A DNA polymerase III holoenzyme-like subassembly from an extreme thermophilic eubacterium

• Published in: Journal of molecular biology 1997-09, Vol.272 (2), p.178-189
• Main Authors: McHenry, Charles S, Seville, Mark, Cull, Millard G
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Antibodies, Bacterial; Antibodies, Monoclonal; Bacterial Proteins - genetics; Base Sequence; Cloning, Molecular; Coenzymes - genetics; Coenzymes - isolation & purification; Cross Reactions; DNA polymerase; DNA Polymerase III - analysis; DNA Polymerase III - genetics; DNA Polymerase III - isolation & purification; DNA replication; Frameshifting, Ribosomal; Genes, Bacterial - genetics; holoenzyme; Molecular Sequence Data; Sequence Analysis; Sequence Analysis, DNA; Sequence Homology, Amino Acid; T. thermophilus; thermophile; Thermus thermophilus - enzymology; Thermus thermophilus - genetics; Thermus thermophilus - immunology
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.1997.1238

We have purified a novel DNA polymerase from Thermus thermophilus. This was enabled by use of general gap filling assays to monitor polymerase activity and cross-reactive monoclonal antibodies against the α catalytic subunit of E. coli DNA polymerase III holoenzyme to distinguish a novel polymerase from the well characterized DNA polymerase I-like Thermus thermophilus DNA polymerase. Two proteins migrating with the polymerase after three chromatographic steps were isolated and subjected to partial amino acid sequencing. The amino termini of both were homologous to the two products of the E. coli dnaX gene, the γ and τ subunits of the DNA polymerase III holoenzyme. Using this information and sequences conserved among dnaX-like genes, we isolated a gene fragment by PCR and used it as a probe to isolate the full length Thermus thermophilus dnaX gene. The deduced amino acid sequence is highly homologous to the DnaX proteins of other bacteria. Examination of the sequence permitted identification of a frameshift site similar to the one used in E. coli to direct the synthesis of the shorter γ DnaX-gene product. Based on this information, we conclude that a conventional replicase exists in extreme thermophilic eubacteria. The general biological and practical technological implications of this finding are discussed.