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 approximately 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.
HIV Drug Resistance Program, National Cancer Institute at Frederick, Frederick, MD 21702, USA.
Chandravanu
C
Dash
Brian J
BJ
Scarth
Christopher
C
Badorrek
Matthias
M
Götte
Stuart F J
SF
Le Grice
D004247:DNA / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D018983:DNA Footprinting; D017931:DNA Primers / Q000737:chemistry*; D054303:HIV Reverse Transcriptase / Q000737:chemistry*; D008958:Models, Molecular; D009943:Organophosphorus Compounds / Q000737:chemistry; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D016914:Ribonuclease H / Q000737:chemistry* / Q000378:metabolism
D017931:DNA Primers; D009943:Organophosphorus Compounds; C032627:methylphosphonic acid; D012313:RNA; D004247:DNA; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H