The conserved active site motif A of Escherichia coli DNA polymerase I is highly mutable.
Escherichia coli DNA polymerase I participates in DNA replication, DNA repair, and genetic recombination; it is the most extensively studied of all DNA polymerases. Motif A in the polymerase active site has a required role in catalysis and is highly conserved. To assess the tolerance of motif A for amino acid substitutions, we determined the mutability of the 13 constituent amino acids Val(700)-Arg(712) by using random mutagenesis and genetic selection. We observed that every residue except the catalytically essential Asp(705) can be mutated while allowing bacterial growth and preserving wild-type DNA polymerase activity. Hence, the primary structure of motif A is plastic. We present evidence that mutability of motif A has been conserved during evolution, supporting the premise that the tolerance for mutation is adaptive. In addition, our work allows identification of refinements in catalytic function that may contribute to preservation of the wild-type motif A sequence. As an example, we established that the naturally occurring Ile(709) has a previously undocumented role in supporting sugar discrimination.
Shinkai
A
A
Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle, Washington 98195-7705, USA.
Patel
P H
PH
Loeb
L A
LA
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D004256:DNA Polymerase I / Q000737:chemistry* / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D019143:Evolution, Molecular; D007532:Isoleucine / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012313:RNA / Q000378:metabolism; D017422:Sequence Analysis, DNA
D007532:Isoleucine; D001224:Aspartic Acid; D012313:RNA; D004256:DNA Polymerase I