A NOVEL KINETIC-ANALYSIS TO CALCULATE NUCLEOTIDE AFFINITY OF PROOFREADING DNA-POLYMERASES - APPLICATION TO PHI-29 DNA-POLYMERASE FIDELITY MUTANTS

Amino acids Tyr(254) and Tyr(390) of phi 29 DNA polymerase belong to o ne of the most conserved regions in eukaryotic-type DNA polymerases, I n this paper we report a mutational study of these two residues to add ress their role in nucleotide selection, This study was carried out by means of a new kinetic analysis that takes advantage of the competiti on between DNA polymerization and 3' --> 5' exonuclease activity to me asure the K-m values for correct and incorrect nucleotides in steady-s tate conditions, This method is valid for any 3' --> 5' exonucleasecon taining DNA polymerase, without any restriction concerning catalytic r ates of nucleotide incorporation. The results showed that the discrimi nation factor achieved by phi 29 DNA polymerase in the nucleotide bind ing step of DNA polymerization is 2.4 x 10(3), that is, a wrong nucleo tide is bound with a 2.4 x 10(3)-fold lower affinity than the correct one, Mutants Y254F, Y390F, and Y390S showed discrimination values of 7 .0 x 10(2), > 1.9 x 10(3), and 2.9 x 10(2), respectively. The reduced accuracy of nucleotide binding produced by mutations Y254F and Y390S l ead us to propose that phi 29 DNA polymerase residues Tyr(254) and Tyr (390), highly conserved in eukaryotic-type DNA polymerases, are involv ed in nucleotide binding selection, thus playing a crucial role in the fidelity of DNA replication. Comparison of the discrimination factors of mutants Y390S and Y390F strongly suggests that the phenyl ring of Tyr(390) is directly involved in checking base-pairing correctness of the incoming nucleotide.