribute to toxicity (33), it is clear that postreplicative mismatch repair, and not base excision repair, is the predominant factor in the cytotoxicity of thioguanine, because a cell line that has lost postreplicative mismatch repair is resistant to 6-thioguanine, despite retaining thymine DNA glycosylase activity (7).…”

Role of DNA mismatch repair and p53 in signaling induction of apoptosis by alkylating agents

“…O 6 MeGs that escape MutS␣ recognition frequently direct the misincorporation of thymine during replication; should the resulting O 6 MeG⅐T mispairs also escape MutS␣ recognition they will produce G⅐C to A⅐T transition mutations in daughter cells (14,66). Only a small fraction of such mutational events will be lethal, and the vast majority of mutant cells will persist in the population; indeed, some of the mutant cells may acquire a growth advantage.…”