title: How does RNase H recognize a DNA.RNA hybrid?
abstract: The mechanism of RNase H substrate recognition is proposed from a model of a chemically modified DNA.RNA hybrid Escherichia coli RNase H complex. Site-directed mutagenesis of the enzyme and substrate titration observed by heteronuclear two-dimensional NMR spectra have been carried out. A model complex has been built, based on free structures of the enzyme and the substrate independently determined by x-ray crystallography and NMR distance geometry, respectively. In addition to steric and electrostatic complementarities between the molecular surfaces of the enzyme and the minor groove of the hybrid in the model, putative hydrogen bonds between the polar groups in the enzyme and 2'-oxygens of the RNA strand of the hybrid fix the hybrid close to the active site of the enzyme. The enzymatic activities of the mutant proteins and the changes in NMR spectra during the course of substrate titration are consistent with the present model. Moreover, the specific cleavage of the RNA strand in DNA.RNA hybrids can be explained as well as cleavage modes in modified heteroduplexes. A mechanism of enzymatic action is proposed.
authors.affiliation: Protein Engineering Research Institute, Osaka, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Nakamura
authors.forename: Y
authors.initials: Y
authors.lastname: Oda
authors.forename: S
authors.initials: S
authors.lastname: Iwai
authors.forename: H
authors.initials: H
authors.lastname: Inoue
authors.forename: E
authors.initials: E
authors.lastname: Ohtsuka
authors.forename: S
authors.initials: S
authors.lastname: Kanaya
authors.forename: S
authors.initials: S
authors.lastname: Kimura
authors.forename: C
authors.initials: C
authors.lastname: Katsuda
authors.forename: K
authors.initials: K
authors.lastname: Katayanagi
authors.forename: K
authors.initials: K
authors.lastname: Morikawa
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004279:DNA, Viral / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012367:RNA, Viral / Q000378:metabolism*; D016914:Ribonuclease H / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D004279:DNA, Viral; D009841:Oligonucleotides; D012367:RNA, Viral; D016914:Ribonuclease H