Integron integrase binds to bulged hairpin DNA.
Gene cassettes are short, monogenic DNA elements that translocate between integrons through site-specific excision and integration. These events require that an integron-coded tyrosine recombinase forms a reactive complex with two sites, at least one of which belongs to the attC class. An attC site can be divided into two pairs of short repeats flanking a palindromic central region. The nucleotide sequence of attC among different cassettes varies extensively, implying that the site contains a structural recognition determinant with low sequence constraints. Oligonucleotides representing many different sequence modifications in either strand of the site were examined for integrase binding by using an electrophoresis mobility shift assay. The inner repeats, a central triplet and two single-nucleotide asymmetries in the site had the strongest influence on binding strength and strand choice. Our data show that the recombinase binds to a bulged hairpin in attC and that the hairpin distortion due to bulging could define the appropriate orientation of the otherwise symmetrical site. This is consistent with the strong bias for binding of recombinase to the bottom-strand oligonucleotides in vitro. Moreover, it was observed that the mobility-shifted complexes persisted under protein-denaturing assay conditions, indicating that a covalent link is indeed formed between integrase and DNA. Upon substitution of the presumed DNA-attacking residue, Y312, with a phenylalanine, DNA binding remained but there was no covalent linkage.
Johansson
Carolina
C
Department of Medical Biochemistry and Microbiology (IMBIM), Uppsala University, Box 582, Biomedical Center, S-751 23 Uppsala, Sweden.
Kamali-Moghaddam
Masood
M
Sundström
Lars
L
D001483:Base Sequence; D001665:Binding Sites; D007446:Chromosome Inversion; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D032023:Integrons; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D016415:Sequence Alignment
D009841:Oligonucleotides; D019000:Phosphotyrosine; D004247:DNA; D019426:Integrases