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The biological consequences of O 6 -methylguanine (m6G) in DNA are well recognized. When template m6G is encountered by DNA polymerases, replication is hindered and trans-lesion replication results in the preferential incorporation of dTMP opposite template m6G. Thus, unrepaired m6G in DNA is both cytotoxic and mutagenic. Yet, cell lines tolerant to m6G in DNA have been isolated, which indicates that some cellular DNA polymerases may replicate m6G-containing DNA with reasonable efficiency. Previous reports suggested that mammalian pol ␤ could not replicate m6G-containing DNA, but we find that pol ␤ can catalyze trans-lesion replication; however, the lesion must reside in the optimal context for pol ␤ activity, single-or short nucleotide gapped substrates. Primed single-stranded DNA templates, with or without template m6G, were poor substrates for pol ␤ as reported in earlier studies. In contrast, trans-lesion replication by bacteriophage T4 DNA polymerase was observed for primed single-stranded DNA templates. Replication of m6G-containing DNA by T4 DNA polymerase required the gp45 accessory protein that clamps the polymerase to the DNA template. The rate-limiting step in replicating m6G-containing DNAs by both DNA polymerases tested was incorporation of dTMP across from the lesion. -DNA methyltransferase repairs m6G residues in DNA; however, 20 -30% of human solid tumor cell lines do not express this repair activity (1). Exposure of cells lacking m6G-DNA methyltransferase to alkylating agents such as MNNG results in high levels of mutations, sister chromatid exchanges, and cell death (reviewed in Ref. 2). Yet, cells unable to repair the m6G damage but tolerant to the killing activities of MNNG have been isolated (reviewed in Refs. 2, 3). As these tolerant cells remain sensitive to the mutagenic effects of alkylating agents, questions about the replication of m6G-containing DNA arise.DNA polymerases are predicted to encounter m6G in three DNA environments: 1) in single-or short nucleotide gaps formed during short patch DNA repair, 2) in lengthy singlestranded regions formed by long patch repair, and 3) at replication forks. Single-nucleotide gaps may be produced by short patch mismatch repair activity that is normally directed to the repair of G:T mispairs that are the result of 5-methylcytosine (5mC) deamination (4). The enzyme that initiates this repair process is a DNA G:T mismatch-specific thymine-DNA glycosylase that removes the mispaired thymine from G:T DNA to generate an apyrimidinic site (5). A G:T thymine-DNA glycosylase also initiates the removal of thymine from m6G:T base pairs (6). The abasic site is further processed to generate a single-nucleotide gap across from guanine for G:T mismatches and across from m6G for m6G:T mismatches. While pol ␤ can efficiently fill in single-nucleotide and small gaps across from undamaged DNA templates (7), pol ␤ activity on single-nucleotide gaps across from m6G has not been reported. Pol ␤ replication is blocked, however, by template m6G in long singlestranded DNA ...

Classical genetic selection was combined with site-directed mutagenesis to study bacteriophage T4 DNA polymerase 3' -S 5' exonuclease activity. A mutant DNA polymerase with very little (<1%) 3' -*5' exonuclease activity was generated. In vivo, the 3' -*5' exonuclease-deficient DNA polymerase produced the highest level ofspontaneous mutation observed in T4, 500-to 1800-fold above that of wild type. The large reduction in 3' -* 5' exonuclease activity appears to be due to two amino acid substitutions: clease motif. Therefore, extrapolation from E. coli DNA polymerase I sequence and structure to other DNA polymerases for which there is no structural information may not be valid.Bacteriophage T4 DNA polymerase is one of the best experimental systems for studying the role of DNA polymerase exonucleolytic proofreading in enhancing DNA replication fidelity (1-10). Wild-type T4 DNA polymerase has a potent 3' 5' exonuclease activity (11), which is important for accurate DNA replication. Mutant DNA polymerases with reduced 3' -* 5' exonuclease activity produce more DNA replication errors (mutator phenotype), whereas mutants with elevated 3' 5' exonuclease activity, relative to polymerizing activity, increase DNA replication accuracy (antimutator phenotype) (3). The mutator phenotype was used to select mutant DNA polymerases with reduced 3' -* 5' exonuclease activity; amino acid substitutions in the mutants were clustered'between T4 DNA polymerase residues 255 and 363 (8)(9)(10)12). Although amino acid changes within this region decreased 3' -* 5' exonuclease activity, the' mutant DNA polymerases still retained significant residual proofreading activity, which suggests that these particular residues do not function catalytically.In the case of Escherichia coli DNA polymerase I (pol I), residues essential for 3' 5' exonuclease activity have been identified. They include four metal ion binding residues; The hypothesis drawn from these sequence comparisons was that many eukaryotic, viral, and bacteriophage DNA polymerases have a conserved 3' -* 5' exonuclease domain similar to that of E. coli pol 1. T4 genetic studies are consistent with this proposal because mutations that reduce 3' -* 5' exonuclease activity are located near the proposed conserved metal ion binding residues. The hypothesis was tested directly in phage 429'DNA polymerase by substituting alanine residues for proposed conserved metal ion binding residues, which resulted in a 1000-fold reduction in 3' -* 5' exonuclease activity without affecting polymerization activity (23). The 429 DNA polymerase result compares favorably with the 105-fold reduction observed when alanine residues were substituted for E. coli pol I residues .We present here in vitro mutagenesis and genetic studies that were designed to test if proposed T4 DNA' polymerase metal ion binding residues, identified on the basis of sequence similarities to those of E. coli pol l (8, 23), are required for 3' 5' exonuclease activity. In 'contrast to the 429 DNA polymerase'studies, alanine subs...

Mutations that conferred sensitivity to the pyrophosphate analog phosphonoacetic acid in bacteriophage T4 DNA polymerase were identified. The mutations were loosely clustered in four regions of the gene. As found for herpes simplex virus DNA polymerase, T4 mutations that altered sensitivity to phosphonoacetic acid also altered sensitivity to nucleotide analogs. Some of the T4 DNA polymerase mutations also altered the ability of the enzyme to translocate from one template position to the next and affected DNA replication fidelity.

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