Divalent Cation Modulation of the Ribonuclease Functions of Human Immunodeficiency Virus Reverse Transcriptase

Nick M. Cirino, Craig E. Cameron, Jeffrey S. Smith, Jason W. Rausch, Monica J. Roth, Stephen J. Benkovic, Stuart F.J. Le Grice

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Abstract

The stimulatory effect of Mg2+ and Mn2+ on the ribonuclease H (RNase H) functions of HIV-1 reverse transcriptase (RT) has been evaluated using a model 90-nt RNA template/36-nt DNA primer. Wild type enzyme exhibits similar endonuclease and directional processing activities in response to both cations, while RNase H* activity (hydrolysis of double-stranded RNA) is only evident in the presence of Mn2+. Enzyme altered at the p66 residue Glu478 (Glu478→Gln478), which participates in metal ion binding, is completely inactive in Mg2+. However, Mn2+ restores specifically its endoribonuclease activity. In the presence of Mn2+, mutant RT also catalyzes specific removal of the tRNA replication primer, eliminating the possibility of contaminating Escherichia coli RNase H in our recombinant enzyme. However, the efficiency with which mutant RT catalyzes transfer of nascent DNA between RNA templates (an event mandating RNase H activity) is severely reduced. These findings raise the possibility that directional processing activity is required to accelerate transfer of nascent DNA between templates during retroviral replication.

Original languageEnglish (US)
Pages (from-to)9936-9943
Number of pages8
JournalBiochemistry
Volume34
Issue number31
DOIs
StatePublished - Aug 1995

All Science Journal Classification (ASJC) codes

  • Biochemistry

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    Cirino, N. M., Cameron, C. E., Smith, J. S., Rausch, J. W., Roth, M. J., Benkovic, S. J., & Le Grice, S. F. J. (1995). Divalent Cation Modulation of the Ribonuclease Functions of Human Immunodeficiency Virus Reverse Transcriptase. Biochemistry, 34(31), 9936-9943. https://doi.org/10.1021/bi00031a016