EVIDENCE THAT AN ARCHAEAL ALPHA-LIKE DNA-POLYMERASE HAS A MODULAR ORGANIZATION OF ITS ASSOCIATED CATALYTIC ACTIVITIES

In this study we report on the evidence that an alpha-like DNA polymer ase purified from the thermoacidophilic archaeon Sulfolobus solfataric us has a modular organization of its associated catalytic activities ( polymerase and 3'-5' exonuclease). This enzyme, a monomer of about 100 kDa whose complete primary structure is available, has a protease hyp ersensitive site that is likely to be cleaved by the action of endogen ous proteases during the purification procedure. As a consequence of t hat, two proteolytic fragments of about 50 and 40 kDa, in addition to the intact 100 M)a molecular species, can be detected upon SDS-PAGE of highly purified S. solfataricus DNA polymerase samples. The amino-ter minal microsequence analysis by Edman degradation has revealed that th e 50- and the 40-kDa polypeptides correspond to the carboxyl- and the amino-terminal portion of the protein molecule, respectively. Using th e bidimensional activity gel assay procedure, recently described by Lo ngley and Mosbaugh (Longley, M. J., and Mosbaugh, D. W. (1991) Biochem istry 30, 2655-2664), we have demonstrated that the 50-kDa fragment re tains a Mg2+ dependent DNA polymerizing activity, whereas the 40-kDa p olypeptide is able to catalyze the excision of mispaired nucleotides a t the 3'-OH terminus of a primer/ template DNA substrate in the presen ce of Mn2+ ions. On the other hand, the 100-kDa protein possess both a ctivities. To date, this is the first report indicating, on the basis of direct functional data, that the polymerization and the 3'-5' exonu clease activity of a family B DNA polymerase can be ascribed to physic ally distinct modules of the enzyme molecule.