Methylphosphonodiester substitution near the conserved CA dinucleotide in the HIV LTR alters both extent of 3'-processing and choice of nucleophile by HIV-1 integrase.
We present evidence suggesting that the 3'-processing activity of HIV-1 integrase is dramatically affected by electrostatic and/or steric perturbations 3' to the conserved CA dinucleotide. When the phosphodiester bond 3' to the scissile phosphodiester is replaced by a methylphosphonodiester linkage, 3'-processing decreases by two orders of magnitude. This block of cleavage can be somewhat overcome by increasing the pH of the reaction. Labeling of the substrates at the 3'-end revealed blockage of water and glycerol, but stimulation of the viral DNA 3'-hydroxyl, acting as the nucleophile with the methylphosphonodiester substrate. Interestingly, a circular trinucleotide was formed using the phosphodiester and methylphosphonodiester substrates when the terminal nucleotide was 3'-deoxyadenosine but not 2'-deoxyadenosine. Mutagenesis of the enzyme active site has previously been shown to alter the choice of nucleophile in the 3'-processing reaction. Taken together, the results in this study suggest that 'mutagenesis' of the DNA backbone can also alter the choice of nucleophile.
Laboratory of Molecular Pharmacology, National Cancer Institute, Bethesda, MD 20892.
A
A
Mazumder
M
M
Gupta
Y
Y
Pommier
D001483:Base Sequence; D017124:Conserved Sequence; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D003839:Deoxyadenosines / Q000737:chemistry / Q000378:metabolism; D004563:Electrochemistry; D005990:Glycerol; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D019426:Integrases; D007700:Kinetics; D008345:Manganese; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000737:chemistry*; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D004279:DNA, Viral; D003839:Deoxyadenosines; D009838:Oligodeoxyribonucleotides; D009943:Organophosphorus Compounds; D008345:Manganese; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C058120:cordycepin; C058118:2'-deoxyadenosine; D005990:Glycerol