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

Genet Anal. 1998 Jan;14(3):75-83. doi: 10.1016/s1050-3862(97)10002-x.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Chlorobi / enzymology
  • Chlorobi / genetics*
  • Cloning, Molecular
  • DNA Polymerase I / genetics*
  • DNA Polymerase I / metabolism
  • Escherichia coli / genetics*
  • Exodeoxyribonucleases / metabolism
  • Gene Expression
  • Genes, Bacterial / genetics*
  • Molecular Sequence Data
  • Phylogeny
  • Recombinant Fusion Proteins
  • Sequence Analysis, DNA
  • Sequence Homology, Amino Acid

Substances

  • Recombinant Fusion Proteins
  • DNA Polymerase I
  • Exodeoxyribonucleases

Associated data

  • GENBANK/Y12328