Family C DNA Polymerase (DNA pol III) Gene of Thermus aquaticus ATCC 25104
Yiping HUANG* and Junetsu ITO
Department of Microbiology and Immunology, College of Medicine, University of Arizona, 1501N. Campbell Ave. Tucson, Arizona 85724, USA
Substantial evidence indicates that the deepest branches in molecular evolutionary treesare occupied by thermophilic or hyper-thermophilic eubacteria and archaea (1). This evidence, together with geological studies suggesting that the hydrosphere was far hotter 3 billion years ago than now (2), strongly implies a hot origin of life on earth. It has been suggested that thermophilic species are the most slowly evolving of the eubacteria and that slowly evolving bacterial lineages tend to retain ancestral characteristics (3). Accordingly, studies with thermophilic organisms may provide useful information not only about the mechanism of thermophilicity of proteins, but also about the early evolution of life on earth.
We have initiated studies of the DNA replication machinery of thermophilic microorganisms. DNA polymerases have been classified globally into four families (A, B, C and X), based on amino acid sequence similarities (4). It is known, based on complete genome sequence analyses of M. jannaschii (archaea) and S. cerevisiae (eukarya) that both archaea and eukarya use family B DNA polymerase for their chromosomal DNA replication. On the other hand, contemporary eubacteria, E. coli, H. influenza, B. subtilis and Cyanobacteria for example, utilize family C DNApolymerase for their chromosomal DNA replication.
We have searched for me family B DNA polymerase in the thermophilic bacterium T. aquatics using PCR technology. The results were clearly negative indicating that T. aquaticus does not have a family B DNA polymerase gene. Instead, we found that T. aquaticus has a family C DNA polymerase gene. Nucleotide sequence analysis shows that this polymerase has a high G+C base composition (66.7%). The amino acid sequence analysis of T. aquaticus DNA polymerase C revealed significant sequence homology with the alpha subunit of Gram-negative E. coli DNA polymerase III (dna E) (62.6%) (5), but has only partial homology with Grampositive B. subtilis DNA polymerase C (family C). Therefore, the T. aquaticus DNA polymerase C appears to be of the Gram-negative type. Surprisingly, however, DNA polymerase C from the high G+C Gram-positive bacteria, such as Micrococcus luteus, Streptomyces griseus and Mycobacteruim tuberculosis, are also highly homologous to the dna E protein of E. coli (unpublished). Evolution of DNA polymerases and a new classification of the family C DNA polymerases will be
