title: Cloning, subcellular localization and functional expression of human RNase HII.
abstract: Recently we showed that the major mammalian RNase H, RNase HI, is evolutionarily related to prokaryotic RNase HII (Frank et al., FEBS-Lett. 421, 23-26, 1998), an enzyme described to be a minor activity in E. coli. As a consequence we addressed the question of whether a human RNase H exists, sharing homology with the main E. coli enzyme, RNase HI. Employing sequence analysis of expressed sequence tags, followed by specific PCR amplification of human cDNA, we cloned, sequenced and expressed a human open reading frame, coding for a 32 kDa protein. Purification of the recombinant His(6)-tagged protein from E. coli extracts using Ni(2+)-chelating chromatography and subsequent renaturation gel assay proved that it is an active RNase H. The properties of this enzyme suggest that it is identical with the human RNase HII, previously purified by one of us (Frank et al., Nucleic Acids Res. 22, 5247-5254, 1994). Studies using a green fluorescent protein-fusion construct reveal that this protein is located in the nucleus.
authors.affiliation: Department of Molecular Genetics, Institute of Tumor Biology and Cancer Research, University of Vienna, Austria.
authors.forename: P
authors.initials: P
authors.lastname: Frank
authors.forename: C
authors.initials: C
authors.lastname: Braunshofer-Reiter
authors.forename: A
authors.initials: A
authors.lastname: Pöltl
authors.forename: K
authors.initials: K
authors.lastname: Holzmann
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000201:enzymology*
chemical_list: D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; C091139:ribonuclease HII; D016914:Ribonuclease H