Regulation of DNA polymerase exonucleolytic proofreading activity: studies of bacteriophage T4 "antimutator" DNA polymerases.

DNA polymerases replicate DNA with high fidelity lab, which demonstrated that single point mutations in the T4 DNA polymerase gene can increase or decrease because of accurate nucleotide incorporation coupled with exonucleolytic proofreading to remove misinmutation rates by 100-fold or more (Drake et al. 1969). Mutational analysis is a powerful method to probe corporated nucleotides. This statement is taken for granted today, in large part, because of groundbreaking enzyme function. Even today with the ready availability of molecular techniques, genetic screens and selections discoveries made 30 years ago that mutations in the DNA polymerase gene of bacteriophage T4 can have are useful because informative mutant enzymes can be identified without structural information or assumpdramatic effects on the fidelity of DNA replication. “Mutator” DNA polymerases (Speyer et al. 1966) are mutant tions about function. Most importantly, since classical genetic methods rely on detection and characterization of mutant DNA polymerases that replicate DNA with less accuracy than the wild-type enzyme, and “antimutator” DNA polyphenotypes in vivo, alterations in function are observed within the context of the living organism. In the case of antimutator merases (Drake and Allen 1968; Drake et al. 1969) are mutant DNA polymerases that replicate DNA with DNA polymerases, these mutants provide a handle to probe DNA polymerase function in vivo. more accuracy, at least for certain types of DNA replicaGenetic and biochemical techniques have continued tion errors. Biochemical characterization revealed that to be used in studies of antimutator DNA polymerases. the balance between the two DNA polymerase functions, This research has led in a number of different directions nucleotide incorporation and the opposing 39→59exonuincluding studies of the evolution of spontaneous muclease activity, was altered in the mutants compared to tation rates (Drake 1990, 1991a, 1991b), the effect of the wild type enzyme. Decreased 39→59exonuclease activantimutator DNA polymerases on frameshift mutageneity was detected for many of the mutator DNA polymerases sis (Kaiser and Ripley 1995), the identification of anti(Muzyczka et al. 1972) while increased 39→59exonuclease mutator DNA polymerases in other organisms (Fijalactivity, relative to polymerase activity, was observed for kowska et al. 1993), and additional studies of DNA the antimutator DNA polymerases (Muzyczka et al. 1972; polymerase function (Clayton et al. 1979; Spacciapoli Gillin and Nossal 1976a,b). The correlation of deand Nossal 1994a,b; Reha-Krantz and Nonay 1994; creased 39→59 exonuclease activity measured in in vitro Reha-Krantz and Wong 1996). The focus of this short assays with decreased accuracy of DNA replication in vivo, review is the regulation of DNA polymerase proofreadand increased 39→59 exonuclease activity with increased ing activity (Stocki et al. 1995). Other aspects of T4 DNA replication fidelity indicates that the 39→59 exoantimutator DNA polymerases are presented in this volnuclease activity of T4 DNA polymerase is an exonucleoume (e.g., Nossal, Wang and Ripley). lytic proofreading activity. T4 antimutator DNA polymerases: Mutations within These observations were made at an exciting time in genes result typically in mutant enzymes with reduced DNA polymerase research. I was fortunate then to be a activity and even loss of function; however, in the case graduate student in Maurice Bessman’s lab at Johns of antimutator DNA polymerases, the mutant enzymes Hopkins University and to see first hand the biochemiappear to be working better, at least with respect to the cal characterization of T4 mutator and antimutator accuracy of DNA replication. Since single point mutaDNA polymerases. But of equal importance was the options can give rise to antimutator DNA polymerases, why portunity to meet Jan Drake and to learn about the have more accurate DNA polymerases not evolved? use of genetics to study the fidelity of DNA replication. Part of the answer must be that there are negative The juxtaposition of biochemistry and genetic analyses consequences of the antimutator phenotype that reduce to elucidate the proofreading role for the 39→59exonuclethe efficiency of the DNA polymerase and the overall ase activity of T4 DNA polymerase made a strong impact fitness of the organism. There is a cost associated with on my graduate studies and continues to do so today. I DNA polymerase proofreading (Fersht et al. 1982). For am still intrigued by the genetic analyses from the Drake example, if T4 antimutator DNA polymerases replicate DNA more slowly than the wild-type enzyme, then fewer phage progeny may be produced. Higher concentraAddress for correspondence: Department of Biological Sciences, CW405 tions of deoxynucleoside triphosphates (dNTPs) are reBioSciences Bldg., University of Alberta, Edmonton, Alberta T6G 2E9, Canada. E-mail: [email protected] quired to support DNA replication by T4 antimutator