PMID: 8605889Mar 1, 1996Paper

Primer-terminus stabilization at the 3'-5' exonuclease active site of phi29 DNA polymerase. Involvement of two amino acid residues highly conserved in proofreading DNA polymerases

The EMBO Journal
Miguel de VegaL Blanco

Abstract

By site-directed mutagenesis in phi29 DNA polymerase, we have analyzed the functional importance of two evolutionarily conserved residues belonging to the 3'-5' exonuclease domain of DNA-dependent DNA polymerases. In Escherichia coli DNA polymerase I, these residues are Thr358 and Asn420, shown by crystallographic analysis to be directly acting as single-stranded DNA (ssDNA) ligands at the 3'-5' exonuclease active site. On the basis of these structural data, single substitution of the corresponding residues of phi29 DNA polymerase, Thr15 and Asn62, produced enzymes with a very reduced or altered capacity to bind ssDNA. Analysis of the residual 3'-5' exonuclease activity of these mutant derivatives on ssDNA substrates allowed us to conclude that these two residues do not play a direct role in the catalysis of the reaction. On the other hand, analysis of the 3'-5' exonuclease activity on either matched or mismatched primer/template structures showed a critical role of these two highly conserved residues in exonucleolysis under polymerization conditions, i.e. in the proofreading of DNA polymerization errors, an evolutionary advantage of most DNA-dependent DNA polymerases. Moreover, in contrast to the dual role in 3'-5' exonucleoly...Continue Reading

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