TY - JOUR
T1 - Ribonuclease H activity during initiation of reverse transcription using tRNAlys/RNA primer/template of human immunodeficiency virus
AU - Artzi, Hanna Ben
AU - Shemesh, Jenny
AU - Zeelon, E.
AU - Amit, Boaz
AU - Kleiman, Lawrance
AU - Gorecki, Marian
AU - Panet, Amos
PY - 1996/1/15
Y1 - 1996/1/15
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0030027993&partnerID=8YFLogxK
U2 - 10.1006/abbi.1996.0026
DO - 10.1006/abbi.1996.0026
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C2 - 8561499
AN - SCOPUS:0030027993
SN - 0003-9861
VL - 325
SP - 209
EP - 216
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 2
ER -