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Site-specific photo-cross-linking between lambda integrase and its DNA recombination target
Margaret J Kovach1, Radhakrishna Tirumalai, Arthur Landy
1Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island 02912, USA.
The Journal of Biological Chemistry
|February 6, 2002
Summary
Researchers studied bacteriophage lambda integrase (Int) by creating DNA with a photoreactive analog. They found a specific DNA modification that efficiently cross-linked Int, revealing insights into DNA recognition by Int recombinases.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Bacteriophage lambda integrase (Int) is a key enzyme in site-specific DNA recombination.
- Int functions as a heterobivalent DNA-binding protein with distinct functional domains.
Purpose of the Study:
- To investigate the DNA-binding and catalytic mechanisms of lambda Int.
- To identify specific DNA-protein interactions within the recombination core site.
Main Methods:
- Construction of core-type DNA oligonucleotides containing a photoreactive analog, 4-thiodeoxythymidine (4-thioT).
- UV irradiation (366 nm) to induce photo-cross-linking between DNA and Int.
- Analysis of cross-linking efficiency, DNA binding, cleavage, and recombination activity.
Main Results:
- A 4-thioT substitution on the DNA strand opposite the Int cleavage site resulted in ~20% photo-induced cross-linking efficiency.
- Int binding and cleavage at the modified site were largely unaffected.
- Site-specific recombination activity was only slightly reduced.
- Photo-cross-linking identified Lys-141 in the central domain as a key residue interacting with the core DNA site.
Conclusions:
- The study elucidates critical DNA-protein interactions in the central domain of lambda Int during recombination.
- Photo-cross-linking provides a powerful tool for mapping protein-DNA interfaces in Int family recombinases.
- Lys-141 is identified as a significant residue involved in core-type DNA recognition.