Coordinated disintegration reactions mediated by Moloney murine leukemia virus integrase.
The protein-DNA and protein-protein interactions important for function of the integrase (IN) protein of Moloney murine leukemia virus (M-MuLV) were investigated by using a coordinated-disintegration assay. A panel of M-MuLV IN mutants and substrate alterations highlighted distinctions between the intermolecular and intramolecular reactions of coordinated disintegration. Mispairing of the crossbone single-strand region and altered long terminal repeat (LTR) positioning affected the intermolecular, but not the intramolecular, reactions of coordinated disintegration. Partial components of the crossbone substrate were coordinated by M-MuLV IN, indicating a reliance on both LTR and target DNA determinants for substrate assembly. The intramolecular reaction was dependent on the presence of either the HHCC domain or a crossbone LTR 5' single-stranded tail. An M-MuLV IN mutant without the HHCC domain (Ndelta105) catalyzed reduced levels of double disintegration but not single disintegration. A separately purified HHCC domain protein (Cdelta232) stimulated double disintegration mediated by Ndelta105, suggesting a role of the N-terminal HHCC domain in stable IN-IN and IN-DNA interactions. Significantly, crossbone substrates lacking the LTR 5' tails were not recognized by the fingerless Ndelta105 protein. Collectively, these data suggest similar roles of the HHCC domain and 5' LTR tail in substrate recognition and modulation of IN activity.
Donzella
G A
GA
Department of Biochemistry, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway 08854, USA.
Jonsson
C B
CB
Roth
M J
MJ
D001483:Base Sequence; D004254:DNA Nucleotidyltransferases / Q000502:physiology*; D004279:DNA, Viral / Q000378:metabolism*; D019426:Integrases; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D016662:Virus Integration
D004279:DNA, Viral; D004254:DNA Nucleotidyltransferases; D019426:Integrases