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

被引:47
作者
Hang, JQ
Rajendran, S
Yang, YL
Li, Y
In, PWK
Overton, H
Parkes, KEB
Cammack, N
Martin, JA
Klumpp, K
机构
[1] Roche Palo Alto LLC, Palo Alto, CA 94304 USA
[2] Roche Discovery Welwyn, Welwyn Garden City, Herts, England
关键词
D O I
10.1016/j.bbrc.2004.03.061
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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-1,3dione) 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. (C) 2004 Elsevier Inc. All rights reserved.
引用
收藏
页码:321 / 329
页数:9
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