Influence of substrate structure on disintegration activity of Moloney murine leukemia virus integrase.
The disintegration activity of Moloney murine leukemia virus (M-MuLV) integrase (IN) was investigated through structural and sequence modifications of a Y substrate that resembles an integration intermediate. The Y substrates, constructed from individual oligonucleotides, contain a single viral long terminal repeat (LTR) joined to a nicked target DNA. Truncation of the double-stranded LTR sequences distal to the conserved 5'-CA-3' dinucleotide progressively diminished disintegration activity. M-MuLV IN was also able to catalyze disintegration of a heterologous double-stranded LTR sequence. Significantly, the activity of M-MuLV IN on single-stranded LTR Y substrates was more dependent on the sequence and length of the LTR strand than that reported for human immunodeficiency virus type 1 (HIV-1) IN. Modifications introduced at the Y-substrate junction demonstrated that the 3'-hydroxyl group at the terminus of the target strand was necessary for efficient joining of the target DNA strands. The presence of a 2'-hydroxyl group at the 3' end of the target strand, as well as a single-nucleotide gap at the LTR-target junction, reduced disintegration activity. The absence of hydroxyl groups on the terminal nucleotide abolished joining of the target strands. The results presented here suggest that M-MuLV IN disintegration activity is dependent on substantially different LTR sequence requirements than those reported for HIV-1 IN and may be mediated primarily through a structural recognition event.
Donzella
G A
GA
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854-5635.
Jonsson
C B
CB
Roth
M J
MJ
D001483:Base Sequence; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D019426:Integrases; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology* / Q000235:genetics; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D013379:Substrate Specificity; D016662:Virus Integration
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases