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Nucleic Acids Research, 2001, Vol. 29, No. 11 2418-2426
© 2001 Oxford University Press

Enzymatic properties of rat DNA polymerase ß mutants obtained by randomized mutagenesis

Adonis Skandalis* and Lawrence A. Loeb1

The Department of Biological Sciences, Brock University, 500 Glenridge Avenue, St Catharines, Ontario L2S 3A1, Canada and 1The Joseph Gottstein Memorial Cancer Research Laboratory, Departments of Pathology and Biochemistry, University of Washington School of Medicine, Box 357705, Seattle, WA 98195-7705, USA

We have used random sequence mutagenesis to generate mutants of DNA polymerase ß 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 ß 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.

* To whom correspondence should be addressed. Tel: +1 905 688 5550; Fax: +1 905 688 1855; Email: askandal{at}spartan.ac.brocku.ca


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