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M9490417.TXT
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1994-09-19
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Document 0417
DOCN M9490417
TI Contributions of DNA polymerase subdomains to the RNase H activity of
human immunodeficiency virus type 1 reverse transcriptase.
DT 9411
AU Smith JS; Gritsman K; Roth MJ; Department of Biochemistry, University of
Medicine and Dentistry; of New Jersey/Robert Wood Johnson Medical
School, Piscataway; 08854.
SO J Virol. 1994 Sep;68(9):5721-9. Unique Identifier : AIDSLINE
MED/94335087
AB Previous studies showed that an isolated human immunodeficiency virus
type 1 (HIV-1) RNase H domain expressed as a fusion protein is highly
active in Mn2+, but activity was dependent on a hexahistidine tag
located at either the carboxyl or amino terminus of the fusion protein
(J. Smith and M. Roth, J. Virol. 67:4037-4049, 1993). It was postulated
that a histidine tag can somehow provide a function normally associated
with the DNA polymerase domain of HIV-1 reverse transcriptase. To
determine the contributions of the DNA polymerase subdomains of HIV-1
reverse transcriptase to its RNase H activity, we have characterized the
activity of isolated RNase H domains which include either portions of
the connection, the entire connection, or both the thumb and connection
as N-terminal extensions. Including increasing lengths of these domains
at the N terminus of the RNase H resulted in a progressive increase in
Mn(2+)-dependent RNase H activity that was independent of a histidine
tag. Activity of the isolated RNase H domains was also stimulated by the
addition of independently purified polymerase subdomains. Further, this
stimulation was shown to be a result of direct physical interactions
between the thumb, connection, and RNase H domains. The connection and
thumb subdomains were shown to contribute to substrate binding. The
fingers and palm subdomains were found to be essential for
Mg(2+)-dependent RNase H activity.
DE Base Sequence DNA Polymerases/CHEMISTRY DNA Primers/CHEMISTRY
HIV-1/*ENZYMOLOGY Molecular Sequence Data Recombinant Fusion Proteins
Reverse Transcriptase/*CHEMISTRY Ribonuclease H, Calf Thymus/*CHEMISTRY
RNA, Transfer, Lys/METABOLISM Structure-Activity Relationship
Substrate Specificity Support, Non-U.S. Gov't Support, U.S. Gov't,
P.H.S. JOURNAL ARTICLE
SOURCE: National Library of Medicine. NOTICE: This material may be
protected by Copyright Law (Title 17, U.S.Code).