Aminoacidic Residues Involved in Determining Processivity of the 3'-5' Exonuclease Function in Family B DNA Polymerase from Sulfolobus solfataricus
Francesca M. PISANI*, Mariarita DE FELICE, Giuseppe MANCO, and Mose ROSSI
Istituto di Biochimica delle Proteine ed Enzimologia, Consiglio, Nazionale delle Ricerche, Via Marconi, 10, 80125-Napoli, Italy
Family B DNA polymerase from the thermoacidophilic archaeon Sulfolobus solfataricus (Sso DNA pol) consists of two relatively protease-resistant domains. By in situ activity gel analysis we found that proteolytic fragments corresponding to the amino- and carboxy-terminal half of the protein molecule retainrd 3'-5' exonuclease and polymerase function, respectively. Herein, we report on the purification and biochemical characterization of two NH2-terminal fragments of family B DNA polymerase from the thermoacidophilic archaeon, which retain 3'-5' exonuclease activity. Both these truncated forms contain the three evolutionarily conserved Exo motifs, but differ in the extent of the carboxyl-terminal deletion. Indeed, protein N438 spans residues 1-438 of Sso DNA pol polypeptide chain, whereas protein N508 (residues 1-508) extends beyond to include a 70-amino-acid segment (Region 1), located between the Exo III sequence and the similarity motif "D--SLYP". Analysis of the kinetic parameters of the 3'-5' exonucleolytic reaction on a 24-mer oligonucleotide reveals that both the truncated proteins possess lower turnover number and reduced affinity with respect to intact Sso DNA pol. However, the Km value of N438 for this substrate was higher in comparison to N508. Consistent with these results, assays for processive 3'-5' exonuclease activity indicate that protein N508, as well as full-length Sso DNA pol, retains a degree of processivity on a 24-base oligonucleotide noticeably higher with respect to N438. This biochemical analysis provides the first direct evidence that in a family B DNA polymerase the region located at the connection between N- and C-terminal domains participates in processivity of the proof-reading function. In addition, to better define amino acids residues within this region involved in nucleic acid substrate interaction, site-directed mutagenesis studies were carried out. The mutant recombinant DNA polymerase forms were purified and their 3'-5' exonuclease activity characterized in comparison with the wild-type enzyme.
