Site-directed mutagenesis of recombinant rat DNA polymerase .beta.: involvement of arginine-183 in primer recognition

Authors:
  • SYSetsuko Yamamoto
    Setsuko Yamamoto
  • KTKiyomi Tanihara
    Kiyomi Tanihara
  • YNYoshio Nishimoto
    Yoshio Nishimoto
  • NLNan Liu
    Nan Liu
  • AMAkio Matsukage
    Akio Matsukage
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Abstract
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 .fwdarw. 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 trisphosphate. A more dramatic change was observed when Arg-183 was replaced by Ala, which resulted in a 99.98% reduction of enzyme activity. Although 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.

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