Helix structure and ends of RNA/DNA hybrids direct the cleavage specificity of HIV-1 reverse transcriptase RNase H.
RNA/DNA hybrids in human immunodeficiency virus (HIV) replication are cleaved by HIV-1 reverse transcriptase (RT) H in locations determined by hybrid structure. Minus strand DNA synthesis is accompanied by cleavage of template viral RNA directed by RT positioned at the growing 3' DNA end. Some RNA remains as oligomers annealed to the new DNA strand and is cut by RTs positioned at the 5' RNA ends. We constructed substrates to the test the hypothesis that internal helix structure, rather than strand end structure, drives the RT to position at 3' DNA and 5' RNA ends. On substrates with an RNA primer recessed on a DNA template, the 5' end of the RNA had a dominant role in the determination of RNase H cleavage positions. If the 5' end region of the RNA could not anneal, cleavage would not occur. Nevertheless, we obtained evidence that helix structure promotes the binding of RT to the end of the helical region closest to the 5' RNA/3' DNA end. When a DNA primer recessed on an RNA template had a 3' unannealed region, cleavage occurred, with RT positioned solely by helical structure at the 5' RNA/3' DNA end of the annealed region of the hybrid. Using substrates having RNA primers annealed to circular DNA templates, we showed that cleavage can be independent of the presence of a DNA 3'end and is directed by the 5' RNA end. Overall, the results suggest that the RT initially binds an internal region of the hybrid and then is driven in the direction to encounter a 3' DNA or 5' RNA end, where it is positioned for catalysts by the strand end. The requirement for two modes of RNA cleavage in viral replication and the unexpected requirement for the 5' RNA end structure are discussed.
Palaniappan
C
C
Department of Biochemistry, University of Rochester, New York 14642, USA.
Fuentes
G M
GM
Rodríguez-Rodríguez
L
L
Fay
P J
PJ
Bambara
R A
RA
D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D054303:HIV Reverse Transcriptase; D007700:Kinetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D012367:RNA, Viral / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins; D016914:Ribonuclease H / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013698:Templates, Genetic
D017931:DNA Primers; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D012367:RNA, Viral; D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H