The Fidelity of Human DNA Polymerase  with and without Exonucleolytic Proofreading and the p55 Accessory Subunit* Received for publication, June 6, 2001, and in revised form, July 30, 2001 Published, JBC Papers in Press, August 14, 2001, DOI 10.1074/jbc.M105230200 Matthew J. Longley, Dinh Nguyen, Thomas A. Kunkel‡, and William C. Copeland§ From the ‡Laboratory of Molecular Genetics and the Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709 Mutations in human mitochondrial DNA influence ag- ing, induce severe neuromuscular pathologies, cause maternally inherited metabolic diseases, and suppress apoptosis. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase  (pol ), we investigated the fidelity of DNA synthesis by hu- man pol . Comparison of the wild-type 140-kDa cata- lytic subunit to its exonuclease-deficient derivative in- dicates pol  has high base substitution fidelity that results from high nucleotide selectivity and exonucleo- lytic proofreading. pol  is also relatively accurate for single-base additions and deletions in non-iterated and short repetitive sequences. However, when copying ho- mopolymeric sequences longer than four nucleotides, pol  has low frameshift fidelity and also generates base substitutions inferred to result from a primer disloca- tion mechanism. The ability of pol  both to make and to proofread dislocation intermediates is the first such ev- idence for a family A polymerase. Including the p55 accessory subunit, which confers processivity to the pol  catalytic subunit, decreases frameshift and base sub- stitution fidelity. Kinetic analyses indicate that p55 pro- motes extension of mismatched termini to lower the fidelity. These data suggest that homopolymeric runs in mitochondrial DNA may be particularly prone to frame- shift mutation in vivo due to replication errors by pol . The human mitochondrial genome (mtDNA) 1 encodes 37 genes required for oxidative phosphorylation or mitochondrial protein synthesis (1). Loss of these essential gene functions clearly induces a multitude of severe metabolic disorders, and mutation of mtDNA is the cause of inheritable mitochondrial diseases (2– 4). Early reports comparing nucleotide substitu- tions in mtDNA from somatic tissues of different primates revealed a 10-fold higher rate of evolution for mtDNA relative to the nuclear genome (5, 6), implying a relatively high muta- tion rate for mtDNA. More recently, the accumulation of dele- tions in mtDNA has been shown to correlate with increasing age (7), and a current longitudinal study strongly supports the age-dependent accumulation of non-inherited point mutations in human mtDNA (8). Recent data suggest that mutations in mtDNA can suppress apoptosis, a situation that would favor the growth of tumor cells (9). Additionally, human somatic cancer cells can acquire a homoplasmic mutant mtDNA geno- type, presumably by mitotic segregation of mutant mitochon- dria during proliferation of tumors (10). The prevalence of mtDNA mutations in a variety of human cancers may be more than a passive association (11). Thilly and co-workers (12, 13) have developed sensitive methods to examine the spectrum of mtDNA mutations that form in human cells in vivo. Molecular genetic analyses such as these are the starting point for study- ing the biochemical mechanisms of mutagenesis of the mito- chondrial genome. Mutations in mtDNA arise from several sources, all of which involve DNA synthesis by the mitochondrial DNA polymerase, pol . Spontaneous replication errors produce mismatches, and replication through unrepaired mismatches can mutate mtDNA. Although Saccharomyces cerevisiae possesses the mis- match repair homolog Msh1 that can stabilize yeast mtDNA (14, 15), evidence for mitochondrial mismatch repair in higher eukaryotes is currently lacking (16). mtDNA chemically dam- aged by hydrolysis, reactive oxygen species, or environmental mutagens contains non-coding or mis-coding lesions (17, 18). Evidence for base excision repair of damaged mtDNA is abun- dant (17, 19 –25), and pol  has a well established role in mitochondrial base excision repair in vitro (26 –28). Replication of DNA templates damaged by platinum adducts may also lead to mutations in mtDNA (29). Because pol  is a component common to each mode of mutagenesis, knowledge of its biosyn- thetic fidelity is critical to understanding mitochondrial mutagenesis. pol  purified from chicken embryos or from pig liver mito- chondria is accurate in vitro, with these enzymes exhibiting error frequencies at a 3-nucleotide mutational target of 3.8  10 -6 per nucleotide and 2.0  10 -6 per nucleotide, respec- tively (30, 31). Both enzymes contain intrinsic 3'- to 5'-exonu- clease activities that prefer mispaired 3' termini. Partial inhi- bition of these exonuclease activities with 20 mM dGMP increases the frequency of errors, suggesting the exonucleases proofread replication errors (30, 31). pol  derived from chicken, pig, Drosophila melanogaster, Xenopus laevis, Saccha- romyces cerevisiae, and human sources copurifies with 3'- to 5'-exonuclease activity (30 –37), and the genes for all known mitochondrial DNA polymerases possess three highly con- served exonuclease motifs common to family A DNA polym- erases (38 – 41). Several lines of evidence show the exonuclease contributes to replication fidelity in vivo. Disruption of the exonuclease motifs in the yeast MIP1 gene generates a mutator phenotype, as exhibited by a several hundred-fold increase in the spontaneous frequency of forming mitochondrial erythro- mycin-resistant mutants (42). Expression of exonuclease-defi- cient pol  fusion proteins in cultured human cells also resulted in the accumulation of point mutations in mitochondrial DNA (43). Also, the loss of exonuclease function of pol  in transgenic mice resulted in the rapid accumulation of point mutations and * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § To whom correspondence should be addressed. Tel.: 919-541-4792; Fax: 919-541-7613; E-mail: copelan1@niehs.nih.gov. 1 The abbreviations used are: mtDNA, mitochondrial DNA; pol , DNA polymerase ; BSA, bovine serum albumin; Exo, exonuclease. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 42, Issue of October 19, pp. 38555–38562, 2001 Printed in U.S.A. This paper is available on line at http://www.jbc.org 38555 by guest on May 20, 2020 http://www.jbc.org/ Downloaded from