MUTATIONAL ANALYSIS OF O29 DNA-POLYMERASE RESIDUES ACTING AS SSDNA LIGANDS FOR 3'-5'-EXONUCLEOLYSIS

Here, three highly conserved amino acid residues have been characteriz ed to function as ssDNA binding ligands at the 3'-5' exonuclease activ e site of empty set29 DNA polymerase. One of these residues, Phe65, be longs to motif fro II, previously described to contain an invariant as partate and an invariant asparagine involved in catalysis and ssDNA bi nding, respectively. The other two residues, Ser122 and Leu123, form a newly identified motif ''(S/T)Lx(2)h'', and are the homologous counte rparts of Pol I residues Asp457 and Met458, and of T4 DNA polymerase r esidues Ser286 and Leu287, the latter three residues shown to contact ssDNA at their corresponding cocrystal 3D structures. Site-directed mu tagenesis and biochemical analysis of eight empty set29 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 polyme rization errors. Extrapolation to the crystal structures of Klenow and T4 DNA polymerases indicates that the invariant aromatic ring contigu ous to the catalytic aspartate of the fro II motif, corresponding to T yr423 in Klenow, Phe218 in T4, and Phe65 in empty set29 DNA polymerase , appears to be critical to orient the ssDNA substrate in a stable con formation to allow 3'-5' exonucleolytic catalysis. This is the first t ime that the functional importance of this invariant residue, belongin g to the fro II motif, has been demonstrated. (C) 1998 Academic Press Limited.