Tyler, J. K. et al. The RCAF complex mediates chromatin assembly during DNA replication and repair. Nature 402, 555- 560

Department of Biology and Center for Molecular Genetics, University of California at San Diego, La Jolla 92093-0347, USA.
Nature (Impact Factor: 41.46). 12/1999; 402(6761):555-560. DOI: 10.1038/990147

ABSTRACT Chromatin assembly is a fundamental biological process that is essential for the replication and maintenance of the eukaryotic genome. In dividing cells, newly synthesized DNA is rapidly assembled into chromatin by the deposition of a tetramer of the histone proteins H3 and H4, followed by the deposition of two dimers of histones H2A and H2B to complete the nucleosome the fundamental repeating unit of chromatin. Here we describe the identification, purification, cloning, and characterization of replication- coupling assembly factor (RCAF), a novel protein complex that facilitates the assembly of nucleosomes onto newly replicated DNA in vitro. RCAF comprises the Drosophila homologue of anti-silencing function 1 protein ASF1 and histones H3 and H4. The specific acetylation pattern of H3 and H4 in RCAF is identical to that of newly synthesized histones. Genetic analyses in Saccharomyces cerevisiae demonstrate that ASF1 is essential for normal cell cycle progression, and suggest that RCAF mediates chromatin assembly after DNA replication and the repair of double-strand DNA damage in vivo.

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    • "A tetramer of histone H3 and H4 and two H2A-H2B dimers form the histone octamer (Kornberg, 1974; Luger et al., 1997; Thomas and Kornberg, 1975). During DNA replication, nucleosome disassembly and reassembly occurs at the replication fork, and histone chaperons CAF-1 and ASF1 are principally responsible for the deposition of histones H3 and H4 onto replicated DNA (Kaufman et al., 1995; Tyler et al., 1999). ASF1 binds a heterodimeric histone H3-H4 complex through its conserved N-terminal domain (aa 1–155) and impedes the formation of the (H3-H4) 2 tetramer both inside the nucleus and in the cytoplasm (English et al., 2005). "
    Protein & Cell 07/2015; 6(9). DOI:10.1007/s13238-015-0190-0 · 3.25 Impact Factor
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    • "Yeast cells lacking ASF1 are sensitive to DNA damaging agents, as it plays important role in checkpoint signaling and genomic stability [13], [17], [18]. In addition to its role as chromatin assembly and disassembly factor, Asf1 is essentially required, in collaboration with histone acetyl-transferases (HATs), for the acetylation of lysine residues of histone H3 at positions 9 and 56 [19], [20]. "
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    ABSTRACT: Genome-wide participation and importance of the histone chaperone Asf1 (Anti-Silencing Function 1) in diverse DNA transactions like replication, repair, heterochromatic silencing and transcription are well documented. Yet its genome-wide targets have not been reported. Using ChIP-seq method, we found that yeast Asf1 associates with 590 unique targets including centromeres, telomeres and condensin-binding sites. It is found selectively on highly transcribed regions, which include replication fork pause sites. Asf1 preferentially associates with the genes transcribed by RNA polymerase (pol) III where its presence affects RNA production and replication-independent histone exchange. On pol II-transcribed genes, a negative correlation is found between Asf1 and nucleosome occupancy. It is not enriched on most of the reported sites of histone exchange or on the genes, which are misregulated in the asf1Δ cells. Interestingly, chromosome-wide distributions of Asf1 and one of the condensin subunits, Brn1 show a nearly identical pattern. Moreover, Brn1 shows reduced occupancy at various condensin-binding sites in asf1Δ cells. These results along with high association of Asf1 with heterochromatic centromeres and telomeres ascribe novel roles to Asf1 in condensin loading and chromatin dynamics.
    PLoS ONE 09/2014; 9(9):e108652. DOI:10.1371/journal.pone.0108652 · 3.23 Impact Factor
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    • "The synergistic activity seen between Hif1p and the cytosolic extract was, in some ways, similar to the increased levels of chromatin assembly that are seen when Asf1p is assayed with either CAF-1 or the HIR complex[16]. Therefore, a candidate gene approach was used and cytosolic extracts were generated from strains in which potential chromatin assembly factors had been deleted. "
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    ABSTRACT: Background Hif1p is an H3/H4-specific histone chaperone that associates with the nuclear form of the Hat1p/Hat2p complex (NuB4 complex) in the yeast Saccharomyces cerevisiae. While not capable of depositing histones onto DNA on its own, Hif1p can act in conjunction with a yeast cytosolic extract to assemble nucleosomes onto a relaxed circular plasmid. Results To identify the factor(s) that function with Hif1p to carry out chromatin assembly, multiple steps of column chromatography were carried out to fractionate the yeast cytosolic extract. Analysis of partially purified fractions indicated that Hif1p-dependent chromatin assembly activity resided in RNA rather than protein. Fractionation of isolated RNA indicated that the chromatin assembly activity did not simply purify with bulk RNA. In addition, the RNA-mediated chromatin assembly activity was blocked by mutations in the human homolog of Hif1p, sNASP, that prevent the association of this histone chaperone with histone H3 and H4 without altering its electrostatic properties. Conclusions These results suggest that specific RNA species may function in concert with histone chaperones to assemble chromatin.
    PLoS ONE 07/2014; 9(7):e100299. DOI:10.1371/journal.pone.0100299 · 3.23 Impact Factor
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