Substrate requirements for secondary cleavage by HIV-1 reverse transcriptase RNase H.
During and after minus-strand DNA synthesis, human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) degrades the RNA genome. To remove RNA left after polymerization, the RT aligns to cut 18 nucleotides in from the 5' RNA end. The enzyme then repositions, making a secondary cut 8 nucleotides from the RNA 5' end. Transfer of the minus strong stop DNA during viral replication requires cleavage of template RNA. Removal of the terminal RNA segment is a special case because the RNA-DNA hybrid forms a blunt end, shown previously to resist cleavage when tested in vitro. We show here that the structure of the substrate extending beyond the RNA 5' end is an important determinant of cleavage efficiency. A short single-stranded DNA extension greatly stimulated the secondary cleavage. Annealing an RNA segment to the DNA extension was even more stimulatory. Recessing the DNA from a blunt end by even one nucleotide caused the RT to reorient its binding, preventing secondary cleavage. The presence of the cap at the 5' end of the viral RNA did not improve the efficiency of secondary cleavage. However, NC protein greatly facilitated the secondary cut on the blunt-ended substrate, suggesting that NC compensates for the unfavorable substrate structure.
Wisniewski
Michele
M
Department of Biochemistry and Biophysics and the Cancer Center, University of Rochester, Rochester, New York 14642, USA.
Chen
Yan
Y
Balakrishnan
Mini
M
Palaniappan
Chockalingam
C
Roques
Bernard P
BP
Fay
Philip J
PJ
Bambara
Robert A
RA
D001483:Base Sequence; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D016914:Ribonuclease H / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D013997:Time Factors; D014158:Transcription, Genetic
D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H