Enzymatic properties of the Caenorhabditis elegans Dna2 endonuclease/helicase and a species-specific interaction between RPA and Dna2

In both budding and fission yeasts, a null mutation of the DNA2 gene is lethal. In contrast, a null mutation of Caenorhabditis elegans dna2(+) causes a delayed lethality, allowing survival of some mutant C.elegans adults to F2 generation. In order to understand reasons for this difference in requirement of Dna2 between these organisms, we examined the enzymatic properties of the recombinant C.elegans Dna2 (CeDna2) and its interaction with replication-protein A (RPA) from various sources. Like budding yeast Dna2, CeDna2 possesses DNA-dependent ATPase, helicase and endonuclease activities. The specific activities of both ATPase and endonuclease activities of the CeDna2 were considerably higher than the yeast Dna2 (∼10- and 20-fold, respectively). CeDna2 endonuclease efficiently degraded a short 5′ single-stranded DNA tail (<10 nt) that was hardly cleaved by ScDna2. Both endonuclease and helicase activities of CeDna2 were stimulated by CeRPA, but not by human or yeast RPA, demonstrating a species-specific interaction between Dna2 and RPA. These and other enzymatic properties of CeDna2 described in this paper may shed light on the observation that C.elegans is less stringently dependent on Dna2 for its viability than Saccharomyces cerevisiae. We propose that flaps generated by DNA polymerase δ-mediated displacement DNA synthesis are mostly short in C.elegans eukaryotes, and hence less dependent on Dna2 for viability

Hetero- and Autoprocessing of the Extracellular Metalloprotease (Mpr) in Bacillus subtilis

Most proteases are synthesized as inactive precursors which are processed by proteolytic cleavage into a mature active form, allowing regulation of their proteolytic activity. The activation of the glutamic-acid-specific extracellular metalloprotease (Mpr) of Bacillus subtilis has been examined. Analysis of Mpr processing in defined protease-deficient mutants by activity assay and Western blotting revealed that the extracellular protease Bpr is required for Mpr processing. pro-Mpr remained a precursor form in bpr-deficient strains, and glutamic-acid-specific proteolytic activity conferred by Mpr was not activated in bpr-deficient strains. Further, purified pro-Mpr was processed to an active form by purified Bpr protease in vitro. We conclude that Mpr is activated by Bpr in vivo, and that heteroprocessing, rather than autoprocessing, is the major mechanism of Mpr processing in vivo. Exchange of glutamic acid for serine in the cleavage site of Mpr (S93E) allowed processing of Mpr into its mature form, regardless of the presence of other extracellular proteases, including Bpr. Thus, a single amino acid change is sufficient to convert the Mpr processing mechanism from heteroprocessing to autoprocessing