Ribonuclease H activity during initiation of reverse transcription using tRNAlys/RNA primer/template of human immunodeficiency virus

Hanna Ben Artzi*, Jenny Shemesh, E. Zeelon, Boaz Amit, Lawrance Kleiman, Marian Gorecki, Amos Panet

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

The specificity of the initial cleavage by the RNaseH activity of HIV-1 reverse transcriptase (RT) during minus strong-stop DNA synthesis was studied using the authentic primer/template tRNAlys/HIV RNA. We observed that concomitant with the initiation of DNA synthesis, RNaseH activity of HIV RT introduced the first endonucleolytic cuts within the U5 region of the HIV RNA template, mainly 1 and 3 bases away from the primer binding site. To analyze whether the cleavage sites were determined by sequence specificity, the authentic U5 region at one of the cleavage sites was mutated. The change of sequence did not alter the initial cleavage pattern of RNaseH. In order to determine the size of the RNA/DNA hybrid that is required for RNaseH activation during reverse transcription initiation, DNA synthesis was limited by dideoxynucleotides. DNA extension of the tRNAlys primer by 17 deoxyribonucleotides but not by 6 deoxyribonucleotides was sufficient to activate the RNaseH site of HIV RT. Taken together, our results indicate that during initiation of minus strong-stop DNA synthesis by HIV RT, the first RNaseH-mediated endonucleolytic cut of the genomic RNA is dictated mainly by the length of the nascent DNA and not by sequence preference.

Original languageEnglish
Pages (from-to)209-216
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume325
Issue number2
DOIs
StatePublished - 15 Jan 1996

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