Molecular cloning of a gene (polA) coding for an unusual DNA polymerase I from Treponema pallidum

The gene coding for the DNA polymerase I from Treponema pallidum, Nichols s train, was cloned and sequenced. Depending on which of the two alternative initiation codons was used, the protein was either 997 or 1015 amino acids long and the predicted protein had a molecular mass of either 112 or 114 kD a, Sequence comparisons with other polA genes showed that all three domains expected in the DNA polymerase I class of enzymes were present in the prot ein (5'-3' exonuclease, 3'-5' exonuclease and polymerase domains). Addition ally, there were four unique insertions of 20-30 amino acids each, not seen ill other DNA polymerase I enzymes. Two of the inserts were near the bound ary of the two exonuclease domains and the other two interrupted the 3'-5' exonuclease domain which is involved in proofreading. The predicted amino-a cid sequence had an exceptionally high content of cysteine (2.4% compared w ith <0.05% for most other sequenced DNA polymerase I enzymes). The polA gen e was further cloned into pProEX(TM)HTa for expression and purification, Th e transformants expressed a protein of 115 kDa, Antibodies raised against s ynthetic peptide fragments of the putative DNA polymerase I recognised the 115-kda band in Western blot analysis. No DNA synthesis activity could be d emonstrated on a primed single-stranded template. Although significant quan tities of the protein were produced in the host Escherichia coli carrying t he plasmid, it was not capable of complementing a polA(-) mutant in the rep lication of a polA-dependent plasmid.