Contributions of DNA polymerase subdomains to the RNase H activity of human immunodeficiency virus type 1 reverse transcriptase

Jeffrey S. Smith, Kira Gritsman, Monica J. Roth

Research output: Contribution to journalArticle

45 Scopus citations

Abstract

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 Mn2+-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 Mg2+-dependent RNase H activity.

Original languageEnglish (US)
Pages (from-to)5721-5729
Number of pages9
JournalJournal of virology
Volume68
Issue number9
DOIs
StatePublished - Sep 1994

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

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