Contributions of DNA polymerase subdomains to the RNase H activity of human immunodeficiency virus type 1 reverse transcriptase.
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.
Smith
J S
JS
Department of Biochemistry, University of Medicine and Dentistry of New Jersey/Robert Wood Johnson Medical School, Piscataway 08854.
Gritsman
K
K
Roth
M J
MJ
D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D008969:Molecular Sequence Data; D012357:RNA, Transfer, Lys / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry*; D011993:Recombinant Fusion Proteins; D016914:Ribonuclease H / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D017931:DNA Primers; D012357:RNA, Transfer, Lys; D011993:Recombinant Fusion Proteins; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H