Role of DNA end distortion in catalysis by avian sarcoma virus integrase.
Retroviral integrase (IN) recognizes linear viral DNA ends and introduces nicks adjacent to a highly conserved CA dinucleotide usually located two base pairs from the 3'-ends of viral DNA (the "processing" reaction). In a second step, the same IN active site catalyzes the insertion of these ends into host DNA (the "joining" reaction). Both DNA sequence and DNA structure contribute to specific recognition of viral DNA ends by IN. Here we used potassium permanganate modification to show that the avian sarcoma virus IN catalytic domain is able to distort viral DNA ends in vitro. This distortion activity is consistent with both unpairing and unstacking of the three terminal base pairs, including the processing site adjacent to the conserved CA. Furthermore, the introduction of mismatch mutations that destabilize the viral DNA ends were found to stimulate the IN processing reaction as well as IN-mediated distortion. End-distortion activity was also observed with mutant or heterologous DNA substrates. However, further analyses showed that using Mn(2+) as a cofactor, processing site specificity of these substrates was also maintained. Our results support a model whereby unpairing and unstacking of the terminal base pairs is a required step in the processing reaction. Furthermore, these results are consistent with our previous observations indicating that unpairing of target DNA promotes the joining reaction.
Katz
R A
RA
Fox Chase Cancer Center, Institute for Cancer Research, Philadelphia, Pennsylvania 19111, USA. R_Katz@fccc.edu
DiCandeloro
P
P
Kukolj
G
G
Skalka
A M
AM
D001358:Avian Sarcoma Viruses / Q000201:enzymology*; D001483:Base Sequence; D002384:Catalysis; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D019426:Integrases / Q000737:chemistry* / Q000494:pharmacology; D008345:Manganese / Q000737:chemistry; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011196:Potassium Permanganate / Q000494:pharmacology; D011485:Protein Binding; D013379:Substrate Specificity; D013997:Time Factors
D011196:Potassium Permanganate; D008345:Manganese; D004247:DNA; D019426:Integrases