Journal Article

Integron integrase binds to bulged hairpin DNA

Carolina Johansson, Masood Kamali-Moghaddam and Lars Sundström

in Nucleic Acids Research

Volume 32, issue 13, pages 4033-4043
Published in print January 2004 | ISSN: 0305-1048
Published online January 2004 | e-ISSN: 1362-4962 | DOI: http://dx.doi.org/10.1093/nar/gkh730
Integron integrase binds to bulged hairpin DNA

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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.

Journal Article.  8208 words.  Illustrated.

Subjects: Chemistry ; Biochemistry ; Bioinformatics and Computational Biology ; Genetics and Genomics ; Molecular and Cell Biology

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