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Nucleic Acids Research Advance Access published online on October 4, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn678
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

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

Chandravanu Dash1, Brian J. Scarth2, Christopher Badorrek1, Matthias Götte2 and Stuart F. J. Le Grice1,*

1HIV Drug Resistance Program, National Cancer Institute at Frederick, Frederick, MD 21702, USA and 2Department of Microbiology and Immunology, McGill University, Montreal, Canada

*To whom correspondence should be addressed. Tel: +301 846 5502; Fax: +301 846 6013; Email: slegrice{at}ncifcrf.gov

Received July 22, 2008. Revised September 22, 2008. Accepted September 23, 2008.

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.


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