Author ORCID Identifier
Candice M. Etson: 0000-0002-9796-269X
Document Type
Article
Publication Date
1-1-2023
Publication Title
International Journal of Molecular Sciences
Abstract
Integration host factor (IHF) is a nucleoid-associated protein involved in DNA packaging, integration of viral DNA and recombination. IHF binds with nanomolar affinity to duplex DNA containing a 13 bp consensus sequence, inducing a bend of ~160° upon binding. We determined that IHF binds to DNA Four-way or Holliday junctions (HJ) with high affinity regardless of the presence of the consensus sequence, signifying a structure-based mechanism of recognition. Junctions, important intermediates in DNA repair and homologous recombination, are dynamic and can adopt either an open or stacked conformation, where the open conformation facilitates branch migration and strand exchange. Using ensemble and single molecule Förster resonance energy transfer (FRET) methods, we investigated IHF-induced changes in the population distribution of junction conformations and determined that IHF binding shifts the population to the open conformation. Further analysis of smFRET dynamics revealed that even in the presence of protein, the junctions remain dynamic as fast transitions are observed for the protein-bound open state. Protein binding alters junction conformational dynamics, as cross correlation analyses reveal the protein slows the transition rate at 1 mM Mg2+ but accelerates the transition rate at 10 mM Mg2+. Stopped flow kinetic experiments provide evidence for two binding steps, a rapid, initial binding step followed by a slower step potentially associated with a conformational change. These measurements also confirm that the protein remains bound to the junction during the conformer transitions and further suggest that the protein forms a partially dissociated state that allows junction arms to be dynamic. These findings, which demonstrate that IHF binds HJs with high affinity and stabilizes junctions in the open conformation, suggest that IHF may play multiple roles in the processes of integration and recombination in addition to stabilizing bacterial biofilms.
Keywords
fluorescence, Holliday Junction, integration host factor, single-molecule FRET
Volume
24
Issue
1
DOI
10.3390/ijms24010580
ISSN
16616596
Rights
© 2022 by the authors.
Recommended Citation
Lin, Shawn H.; Zhao, Dacheng; Deng, Vivian; Birdsall, Veronica K.; Ho, Suzanne; Buzovetsky, Olga; Etson, Candice M.; and Mukerji, Ishita, "Integration Host Factor Binds Dna Holliday Junctions" (2023). Physics: Faculty Publications, Smith College, Northampton, MA.
https://scholarworks.smith.edu/phy_facpubs/115
Comments
Archived as published.