Role of the "YxGG/A" motif of O29 DNA polymerase in protein-primed replication

We have analyzed the functional significance of the o29 DNA polymerase "YxG G/A" motif in initiation and replication reactions involving the terminal p rotein (TP) as a primer. This motif, located between the proposed limits of the polymerase and exonuclease domains, has been shown to be very importan t for the coordination between synthesis and degradation in o29 DNA polymer ase. Mutations in this region affected the polymerization/exonucleolysis (p ol/exo) balance, due to its import ance for DNA template binding stability at both active sites. Here, we show that the YxGG/A motif of o29 DNA polyme rase is necessary for the formation of a stable complex between TP and o29 DNA polymerase, affecting initiation and transition during replication of o 29 TP-DNA. The phenotypes in TP-primed reactions in nine of 11 mutant polym erases, showed reduced initiation and/or replication activities using TP-DN A as template. High dATP concentrations allowed the reduced initiation acti vities of some of these mutant polymerases to reach the wild-type level. Th e reduction in their affinity for the initiating nucleotide is likely due t o their reduced interaction with the TP. Besides, the YxGG/A motif of o29 D NA polymerase controls the pol/exo balance in the transition step immediate ly after TP-primed initiation, before DNA polymerase and TP dissociate. Thu s, from the first elongation step, the phenotypes of the mutant polymerases parallel those obtained in DNA-primed replication: wild-type, high and low pol/exo balance. A detailed analysis of different transition intermediates suggests that mutants at the YxGG/A motif switch from interaction with TP to DNA once the TP has been extended with six nucleotides. (C) 1999 Academi c Press.