Characterization of enzymatic properties of the Caenorhabditis elegans Dna2 protein, a homologue of Saccharomyces cerevisiae Dna2

초록

The Dna2 protein, known as an endonuclease and helicase, is highly conserved in all eukaryotes that suggesting its critical role in DNA metabolism. The Dna2 protein was well studied in S. cerevisiae, a unicellular organism, and shown to play an important role in Okazaki fragment processing on the lagging strand. To understand a role of Dna2 protein in multicellular organisms, we carried out a comparative study about C. elegans Dna2 (CeDna2), homologue of S. cerevisiae Dna2 (ScDna2). The full-length cDNA of C. elegans Dna2 was constructed by joining a cDNA clone containing 5'-end and EST clone, yk46a12 lacking the 5'-end 88 nucleotides. Using this cDNA and derivatives altered by site-directed mutagenesis, we prepared recombinant baculoviruses that produce either wild-type or mutant CeDna2 proteins fused to FLAG and polyhistidine epitopes at its N-terminal. Using affinity columns CeDna2 proteins were purified to near homogeneity from insect cells. Using wild type and mutant CeDna2 enzymes we determined biochemical properties of DNA-dependent ATPase, DNA helicase, and endonuclease activities.