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Nucleic Acids Research, 1994, Vol. 22, No. 21 4441-4448
© 1994

ENZYMOLOGY

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

Abhijit Mazumder, Malini Gupta and Yves Pommier*

Laboratory of Molecular Pharmacology, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute Building 37, Room 5C25, Bethesda, MD 20892, USA

*To whom correspondence should be addressed

Received July 12, 1994. Revised August 22, 1994. Accepted August 22, 1994.

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 ethylphosphonodiester 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.


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