Site-directed mutagenesis of recombinant rat DNA polymerase beta: involvement of arginine-183 in primer recognition.
By site-directed mutagenesis using synthetic oligonucleotides, amino acid residues 181Phe-Arg-Arg183 of recombinant rat DNA polymerase beta were replaced by other amino acids to clarify the roles of these residues in the DNA synthesizing reaction. Replacement of Phe-181 by alanine reduced the enzyme activity only 30%. Replacement of Arg-182 by alanine and glutamine resulted in reduction of the activity by about 67% and 95%, respectively. The Arg-182----Gln replacement increased the binding strength to single-stranded DNA but did not significantly change the Km's for the primer and dTTP, suggesting that Arg-182 is involved in modulation of binding to the template rather than to the primer or deoxyribonucleoside triphosphate. Replacement of Arg-183 by Gln resulted in reduction of the activity by about 95%, and this change, although causing little change in binding strength to single-stranded DNA, resulted in a 3-4-fold increase in the Km's for the primer and deoxyribonucleoside triphosphate. A more dramatic change was observed when Arg-183 was replaced by Ala, which resulted in a 99.98% reduction of enzyme activity. Although the Km for deoxyribonucleoside triphosphate of this mutant enzyme was hardly changed, that for the primer increased 159-fold. Therefore, it is concluded that Arg-183 occupies an important part of the primer recognition site of DNA polymerase beta.
Date
T
T
Department of Biochemistry, Kanazawa Medical University, Ishikawa, Japan.
Yamamoto
S
S
Tanihara
K
K
Nishimoto
Y
Y
Liu
N
N
Matsukage
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D004256:DNA Polymerase I / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009694:Nucleic Acid Precursors / Q000235:genetics; D009841:Oligonucleotides / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D009694:Nucleic Acid Precursors; D009841:Oligonucleotides; D011994:Recombinant Proteins; D001120:Arginine; D004256:DNA Polymerase I