Transcription elongation past O6-methylguanine by human RNA polymerase II and bacteriophage T7 RNA polymerase

Methylating agents are among the array of chemicals that damage the genome. They react with nucleophilic sites in DNA, adding methyl groups to the purines, pyrimidines and the phosphodiester backbone, resulting in modifications that compromise the fidelity and efficiency of replication and transcription. These agents include exogenous compounds like N-methyl-N-nitrosourea, methyl methane sulfonate and N-methyl-N′-nitro-nitrosoguanidine (1), along with endogenous biochemical agents like S-adenosylmethionine (2) and metabolically nitrosated amines and amides such as betaine and choline (3). Nitrates and nitrites found in food and beverages can also form methylating agents in cells (4). Hence, aberrant DNA methylation is an unavoidable consequence of metabolism and exposure to the surrounding environment (5,6). Approximately 12 different methylated DNA lesions have been identified, with the relative amount of each depending largely on the chemical nature of the reactive alkylating species. The most frequent lesions occur at the N7- or O6-positions of guanine and the N3 position of adenine (6,7). O6-Methylguanine (O6-meG) (Figure 1) is a stable DNA lesion formed by the reaction of methylating agents, including the chemotherapeutic agents procarbazine and temozolomide, with guanine (8,9). O6-MeG is highly mutagenic since it often instructs DNA polymerases to incorporate thymine instead of cytosine, resulting in GC to AT transitions (10–12). The highly mutagenic nature of O6-meG offers an explanation as to why methylating agents that produce it are carcinogenic (13). But O6-meG can also block replication (14), inducing chromosomal aberrations, sister chromatid exchange and intra-chromosomal homologous recombination (15–17). Figure 1. Structures of the four possible conformations of O6-meG. The glycosidic torsion angle (χ) defined by the atoms O4′–C1′–N9–C4 is shown in black and the torsion angle (κ) defined by the atoms N1–C6– ... O6-MeG is repaired in a non-enzymatic, stoichiometric fashion by the direct reversal protein O6-methylguanine–DNA methyltransferase (MGMT) (18). MGMT transfers the methyl group from the lesion to a cysteine residue within itself, rendering it incapable of continued repair; hence, the number of O6-meG lesions that can be cleared from a cell's genome is limited by the available MGMT molecules, unless more of the protein is made. In fact, variations in MGMT levels among cell types result in a differential ability of organs and tissues to repair O6-meG via direct reversal, leading to the accumulation of O6-meG after MGMT is depleted (19,20). Replication past O6-meG by several DNA polymerases has been well studied (7,10,14,21–24); however, little is known about its effect on transcription elongation. In Escherichia coli, RNA polymerase bypasses O6-meG with high efficiency, often misincorporating uracil opposite the lesion. The resulting alterations to the transcripts have been termed transcriptional mutagenesis (TM) (25). Here, we examined transcriptional processing of O6-meG by bacteriophage T7 RNA polymerase (T7 RNAP) and human RNA polymerase II (hRNAPII). These two polymerases differ significantly in the architecture of their active sites and behave differently when facing DNA lesions (26–28). It is shown here for the first time that O6-meG poses a minimal hurdle to transcription by T7 RNAP, but it does impede hRNAPII transcription to a significant extent. Consistent with the previous study with E. coli RNA polymerase (25), bypass of O6-meG causes significant sequence alterations in the RNA transcripts, with predominant misincorporation of uracil by both polymerases. Computer modeling was utilized to provide a structural explanation of the experimental results. These models showed that the altered Watson–Crick hydrogen-bonding edge of O6-meG (Figure 1) underlies the observed selectivity for uracil as well as cytosine incorporation during transcription past the lesion, with hydrogen-bonding possibilities between O6-meG and uracil or cytosine that have been inferred in NMR solution studies (29–32) and observed in X-ray crystal structures of O6-meG (22,33–36). In addition, the more crowded active site region of hRNAPII explained the greater blocking effect of the lesion in this polymerase.