Examining the ribonuclease H primer grip of HIV-1 reverse transcriptase by charge neutralization of RNA/DNA hybrids

The crystal structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) bound to an RNA/DNA hybrid reveals an extensive network of contacts with the phosphate backbone of the DNA strand ~4-9 bp downstream from the ribonuclease H (RNase H) catalytic center. Collectively design...

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Bibliographic Details
Published in:Nucleic acids research 2008-11, Vol.36 (20), p.6363-6371
Main Authors: Dash, Chandravanu, Scarth, Brian J, Badorrek, Christopher, Götte, Matthias, Le Grice, Stuart F.J
Format: Article
Language:English
Subjects:
Bacillus halodurans
DNA - biosynthesis
DNA - chemistry
DNA - metabolism
DNA Footprinting
DNA Primers - chemistry
HIV Reverse Transcriptase - chemistry
Human immunodeficiency virus 1
Models, Molecular
Nucleic Acid Enzymes
Organophosphorus Compounds - chemistry
Ribonuclease H - chemistry
Ribonuclease H - metabolism
RNA - chemistry
RNA - metabolism
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Summary:The crystal structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) bound to an RNA/DNA hybrid reveals an extensive network of contacts with the phosphate backbone of the DNA strand ~4-9 bp downstream from the ribonuclease H (RNase H) catalytic center. Collectively designated as 'the RNase H primer grip', this motif contains a phosphate binding pocket analogous to the human and Bacillus halodurans RNases H. The notion that the RNase H primer grip mediates the trajectory of RNA/DNA hybrids accessing the RNase H active site suggests that locally neutralizing the phosphate backbone may be exploited to manipulate nucleic acid flexibility. To examine this, we introduced single and tandem methylphosphonate substitutions through the region of the DNA primer contacted by the RNase H primer grip and into the RNase H catalytic center. The ability of mutant hybrids to support RNase H and DNA polymerase activity was thereafter examined. In addition, site-specific chemical footprinting was used to evaluate movement of the DNA polymerase and RNase H domains. We show here that minor alteration to the RNase H primer can have a dramatic effect on enzyme positioning, and discuss these findings in light of recent crystallography of human RNase H containing an RNA/DNA hybrid.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkn678