Mutational specificity of animal cell DNA polymerases

Since DNA polymerases are involved in DNA replication, recombination, and repair, the frequency with which these enzymes commit errors during synthesis is likely to be an important factor in controlling mutation rates in cells. The fidelity of DNA polymerases was originally studied by following misincorporation using synthetic nucleic acid templates containing only one or two bases. Later, by assaying for reversion of an amber codon after copying phi X174 single-stranded DNA molecules, the base substitution accuracy of in vitro DNA synthesis on natural DNA was determined. Most recently, a forward mutation assay has been developed that uses gap-filling synthesis on an M13mp2 DNA template, thus permitting the detection of a variety of different errors during DNA synthesis on natural DNA templates. Detailed mutational spectra for animal cell polymerases-alpha, beta, and gamma have been determined and demonstrate that a variety of errors can be generated by these purified enzymes. The frequencies of base mispairs, base additions, and deletion errors by DNA polymerases vary widely and depend on both the DNA sequence and the enzyme used. An understanding of the mechanisms by which DNA polymerases avoid or generate various mutations depends on the definition of the parameters that influence the frequency and specificity of particular errors. Future experiments will combine the use of the methods available to measure fidelity with advances in DNA replication enzymology and should lead to exciting new insights into the mechanisms of spontaneous mutagenesis.