Evidence that an archaeal alpha-like DNA polymerase has a modular organization of its associated catalytic activities.

Abstract:

In this study we report on the evidence that an alpha-like DNA polymerase purified from the thermoacidophilic archaeon Sulfolobus solfataricus 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 hypersensitive site that is likely to be cleaved by the action of endogenous proteases during the purification procedure. As a consequence of that, two proteolytic fragments of about 50 and 40 kDa, in addition to the intact 100-kDa molecular species, can be detected upon SDS-PAGE of highly purified S. solfataricus DNA polymerase samples. The amino-terminal microsequence analysis by Edman degradation has revealed that the 50- and the 40-kDa polypeptides correspond to the carboxyl- and the amino-terminal portion of the protein molecule, respectively. Using the bidimensional activity gel assay procedure, recently described by Longley and Mosbaugh (Longley, M. J., and Mosbaugh, D. W. (1991) Biochemistry 30, 2655-2664), we have demonstrated that the 50-kDa fragment retains a Mg(2+)-dependent DNA polymerizing activity, whereas the 40-kDa polypeptide is able to catalyze the excision of mispaired nucleotides at the 3'-OH terminus of a primer/template DNA substrate in the presence of Mn2+ ions. On the other hand, the 100-kDa protein possess both activities. To date, this is the first report indicating, on the basis of direct functional data, that the polymerization and the 3'-5' exonuclease activity of a family B DNA polymerase can be ascribed to physically distinct modules of the enzyme molecule.

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