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 modifi cations of a Y substrate that resembles an integration intermediate. T he Y substrates, constructed from individual oligonucleotides, contain a single viral long terminal repeat (LTR) joined to a nicked target D NA. Truncation of the double-stranded LTR sequences distal to the cons erved 5'-CA-3' dinucleotide progressively diminished disintegration ac tivity. M-MuLV IN was also able to catalyze disintegration of a hetero logous 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 imm unodeficiency virus type 1 (HIV-1) IN. Modifications introduced at the Y-substrate junction demonstrated that the 3'-hydroxyl group at the t erminus of the target strand was necessary for efficient joining of th e target DNA strands. The presence of a 2'-hydroxyl group at the 3' en d of the target strand, as well as a single-nucleotide gap at the LTR- target junction, reduced disintegration activity. The absence of hydro xyl groups on the terminal nucleotide abolished joining of the target strands. The results presented here suggest that M-MuLV IN disintegrat ion activity is dependent on substantially different LTR sequence requ irements than those reported for HIV-1 IN and may be mediated primaril y through a structural recognition event.