The functional significance of the conserved motif 'YxGG/A', located between the 3'-5' exonuclease and polymerization domains of eukaryotic-type DNA polymerases, has been studied by site-directed mutagenesis in phi29 DNA polymerase. Single substitutions at this region were obtained, and 11 phi29 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 DNA structure, giving rise to three different mutant phenotypes: (i) favored polymerization (high pol/exo ratio); (ii) favored exonucleolysis (low pol/exo ratio); and (iii) favored exonucleolysis and null polymerization. Interestingly, these three different phenotypes could be obtained by mutating a single amino acid at the 'YxGG/A' motif. All different 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 or an increased accessibility to the exonuclease active site. These results allow us to propose that this motif, located in the connecting region 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.