title: Activity of the isolated HIV RNase H domain and specific inhibition by N-hydroxyimides.
abstract: This report describes a procedure to generate enzymatically active, isolated HIV RNase H domain. In contrast to previously described preparations, the RNA cleavage activity of the untagged RNase H domain was surprisingly similar to that of the full-length HIV-RT protein. Signature cleavages at 18 and 9 nucleotides downstream of a recessed RNA 5'-end were retained with the isolated RNase H domain. Activity was strongly decreased by deletion of 3 amino acids from the C-terminus, consistent with an important structural or functional role of the C-terminal alpha-helix. A prototype N-hydroxyimide (2-hydroxy-4H-isoquinoline-1,3-dione) was found to inhibit the activity of the isolated HIV RNase H domain as well as the RNase H activity of full-length HIV reverse transcriptase. In contrast, the compound did not significantly inhibit the structurally closely related Escherichia coli RNase HI. Specific binding of N-hydroxyimide compounds to the isolated RNase H domain was observed by protein fluorescence quenching.
authors.affiliation: Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304, USA.
authors.forename: Julie Qi
authors.initials: JQ
authors.lastname: Hang
authors.forename: Surendran
authors.initials: S
authors.lastname: Rajendran
authors.forename: Yanli
authors.initials: Y
authors.lastname: Yang
authors.forename: Yu
authors.initials: Y
authors.lastname: Li
authors.forename: Philippe Wong Kai
authors.initials: PW
authors.lastname: In
authors.forename: Hilary
authors.initials: H
authors.lastname: Overton
authors.forename: Kevin E B
authors.initials: KE
authors.lastname: Parkes
authors.forename: Nick
authors.initials: N
authors.lastname: Cammack
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Martin
authors.forename: Klaus
authors.initials: K
authors.lastname: Klumpp
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry; D054303:HIV Reverse Transcriptase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D007094:Imides / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D016914:Ribonuclease H / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D007094:Imides; D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H