title: Ribonuclease H from K562 human erythroleukemia cells. Purification, characterization, and substrate specificity.
abstract: The major ribonuclease H from K562 human erythroleukemia cells has been purified more than 4,000-fold. This RNase H, now termed RNase H1, is an endoribonuclease whose products contain 5'-phosphoryl and 3'-hydroxyl termini. The enzyme has a native molecular weight of 89,000 based on its sedimentation and diffusion coefficients. Human RNase H1 has an absolute requirement for a divalent cation. Maximal activity is obtained with either 10 mM Mg2+, 5 mM Co2+, or 0.5 mM Mn2+. The pH optimum is between 8.0 and 8.5 in the presence of 10 mM Mg2+. The isoelectric point is 6.4. RNase H1 lacks double-stranded and single-stranded RNase and DNase activities, and it will not hydrolyze the DNA moiety of an RNA.DNA heteroduplex. Unlike the Escherichia coli enzyme, which requires a heteroduplex that contains at least four consecutive ribonucleotides for activity, human RNase H1 can hydrolyze a DNA.RNA.DNA/DNA heteroduplex that contains a single ribonucleotide. Cleavage occurs at the 5' phosphodiester of this residue. This substrate specificity suggests that human RNase H1 could play a role in ribonucleotide excision from genomic DNA during replication.
authors.affiliation: Department of Biochemistry, University of Iowa, Iowa City 52242.
authors.forename: P S
authors.initials: PS
authors.lastname: Eder
authors.forename: J A
authors.initials: JA
authors.lastname: Walder
mesh_terms: D001483:Base Sequence; D002412:Cations; D002853:Chromatography, Liquid; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007525:Isoelectric Focusing; D004915:Leukemia, Erythroblastic, Acute / Q000201:enzymology*; D008969:Molecular Sequence Data; D012313:RNA / Q000378:metabolism; D016914:Ribonuclease H; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
chemical_list: D002412:Cations; D012313:RNA; D004247:DNA; D004722:Endoribonucleases; D016914:Ribonuclease H