A thumb subdomain mutant of the large fragment of Escherichia coli DNA polymerase I with reduced DNA binding affinity, processivity, and frameshift fidelity.
In Klenow fragment DNA polymerase, a flexible 50-amino acid subdomain at the tip of the thumb which includes two alpha helices has been suggested to interact with the duplex template-primer (Beese, L.S., Derbyshire, V. and Steitz, T.A. (1993) Science 260, 352-355). The present study investigates the properties of Klenow polymerase containing a 24-amino acid deletion (residues 590-613) that removes a portion of the tip of the thumb. The mutant polymerase has relatively normal dNTP binding and catalytic rate. However, its DNA binding affinity is reduced by more than 100-fold relative to the intact polymerase and its ability to conduct processive synthesis is also reduced. Although the mutant polymerase has relatively normal base substitution fidelity, it has strongly reduced frameshift fidelity, being especially error-prone for single nucleotide addition errors in homopolymeric runs. The addition error rate increases as the length of the reiterated sequence increases, indicative of errors initiated by template-primer strand slippage. These observations suggest a role for the tip of the thumb of Klenow polymerase in determining DNA binding, processivity and frameshift fidelity, perhaps by tracking the minor groove of the duplex DNA. The results are discussed in light of remarkably similar observations with T7 DNA polymerase in the presence or absence of thioredoxin, an accessory subunit that affects these same properties.
Minnick
D T
DT
Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Astatke
M
M
Joyce
C M
CM
Kunkel
T A
TA
D001483:Base Sequence; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D004274:DNA, Recombinant / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D016368:Frameshift Mutation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011499:Protein Processing, Post-Translational
D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D004256:DNA Polymerase I