GENE CLONING AND CHARACTERIZATION OF RECOMBINANT RNASE HII FROM A HYPERTHERMOPHILIC ARCHAEON

We have cloned the gene encoding RNase HII (RNase HIIPk) from the hype rthermophilic archaeon Pyrococcus kodakaraensis KOD1 by screening of a library for clones that suppressed the temperature-sensitive growth p henotype of an mh mutant strain of Escherichia coli. This gene was exp ressed in an mh mutant strain off. call, the recombinant enzyme was pu rified, and its biochemical properties were compared with those of E. coli RNases HI and HII, RNase HIIPk is composed of 228 amino acid resi dues (molecular weight, 25,799) and acts as a monomer, Its amino acid sequence showed little similarity to those of enzymes that are members of the RNase HI family of proteins but showed 40, 31, and 25% identit ies to those of Methanococcus jannaschii, Saccharomyces cerevisiae, an d E. coli RNase HII proteins, respectively. The enzymatic activity,vas determined at 30 degrees C and pH 8.0 by use of an M13 DNA-RNA hybrid as a substrate. Under these conditions, the most preferred metal ions were Co2+ for RNase HIIPk, Mn2+ for E. coli RNase HII, and Mg2+ for E . coli RNase HI. The specific activity of RNase HIIPk determined in th e presence of the most preferred metal ion was 6.8-fold higher than th at off. coli RNase HII and 4.5-fold lower than that off. coli RNase HI , Like E. coli RNase HI, RNase HIIPk and E. call RNase HII cleave the RNA strand of an RNA-DNA hybrid endonucleolytically at the P-O3' bond. In addition, these enzymes cleave oligomeric substrates in a similar manner. These results suggest that RNase HIIPk and E. coli RNases HI a nd HII are structurally and functionally related to one another.