Evidence for a dual functional role of a conserved histidine in RNA·DNA heteroduplex cleavage by human RNase H1.
Ribonuclease H1 is a conserved enzyme that cleaves the RNA strand of RNA·DNA heteroduplexes and has important functions in the nuclear and mitochondrial compartments. The therapeutic action of antisense oligodeoxynucleotides involves the recruitment of RNase H1 to cleave disease-relevant RNA targets. Recombinant human (Hs) RNase H1 was purified from a bacterial expression host, and conditions were identified that provided optimal oligonucleotide-directed RNA cleavage in vitro. Hs-RNase H1 exhibits optimal catalytic activity in pH 7.5 HEPES buffer and a salt (KCl) concentration of ~ 100-150 mm. Mg(2+)  best supports Hs-RNase H1 with an optimal concentration of 10 mm, but at higher concentrations inhibits enzyme activity. Mn(2+)  and Co(2+)  also support catalytic activity, while Ni(2+)  and Zn(2+)  exhibit only modest activities as cofactors. The optimized assay was used to show that an antisense oligonucleotide, added in substoichiometric amounts to initiate RNA cleavage, supports up to 30 rounds of reaction in 30 min. Mutation to alanine of the conserved histidine at position 264 causes an ~ 100-fold decrease in k(cat)  under multiple-turnover conditions, but does not alter K(m) . Under single-turnover conditions, the H264A mutant exhibits a 12-fold higher exponential time constant for substrate cleavage. The defective activity of the H264A mutant is not rescued in either assay condition by higher Mg(2+)  concentrations. These data implicate the H264 side chain in phosphodiester hydrolysis as well as in product release, and are consistent with a proposed model in which the H264 side chain interacts with a divalent metal ion to support catalysis.
Department of Chemistry, Temple University, Philadelphia, PA 19122, USA.
Nageswara R
NR
Alla
Allen W
AW
Nicholson
D002384:Catalysis; D002413:Cations, Divalent; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009692:Nucleic Acid Heteroduplexes; D059748:Proteolysis; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism*
D002413:Cations, Divalent; D009692:Nucleic Acid Heteroduplexes; D011994:Recombinant Proteins; D006639:Histidine; D012313:RNA; D004247:DNA; D016914:Ribonuclease H; C078856:ribonuclease HI