Domain Organization and Biochemical Features of Sulfolobus solfataricus DNA Polymerase
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) is a monomer of about 100 kDa with two associated catalytic functions: 3'-5' exonuclease and DNA polymerase activities (1). By partial proteolysis experiments carried out on the homogeneous recombinant protein these two enzymatic functions were demonstrated to be organized in a modular fashion (2). To further investigate the domain organization of Sso DNA pol, truncated forms of the enzyme corresponding to the N- (3'--5' exonuclease) or to the C-terminal (polymerase) half of the protein molecule were constructed. In particular, two NH2- terminal fragments, which retain 3 t-5' exonuclease activity, referred to as N508 (residues 1-508) and N438 (residues 1-438), were over-expressed in E. coli, purified and biochemically characterized. 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 direct evidence that in this family B DNA polymerase the region located at the connection between the N- and C-terminal domains is involved in DNA substrate interaction. These results are in line with the mapping of DNA-induced conformational changes carried out by the limited proteolysis approach. Indeed, the comparison of the protease cleavage map in the absence/presence of nucleic acids ligands allowed us to identify a highly flexible central region of the polypeptide chain (Region 1 ), Which is protected from the proteolytic attack upon nuceic acid binding. More recently, the involvement of this conformationally mobile protein subdomain in DNA substrate interaction has been tested by site-directed mutagenesis studies.
1. Pisani, F. M. and Rossi, M. (1994) J. Biol. Chem., 269, 7887-7892.
2. Pisani, F. M., Manco, G., Carratore, V. and Rossi, M. (1996) Biochemistry, 35, 9158-9166.
