Enzymatic properties of rat DNA polymerase beta mutants obtained by randomized mutagenesis.


We have used random sequence mutagenesis to generate mutants of DNA ...
We have used random sequence mutagenesis to generate mutants of DNA polymerase beta in an effort to identify amino acid residues important for function, catalytic efficiency and fidelity of replication. A library containing 100 000 mutants at residues 274-278 in the N-helix of the thumb subdomain of the polymerase was constructed and screened for polymerase activity by genetic complementation. The genetic screen identified 4000 active pol beta mutants, 146 of which were sequenced. Each of the five positions mutagenized tolerated substitutions, but residues G274 and F278 were only found substituted in combination with mutations at other positions. The least conserved residue, D276, was replaced by a variety of amino acids and, therefore, does not appear to be essential for function. Steady-state kinetic analysis, however, demonstrated that D276 may be important for catalytic efficiency. Mutant D276E exhibited a 25-fold increase in catalytic efficiency over the wild-type enzyme but also a 25-fold increase in G:T misincorporation efficiency. We present a structural model that can account for the observations and we discuss the implications of this study for the question of enzyme optimization by natural selection.




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