Mutational analysis of phi29 DNA polymerase residues acting as ssDNA ligands for 3'-5' exonucleolysis.

Abstract:

Here, three highly conserved amino acid residues have been characterized to function as ssDNA binding ligands at the 3'-5' exonuclease active site of phi29 DNA polymerase. One of these residues, Phe65, belongs to motif Exo II, previously described to contain an invariant aspartate and an invariant asparagine involved in catalysis and ssDNA binding, respectively. The other two residues, Ser122 and Leu123, form a newly identified motif "(S/T)Lx2h", and are the homologous counterparts of Pol I residues Asp457 and Met458, and of T4 DNA polymerase residues Ser286 and Leu287, the latter three residues shown to contact ssDNA at their corresponding cocrystal 3D structures. Site-directed mutagenesis and biochemical analysis of eight phi29 DNA polymerase mutant proteins at residues Phe65, Ser122 and Leu123 indicated their functional importance for: (1) a stable interaction with ssDNA; (2) 3'-5' exonucleolysis of ssDNA substrates; (3) proofreading of DNA polymerization errors. Extrapolation to the crystal structures of Klenow and T4 DNA polymerases indicates that the invariant aromatic ring contiguous to the catalytic aspartate of the Exo II motif, corresponding to Tyr423 in Klenow, Phe218 in T4, and Phe65 in phi29 DNA polymerase, appears to be critical to orient the ssDNA substrate in a stable conformation to allow 3'-5' exonucleolytic catalysis. This is the first time that the functional importance of this invariant residue, belonging to the Exo II motif, has been demonstrated.

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