Characterization of the native and recombinant catalytic subunit of human DNA polymerase gamma: identification of residues critical for exonuclease activity and dideoxynucleotide sensitivity.
The human DNA polymerase gamma catalytic subunit was overexpressed in recombinant baculovirus-infected insect cells, and the 136 000 Da protein was purified to homogeneity. Application of the same purification protocol to HeLa mitochondrial lysates permitted isolation of native DNA polymerase gamma as a single subunit, allowing direct comparison of the native and recombinant enzymes without interference of other polypeptides. Both forms exhibited identical properties, and the DNA polymerase and 3' --> 5' exonuclease activities were shown unambiguously to reside in the catalytic polypeptide. The salt sensitivity and moderate processivity of the isolated catalytic subunit suggest other factors could be required to restore the salt tolerance and highly processive DNA synthesis typical of gamma polymerases. To facilitate our understanding of mitochondrial DNA replication and mutagenesis as well as cytotoxicity mediated by antiviral nucleotide analogues, we also constructed two site-directed mutant proteins of the human DNA polymerase gamma. Substituting alanine for two essential acidic residues in the exonuclease motif selectively eliminated the 3' --> 5' exonucleolytic function of the purified mutant polymerase gamma. Replacement of a tyrosine residue critical for sugar recognition with phenylalanine in polymerase motif B reduced dideoxynucleotide inhibition by a factor of 5000 with only minor effects on overall polymerase function.
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
M J
MJ
Longley
P A
PA
Ropp
S E
SE
Lim
W C
WC
Copeland
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D002384:Catalysis; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005092:Exonucleases / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D013379:Substrate Specificity / Q000235:genetics
D000596:Amino Acids; D003854:Deoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases