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HERO ID
8455640
Reference Type
Journal Article
Title
Two-metal ion mechanism of RNA cleavage by HIV RNase H and mechanism-based design of selective HIV RNase H inhibitors
Author(s)
Klumpp, K; Hang, JQ; Rajendran, S; Yang, Y; Derosier, A; Wong Kai In, P; Overton, H; Parkes, KE; Cammack, N; Martin, JA; ,
Year
2003
Is Peer Reviewed?
1
Journal
Nucleic Acids Research
ISSN:
0305-1048
EISSN:
1362-4962
Publisher
OXFORD UNIV PRESS
Location
OXFORD
Page Numbers
6852-6859
Language
English
PMID
14627818
DOI
10.1093/nar/gkg881
Web of Science Id
WOS:000186802500023
URL
https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/gkg881
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Abstract
Human immunodeficiency virus (HIV) RNase H activity is essential for the synthesis of viral DNA by HIV reverse transcriptase (HIV-RT). RNA cleavage by RNase H requires the presence of divalent metal ions, but the role of metal ions in the mechanism of RNA cleavage has not been resolved. We measured HIV RNase H activity associated with HIV-RT protein in the presence of different concentrations of either Mg2+, Mn2+, Co2+ or a combination of these divalent metal ions. Polymerase-independent HIV RNase H was similar to or more active with Mn2+ and Co2+ compared with Mg2+. Activation of RNase H by these metal ions followed sigmoidal dose-response curves suggesting cooperative metal ion binding. Titration of Mg2+-bound HIV RNase H with Mn2+ or Co2+ ions generated bell-shaped activity dose-response curves. Higher activity could be achieved through simultaneous binding of more than one divalent metal ion at intermediate Mn2+ and Co2+ concentrations, and complete replacement of Mg2+ occurred at higher Mn2+ or Co2+ concentrations. These results are consistent with a two-metal ion mechanism of RNA cleavage as previously suggested for a number of polymerase-associated nucleases. In contrast, the structurally highly homologous RNase HI from Escherichia coli is most strongly activated by Mg2+, is significantly inhibited by submillimolar concentrations of Mn2+ and most probably cleaves RNA via a one-metal ion mechanism. Based on this difference in active site structure, a series of small molecule N-hydroxyimides was identified with significant enzyme inhibitory potency and selectivity for HIV RNase H.
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