DNA substrate requirements for different activities of the human immunodeficiency virus type 1 integrase protein.
The integrase protein (IN) of human immunodeficiency virus type 1 removes two nucleotides from both 3' ends of the viral DNA (donor cleavage) and subsequently couples the newly generated 3' OH groups to phosphates in the target DNA (integration). The sequence requirements of IN for cleavage as well as for integration of viral DNA substrates have previously been studied by mutational analyses and by adduct interference assays. We extended these studies by analysis of heteroduplex oligonucleotide substrates and by missing-base analysis. We found for some base pairs that mutation of only one of the two bases and not the other affected IN activity. These base pairs center around the cleavage site. Besides donor cleavage and integration, IN can also perform "intermolecular disintegration," which has been described as the reversal of the integration reaction. We found that this reaction is independent of viral DNA sequences. In addition, the optimum spacing between the integration sites in intermolecular disintegration does not reflect the spacing found in vivo. These results indicate that this reaction is not the exact reversal of integration but rather is a sequence-independent phosphoryl transfer reaction between gapped DNA duplex molecules.
Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam.
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van den Ent
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Plasterk
D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis; D004252:DNA Mutational Analysis; D004254:DNA Nucleotidyltransferases / Q000096:biosynthesis / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D019426:Integrases; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D013379:Substrate Specificity; D016662:Virus Integration
D002352:Carrier Proteins; D004279:DNA, Viral; D058619:Maltose-Binding Proteins; D009692:Nucleic Acid Heteroduplexes; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases