Quantifying ATP-Independent Nucleosome Chaperone Activity with Single-Molecule Methods.

Autor:	McCauley MJ; Department of Physics, Northeastern University, Boston, MA, USA., Joshi J; Department of Physics, Northeastern University, Boston, MA, USA., Becker N; Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, Rochester, MN, USA., Hu Q; Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, Rochester, MN, USA., Botuyan MV; Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, Rochester, MN, USA., Rouzina I; Department of Chemistry and Biochemistry, Ohio State University, Columbus, OH, USA., Mer G; Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, Rochester, MN, USA., James Maher L 3rd; Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, Rochester, MN, USA., Williams MC; Department of Physics, Northeastern University, Boston, MA, USA. ma.williams@northeastern.edu.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2024; Vol. 2694, pp. 29-55.
DOI:	10.1007/978-1-0716-3377-9_2
Abstrakt:	The dynamics of histone-DNA interactions govern chromosome organization and regulates the processes of transcription, replication, and repair. Accurate measurements of the energies and the kinetics of DNA binding to component histones of the nucleosome under a variety of conditions are essential to understand these processes at the molecular level. To accomplish this, we employ three specific single-molecule techniques: force disruption (FD) with optical tweezers, confocal imaging (CI) in a combined fluorescence plus optical trap, and survival probability (SP) measurements of disrupted and reformed nucleosomes. Short arrays of positioned nucleosomes serve as a template for study, facilitating rapid quantification of kinetic parameters. These arrays are then exposed to FACT (FAcilitates Chromatin Transcription), a non-ATP-driven heterodimeric nuclear chaperone known to both disrupt and tether histones during transcription. FACT binding drives off the outer wrap of DNA and destabilizes the histone-DNA interactions of the inner wrap as well. This reorganization is driven by two key domains with distinct function. FD experiments show the SPT16 MD domain stabilizes DNA-histone contacts, while the HMGB box of SSRP1 binds DNA, destabilizing the nucleosome. Surprisingly, CI experiments do not show tethering of disrupted histones, but increased rates of histone release from the DNA. SI experiments resolve this, showing that the two active domains of FACT combine to chaperone nucleosome reassembly after the timely release of force. These combinations of single-molecule approaches show FACT is a true nucleosome catalyst, lowering the barrier to both disruption and reformation. (© 2024. The Author(s).)
Databáze:	MEDLINE
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