Abstract
The site-specific recombinase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein and is composed of three domains as follows: an amino-terminal domain that binds with high affinity to "arm-type" sequences within the recombination target DNA (att sites), a carboxyl-terminal domain that contains all of the catalytic functions, and a central domain that contributes significantly to DNA binding at the "core-type" sequences where DNA cleavage and ligation are executed. We constructed a family of core-type DNA oligonucleotides, each of which contained the photoreactive analog 4-thiodeoxythymidine (4-thioT) at a different position. When tested for their respective abilities to promote covalent cross-links with Int after irradiation with UV light at 366 nm, one oligonucleotide stood out dramatically. The 4-thioT substitution on the DNA strand opposite the site of Int cleavage led to photo-induced cross-linking efficiencies of approximately 20%. The efficiency and specificity of Int binding and cleavage at this 4-thioT-substituted core site was shown to be largely uncompromised, and its ability to participate in a full site-specific recombination reaction was reduced only slightly. Identification of the photo-cross-linked residue as Lys-141 in the central domain provides, along with other results, several insights about the nature of core-type DNA recognition by the bivalent recombinases of the lambda Int family.
Highlights
The integrase (Int)1 protein of bacteriophage , which was first purified by Kikuchi and Nash [1], belongs to a large family of site-specific recombinases that rearrange DNA sequences having little or no sequence homology. Int is a heterobivalent site-specific DNA-binding protein and a type I topoisomerase that catalyzes the insertion and excision of the viral genome into and out of the host Escherichia coli genome
DNA cleavage is mediated by a tyrosine hydroxyl that attacks the scissile phosphate, forming a 3Ј-phosphotyrosine link to the nicked DNA
For other family members, such as and HP1 integrases, the att sites are more complex and contain additional protein-binding sites in viral DNA sequences that compose flanking “arms.” Some of these flanking sites bind to DNA bending accessory proteins like IHF, Xis, and Fis, whereas others bind to the amino-terminal domain of Int, resulting in a higher order complex where Int bridges the core and arm sequences of a sharply bent att DNA
Summary
Integrase; 4-thioT, 4-thiodeoxythymidine; nt, nucleotide; HPLC, high pressure liquid chromatography; CB, core binding; MOPS, 4-morpholinepropanesulfonic acid. The Cre/loxA cocrystal structure indicated that upon binding to lox (core-type) DNA, the catalytic domain buries ϳ50% more solvent-accessible surface area at the DNA interface than the amino-terminal (CB-analogous) domain [19]. To explore further these apparent dichotomies, we looked for a different chemistry with which to probe the Int-core DNA interface(s). An additional bonus of these experiments is the very high efficiency with which the photo-cross-link is generated, providing a potentially useful handle for dissecting the higher order organization of the large complex structures associated with this site-specific recombination pathway
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
