Characterization of Mg(2+)-dependent 3'-processing activity for human immunodeficiency virus type 1 integrase in vitro: real-time kinetic studies using fluorescence resonance energy transfer.
Human immunodeficiency virus type 1 integrase (HIV-1 IN) catalyzes the integration of HIV-1 DNA into the host chromosome. In vitro reactions with endogenous viral DNA require Mg2+ as the metal cofactor, whereas in vitro studies performed with short oligonucleotide substrates utilize Mn2+. In this study, we report that the donor processing activity of HIV-1 IN alters depending on the structure and length of the oligonucleotide substrates. Increases in the length of the substrate cause alterations in the efficiency of Mg(2+)-dependent donor processing activity, thereby reconciling this discrepancy between the in vivo and in vitro HIV-1 IN mediated reactions. We have also found that the 3'-processing activity of HIV-IN is responsible for cleaving the junction between the viral and target sequences of the recombination intermediate. Its mechanism differs from the previously described disintegration reaction in that the donor strands are regenerated without a joining reaction of the target strands. Kinetic studies of 3'-processing activity suggest that the kcat (0.24/h) is very low. This implies that HIV-1 IN remains as a complex with the processed DNA prior to the strand transfer reaction.
Lee
S P
SP
Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D.C. 20007, USA.
Kim
H G
HG
Censullo
M L
ML
Han
M K
MK
D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D019426:Integrases; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D004247:DNA; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D008274:Magnesium