A DNA polymerase III holoenzyme-like subassembly from an extreme thermophilic eubacterium.
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 alpha 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 gamma and tau 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 gamma 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.
McHenry
C S
CS
Department of Biochemistry and Molecular Genetics and the Molecular Biology Program, University of Colorado Health Sciences Center, Denver CO 80262, USA.
Seville
M
M
Cull
M G
MG
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003067:Coenzymes / Q000235:genetics / Q000302:isolation & purification*; D003429:Cross Reactions; D004258:DNA Polymerase III / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D018965:Frameshifting, Ribosomal; D005798:Genes, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D017421:Sequence Analysis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics / Q000276:immunology
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D003067:Coenzymes; C103076:DnaX protein, Bacteria; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III