Cloning, sequence analysis and expression in E. coli of the DNA polymerase I gene from Chloroflexus aurantiacus, a green nonsulfur eubacterium.
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.
Tvermyr
M
M
Department of Biochemistry, University of Oslo, Norway.
Kristiansen
B E
BE
Kristensen
T
T
D000595:Amino Acid Sequence; D019414:Chlorobi / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D004256:DNA Polymerase I / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D011993:Recombinant Fusion Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D011993:Recombinant Fusion Proteins; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases