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

Biochem Biophys Res Commun. 2004 Apr 30;317(2):321-9. doi: 10.1016/j.bbrc.2004.03.061.


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.

MeSH terms

  • 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
  • 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


  • Enzyme Inhibitors
  • Imides
  • Recombinant Proteins
  • HIV Reverse Transcriptase
  • Ribonuclease H