Activity of the isolated HIV RNase H domain and specific inhibition by N-hydroxyimides

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 an...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 2004-04, Vol.317 (2), p.321-329
Main Authors: Hang, Julie Qi, Rajendran, Surendran, Yang, Yanli, Li, Yu, Wong Kai In, Philippe, Overton, Hilary, Parkes, Kevin E.B., Cammack, Nick, Martin, Joseph A., Klumpp, Klaus
Format: Article
Language:English
Subjects:
Amino Acid Substitution
Binding Sites
Enzyme Activation
Enzyme Inhibitors - chemistry
HIV Reverse Transcriptase - biosynthesis
HIV Reverse Transcriptase - chemistry
HIV Reverse Transcriptase - genetics
HIV-1 - enzymology
HIV-1 - genetics
Human immunodeficiency virus
Hydrolysis
Imides - chemistry
Protein Binding
Protein Structure, Tertiary
Recombinant Proteins - biosynthesis
Recombinant Proteins - chemistry
Ribonuclease H - biosynthesis
Ribonuclease H - chemistry
Ribonuclease H - genetics
Ribonuclease H - isolation & purification
Sensitivity and Specificity
Structure-Activity Relationship
Substrate Specificity
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Summary: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 α-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.
ISSN:0006-291X
1090-2104
DOI:10.1016/j.bbrc.2004.03.061