Cloning, sequence analysis and expression in E. coli of the DNA polymerase I gene from Chloroflexus aurantiacus, a green nonsulfur eubacterium.

Abstract:

We have cloned and sequenced the polA gene from Chloroflexus aurantiacus, a green nonsulfur eubacterium, and expressed the recombinant protein in Escherichia coli. One open reading frame encodes a protein with 942 amino acids showing 38% identity with DNA polymerase I from E. coli. Sequence alignments with other members of DNA polymerase family A and analysis of the separate domains show that the central 3'-5' exonuclease domain is 30% identical to the corresponding E. coli domain and that three sequence motifs associated with 3'-5' exonuclease activity are conserved. Also, a protein fraction from E. coli expressing the Chloroflexus polymerase contains a thermostable 3'-5' exonucleolytic activity, indicating that this activity is present in the enzyme, in agreement with the sequence analysis. The N-terminal 5'-3' exonuclease domain and the C-terminal polymerase domain show 31 and 46% identity, respectively, with the corresponding E. coli domains and all sequence motifs associated with these two enzymatic activities also are conserved. Since several DNA polymerase I enzymes lack the proofreading activity associated with the central domain it has been suggested that the ancestral polA gene contained only the two more conserved N- and C-terminal domains and that the proofreading 3'-5' exonuclease domain was introduced later in those eubacterial branches that have this activity. Our data indicate a different scenario where the ancestral polA gene contained both the exonucleolytic activities in addition to the polymerase activity and where several eubacterial branches lost the polymerase-associated proofreading activity during evolution.

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