Chromatin is made up of nucleosomes, where each nucleosome core particle consists of ~147 bp of DNA wrapped around an octamer of histone proteins. A canonical histone octamer contains two H2A-H2B dimers and one H3-H4 tetramer. H2A.Z is a well-conserved histone variant in eukaryotes and is essential for viability in metazoans. It is found at +1 nucleosomes next to transcription start sites, DNA replication origins and sites of DNA damage. H2A.Z is enzymatically deposited by SWR1 through an ATP-dependent reaction known as histone exchange. SWR1 is a one-megadalton chromatin remodeling complex consisting of 14 different protein subunits, with homologous complexes in human known as SRCAP and p400. SWR1 catalyzes histone exchange by evicting an H2A-H2B dimer from the nucleosome and inserting an H2A.Z-H2B dimer. Despite this knowledge, the molecular mechanism of histone exchange remains elusive. Here, we developed a 3-color single-molecule FRET assay to visualize how SWR1 manipulates nucleosomal DNA and histones in real-time during histone exchange. By strategically placing fluorophores on DNA and histones, we were able to simultaneously monitor DNA unwrapping and the eviction of H2A-H2B on single nucleosome substrates. We observed two types of DNA unwrapping events – unproductive DNA unwrapping which occurs prior to histone exchange and is caused by SWR1 binding alone, and productive DNA unwrapping which happens during histone exchange and requires the additional presence of H2A.Z-H2B and ATP. Unexpectedly, after eviction from the nucleosome, H2A-H2B stays associated with the SWR1-nucleosome complex and can be subsequently removed by a histone chaperone. Our results provide unprecedented details of how SWR1 manipulates the nucleosomal DNA during histone exchange and reveal the lifetimes of several previously unknown reaction intermediates. We believe this multi-color FRET approach can be applied to study to the kinetics of many other chromatin-related processes at the single-molecule level.

Real-time DNA and histone dynamics during SWR1-catalyzed histone H2A.Z exchange

ACS Fall 2021

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