title: Preliminary study on the DNA-binding properties of phage ΦC31 integrase.
abstract: ΦC31 integrase is a member of the large serine subfamily and is required for the recombination of the phage genome into the host chromosome, either to establish or exit from the lysogenic state. This enzyme can also mediate site-specific integration in mammalian cells in a cofactor-independent manner and has been considered as a potentially powerful tool for gene therapy. It has previously been reported that DAXX interacts with ΦC31 integrase and markedly inhibits its integration efficiency, and the 451RFGK454 tetramer of ΦC31 integrase has been identified as the interacting motif. Here, we report that both the deletion of the tetramer or the replacement of Arg with His greatly reduced the recombination activity of the ΦC31 integrase. Electrophoretic mobility shift assays further demonstrated that the DNA-binding ability and binding specificity of the two mutants were dramatically reduced. Bioinformatic analysis indicated a probable helix-turn-helix-like DNA-binding motif between residues 415-525, a region that contains the tetramer motif. However, neither truncated Int(415-525) nor Int(△415-525) alone could bind to the attB target sequence. Results of a circular dichroism spectroscopy assay indicated that Int(415-525) did not fold correctly into a helix-turn-helix-like structure, which may be one of the reasons for its lack of DNA-binding ability. Thus, the identification and confirmation of four key amino acids in the DNA-binding specificity and recombination activity of ΦC31 integrase provide information about the domain structure and function of the large C-terminal region and suggest important implications for the more efficient use of integrase in gene transfer and gene therapy.
authors.affiliation: State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, 200433, China.
authors.forename: Zhihui
authors.initials: Z
authors.lastname: Li
authors.forename: Yuxiang
authors.initials: Y
authors.lastname: Fang
authors.forename: Rencheng
authors.initials: R
authors.lastname: Wang
authors.forename: Jinglun
authors.initials: J
authors.lastname: Xue
authors.forename: Jinzhong
authors.initials: J
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D001287:Attachment Sites, Microbiological; D001435:Bacteriophages / Q000201:enzymology*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019426:Integrases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D014764:Viral Proteins; D019426:Integrases