title: Yeast CAF-1 assembles histone (H3-H4)2 tetramers prior to DNA deposition.
abstract: Following acetylation, newly synthesized H3-H4 is directly transferred from the histone chaperone anti-silencing factor 1 (Asf1) to chromatin assembly factor 1 (CAF-1), another histone chaperone that is critical for the deposition of H3-H4 onto replicating DNA. However, it is unknown how CAF-1 binds and delivers H3-H4 to the DNA. Here, we show that CAF-1 binds recombinant H3-H4 with 10- to 20-fold higher affinity than H2A-H2B in vitro, and H3K56Ac increases the binding affinity of CAF-1 toward H3-H4 2-fold. These results provide a quantitative thermodynamic explanation for the specific H3-H4 histone chaperone activity of CAF-1. Surprisingly, H3-H4 exists as a dimer rather than as a canonical tetramer at mid-to-low nanomolar concentrations. A single CAF-1 molecule binds a cross-linked (H3-H4)2 tetramer, or two H3-H4 dimers that contain mutations at the (H3-H4)2 tetramerization interface. These results suggest that CAF-1 binds to two H3-H4 dimers in a manner that promotes formation of a (H3-H4)2 tetramer. Consistent with this idea, we confirm that CAF-1 synchronously binds two H3-H4 dimers derived from two different histone genes in vivo. Together, the data illustrate a clear mechanism for CAF-1-associated H3-H4 chaperone activity in the context of de novo nucleosome (re)assembly following DNA replication.
authors.affiliation: Howard Hughes Medical Institute and the Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Duane D
authors.initials: DD
authors.lastname: Winkler
authors.forename: Hui
authors.initials: H
authors.lastname: Zhou
authors.forename: Mohd A
authors.initials: MA
authors.lastname: Dar
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Zhang
authors.forename: Karolin
authors.initials: K
authors.lastname: Luger
mesh_terms: D000107:Acetylation; D000818:Animals; D056504:Chromatin Assembly Factor-1 / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D017384:Sequence Deletion; D013816:Thermodynamics; D014982:Xenopus laevis; D015003:Yeasts / Q000378:metabolism
chemical_list: D056504:Chromatin Assembly Factor-1; D005656:Fungal Proteins; D006657:Histones; D004247:DNA