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 initiated a comparative study between C. elegans Dna2 (CeDna2) and S. cerevisiae Dna2 (ScDna2). For this purpose, the full-length cDNA encoding CeDna2 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 with an amino acid change by site-directed mutagenesis, we prepared recombinant baculoviruses that produce wild-type, helicase-negative, and nuclease negative CeDna2 proteins fused to FLAG and polyhistidine epitopes at its N-terminus. In this presentation, we report biochemical properties of DNA-dependent ATPase, DNA helicase, and endonuclease activities using these proteins.