Abstract
Human GEN1 is a cytosolic homologous recombination protein that resolves persisting four-way Holliday junctions (HJ) after the dissolution of the nuclear membrane. GEN1 dimerization has been suggested to play key role in the resolution of the HJ, but the kinetic details of its reaction remained elusive. Here, single-molecule FRET shows how human GEN1 binds the HJ and always ensures its resolution within the lifetime of the GEN1-HJ complex. GEN1 monomer generally follows the isomer bias of the HJ in its initial binding and subsequently distorts it for catalysis. GEN1 monomer remains tightly bound with no apparent dissociation until GEN1 dimer is formed and the HJ is fully resolved. Fast on- and slow off-rates of GEN1 dimer and its increased affinity to the singly-cleaved HJ enforce the forward reaction. Furthermore, GEN1 monomer binds singly-cleaved HJ tighter than intact HJ providing a fail-safe mechanism if GEN1 dimer or one of its monomers dissociates after the first cleavage. The tight binding of GEN1 monomer to intact- and singly-cleaved HJ empowers it as the last resort to process HJs that escape the primary mechanisms.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1935-1949 |
| Number of pages | 15 |
| Journal | Nucleic Acids Research |
| Volume | 47 |
| Issue number | 4 |
| DOIs | |
| State | Published - Dec 22 2018 |
Bibliographical note
KAUST Repository Item: Exported on 2020-10-01Acknowledgements: ACKNOWLEDGEMENTS: We thank Prof. Yuji Iwata for his help in the initial stage of cloning GEN1. We thank Yujing Ouyang for the preparation of the functionalized cover slips. We thank Dr Susan Tsutakawa, Prof. John Tainer and Dr Maged Serag for thoughtful discussions. FUNDING: King Abdullah University of Science and Technology through core funding and Competitive Research Award [CRG3 to S.M.H.]. Funding for open access charge: King Abdullah University of Science and Technology through CRG3.