A DNA-BINDING MOTIF COORDINATING SYNTHESIS AND DEGRADATION IN PROOFREADING DNA-POLYMERASES

The functional significance of the conserved motif 'YxGG/A', located b etween the 3'-5' exonuclease and polymerization domains of eukaryotic- type DNA polymerases, has been studied by site-directed mutagenesis in phi 29 DNA polymerase. Single substitutions at this region were obtai ned, and 11 phi 29 DNA polymerase mutant derivatives were overproduced in Escherichia coli and purified to homogeneity. Nine mutants showed an altered polymerase/3'-5' exonuclease balance on a template/primer D NA structure, giving rise to three different mutant phenotypes: (i) fa vored polymerization (high pol/exo ratio); (ii) favored exonucleolysis (low pol/exo ratio); and (iii) favored exonucleolysis and null polyme rization. Interestingly, these three different phenotypes could be obt ained by mutating a single amino acid at the 'YxGG/A' motif. All diffe rent phenotypes could be directly related to defects in DNA binding at a particular active site. Thus, a high pol/exo ratio was related to a poor stability at the 3'-5' exonuclease active site. On the contrary, a low pol/exo ratio or null polymerization capacity was related to a poor stability at the polymerization active site and either a normal o r an increased accessibility to the exonuclease active site. These res ults allow us to propose that this motif, located in the connecting re gion between the N-terminal and C-terminal domains, has a primary role in DNA binding, playing a critical role in the coordination or cross- talk between synthesis and degradation.