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.
Mazumder
A
A
Laboratory of Molecular Pharmacology, National Cancer Institute, Bethesda, MD 20892.
Gupta
M
M
Pommier
Y
Y
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