Tyler, J. K. et al. The RCAF complex mediates chromatin assembly during DNA replication and repair. Nature 402, 555- 560
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.
- SourceAvailable from: Namgyu Lee
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- "As expected, histone proteins associated with chromatin assembly such as HIST1H1D, H2AFZ, and H2AFY were enriched in our proteomic analysis (Fig. 2E), which is consistent with the previous reports that nuclear sirtuins could regulate chromatin structures by deacetylating histone proteins   , and both SIRT6 and SIRT7 were preferentially localized in the chromatin region  . Moreover, chromatin assembly was shown to be coupled to the DNA repair system  . SIRT6 regulates chromatin assembly by recruiting SNF2H to DNA break sites and changes chromatin structure by deacetylating H3K56 to promote the recruitment of DNA repair factors . "
ABSTRACT: Sirtuins are NAD+-dependent deacetylases that regulate a range of cellular processes. Although diverse functions of sirtuins have been proposed, those functions of SIRT6 and SIRT7 which are mediated by their interacting proteins remain elusive. In the present study, we identified SIRT6- and SIRT7-interacting proteins, and compared their interactomes to investigate functional links. Our interactomes revealed 136 interacting proteins for SIRT6 and 233 for SIRT7 while confirming 7 and 111 proteins identified previously for SIRT6 and SIRT7, respectively. Comparison of SIRT6 and SIRT7 interactomes under the same experimental conditions disclosed 111 shared proteins, implying related functional links. The interaction networks of interactomes indicated biological processes associated with DNA repair, chromatin assembly and aging. Interactions of two highly acetylated proteins, nucleophosmin (NPM1) and nucleolin, with SIRT6 and SIRT7 were confirmed by co-immunoprecipitation. NPM1 was found to be deacetylated by both SIRT6 and SIRT7. In senescent cells, the acetylation level of NPM1 was increased in conjunction with decreased levels of SIRT6 and SIRT7, suggesting that the acetylation of NPM1 could be regulated by SIRT6 and SIRT7 in the aging process. Our comparative interactomic study of SIRT6 and SIRT7 implies important functional links to aging by their associations with interacting proteins.This article is protected by copyright. All rights reservedProteomics 07/2014; 14(13-14). DOI:10.1002/pmic.201400001 · 3.97 Impact Factor
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- "Genes Genom strains lacking Vps15 or Vps34 exhibited high sensitivity to HU to a degree comparable with that of the asf1D strain, which is significantly sensitive to HU (Tyler et al. 1999). However, the ESCRT deletion strains and the strain deficient in ESCRT-III adaptor protein Bro1 showed no sensitivity to HU (Fig. 1b). "
ABSTRACT: Endosomal sorting complex required for transport (ESCRT) is involved in membrane protein degradation through the recognition and sorting of ubiquitylated cargo proteins into the multivesicular body before fusion with the lysosome/vacuole. However, recent studies have challenged this canonical cellular function of ESCRT and have implicated a role for this machinery in multiple intracellular pathways. Here, we provide evidence that ESCRT complexes contribute to the regulation of transcription elongation in Saccharomyces cerevisiae. Most strains deficient in each subunit of ESCRT-0, -I, -II, and -III showed significant sensitivity to 6-azauracil or mycophenolic acid, a phenotype associated with transcription elongation defects. Moreover, these deletion strains significantly reduced transcription activation through Gcn4, a regulator of the general amino acid control. The transcription factor Rim101, which is proteolytically activated through the multimerized component Vps32/Snf7 of ESCRT-III and its collaborative proteins, was not associated with transcription elongation or Gcn4 activation. In addition, we observed that ESCRT complexes were crosslinked at the 3′ region of the coding sequence in the actively transcribed gene. In summary, these results suggest that ESCRT complexes promote genes transcription during the late stages of elongation and are required for transcription activation through Gcn4.Genes & genomics 06/2014; 36(3). DOI:10.1007/s13258-013-0171-8 · 0.57 Impact Factor
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- "Identified as a single protein in yeast (Le et al. 1997), ASF1 exists as two paralogs in most vertebrates, termed ASF1a and ASF1b in mammals. Among its conserved functions, ASF1 synergizes with two H3–H4 cochaperones: chromatin assembly factor 1 (CAF-1, consisting of three subunits p48, p60, and p150) and histone regulator A (HIRA, homolog of the Hir complex in yeast) to channel the replicative H3.1 and replacement H3.3 histone variants in distinct deposition pathways (Tyler et al. 1999; Mello et al. 2002; Tagami et al. 2004; Green et al. 2005; De Koning et al. 2007; Mousson et al. 2007; Yamane et al. 2011). Although conservation of the ASF1 core binding domain for histones H3–H4 and cochaperones suggests common properties, experimental evidence suggests that the human paralogs are not functionally equivalent (Tagami et al. 2004; Tamburini et al. 2005; English et al. 2006; Tang et al. 2006; De Koning et al. 2007; Mousson et al. 2007; Natsume et al. 2007). "
ABSTRACT: Gene duplication is regarded as the main source of adaptive functional novelty in eukaryotes. Processes such as neo- and sub-functionalization impact the evolution of paralogous proteins where functional divergence is frequently key to retain the gene copies. Here we examined anti-silencing function 1 (ASF1), a conserved eukaryotic H3-H4 histone chaperone, involved in histone dynamics during replication, transcription, and DNA repair. While yeast feature a single ASF1 protein, two paralogs exist in most vertebrates, termed ASF1a and ASF1b, with distinct cellular roles in mammals. To explain this division of tasks, we integrated evolutionary and comparative genomic analyses with biochemical and structural approaches. First, we show that a duplication event at the ancestor of jawed vertebrates, followed by ASF1a relocation into an intron of MCM9 gene at the ancestor of tetrapods, provided a different genomic environment for each paralog with marked differences of GC content and DNA replication timing. Second, we found signatures of positive selection in the N- and C-terminal regions of ASF1a and ASF1b. Third, we demonstrate that regions outside the primary interaction surface are key for the preferential interactions of the human paralogs with distinct H3-H4 chaperones. Based on these data, we propose that ASF1 experienced subfunctionalization shaped by the adaptation of the genes to their respective genomic context, reflecting a case of genomic-context driven escape from adaptive conflict.Molecular Biology and Evolution 05/2013; 30(8). DOI:10.1093/molbev/mst086 · 14.31 Impact Factor