The naked truth about eukaryotic DNA polymerase errors

Abstract

The frequency of accumuhted spontaneous mutations in animal cells is of the order of 10 -o errors or less per base pair replicated 1. This remarkably low mutation rate represents the balance of unrepaired errors occurring during several aspects of DNA metabolism, including replication, repair, recombination and transposition. Each such process involves the copying of a nucleotide sequence template by a DNA pelymerase. The accuracy or fidelity of this template-copying process by individual DNA polymerase enzymes makes a key contribution to the overall spontaneous mutation rate. Up to 1982, two methods bad been used to assay the fidelity of DNA synthesis in vitro t. The earliest procedures were based on the misincorporation of nucleotides by DNA polymerases acting on synthetic polynucleotide templates of defined base composition (one or two bases represented). Later, measurements were made of the frequency of reversion of specific amber mutations in q>X174 DNA, a natural template, applying one of several strategies x for DNA synthesis, each followed by transfeedon of the product phage DNA into permissive and non-permissive bacteria. The method using ~X174 DNA can detect error frequencies as low as 10 -6 , but the reversion events scored can occur only through a limited subset of base substitutions. In order to provide a much more detailed picture of the nature and scope of errors made by eukaryotic DNA polymerases, Kankel z has developed an approach based on the copying by DNA polymerases in vitro of a target 250-base single-stranded DNA segment of the bacteriophage de.fivafive Ml3mp2. A ~ppod doublestranded circle is prepared in which the target DNA is exposed as a singiestranded region, coding for the ~peptide of ~gMactosidase. Thus, the erroneous copying of this nucleotide sequence can be readily detected by transfecting the M13mp2 DNA molecules producedintoE, coliandsUd~ng the resultant phage plaques with the chromogenic substrate X-gal. While ondamaged M13mp2 molecules yield dark blue plaques, phage DNA carrying mutations in the target sequence show characteristic hues ranging from light blue to colourless, depending on the introduced mutation. Moreover, the individual mutated target DNA segments can be readily sequenced. From the sequence analysis of a large number of mutants a detailed error profile can be established for a given DNA polymerase acting under defined conditions, in terms of the spoctnun of mutations that are engendered and the frequencies of individual events or classes of events. An on t l~ of the technique is given in Fig. 1. In a series of papers published over the past two years z-~, Kankel has presented complete descriptions of the error profiles of three DNA polymerases (~, ~ andy) from various higher eukaryotes (mammals and birds) acting on the Ml3mp2 singlesU-anded DNA target sequence. (Pnl-~ is considered to be the replicative enzyme acing on chromosomal DNA, POl-p is a nuclear repair enzyme, whilst pol-¥ is the enzyme involved in replication of mitochondrial DNA). The resolution of this system (summarized in Ref. 6) is enormously improved over that previously obtainable with ~Xl?4 amber-reversion assays. It became pos~le to identify the propensity of each enzyme to commit particnlar base-substitution ergots (Le. those leading to a detectable mutant phenotype) at a large number of individual nudeotidea in the target sequence. Moreover, the data indicated the frequency of single base deletions at particular locations in the target sequence and revealed the nature and frequency of more extensive deletions in the newly synthesized DN~ Some implications of the new body of data in terms of the activities of mammalian DNA poly-