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Structural basis of directionality control in large serine integrases
Heewhan Shin1, Ying Pigli1, Tania Peña Reyes1
1Department of Biochemistry & Molecular Biology, The University of Chicago; Chicago IL, 60637, USA.
Biorxiv : the Preprint Server for Biology
|January 13, 2025
Summary
Large serine integrases (LSIs) mediate DNA recombination, but recombination directionality factors (RDFs) can reverse this. New cryo-EM structures reveal how RDFs control LSI reactions, guiding genome editing tool development.
Area of Science:
- Molecular Biology
- Structural Biology
- Genetics
Background:
- Large serine integrases (LSIs) are enzymes that catalyze site-specific DNA recombination.
- The directionality of LSI-mediated reactions is controlled by recombination directionality factors (RDFs).
- A lack of structural data has limited understanding of how directionality is controlled.
Purpose of the Study:
- To elucidate the mechanism of directionality control in large serine integrase-mediated DNA recombination.
- To provide structural insights into the role of recombination directionality factors (RDFs) in controlling integrase activity.
Main Methods:
- Cryo-electron microscopy (cryo-EM) was used to determine the structures of SPbeta integrase-DNA complexes.
- Six distinct complexes were resolved along both the integrative and excisive reaction pathways.
- High-resolution structures (4.16-7.18Å) were obtained for key reaction intermediates.
Main Results:
- The structures reveal how RDFs reposition a key integrase subdomain.
- This repositioning dictates the formation of synaptic complexes for recombination initiation.
- RDFs also lock product complexes, preventing reversal of the DNA recombination reaction.
Conclusions:
- The study provides unprecedented structural insights into LSI reaction mechanism and directionality control.
- Mechanistic understanding derived from these structures can inform the engineering of novel genome editing tools.
- The findings offer a framework for developing more efficient and versatile DNA manipulation technologies.
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