Activation of the DNA Damage Checkpoint in Yeast Lacking the Histone Chaperone Anti-Silencing Function 1

Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center at Fitzsimons, P.O. Box 6511, Aurora, CO 80045, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 01/2005; 24(23):10313-27. DOI: 10.1128/MCB.24.23.10313-10327.2004
Source: PubMed


The packaging of the eukaryotic genome into chromatin is likely to be important for the maintenance of genomic integrity. Chromatin structures are assembled onto newly synthesized DNA by the action of chromatin assembly factors, including anti-silencing function 1 (ASF1). To investigate the role of chromatin structure in the maintenance of genomic integrity, we examined budding yeast lacking the histone chaperone Asf1p. We found that yeast lacking Asf1p accumulate in metaphase of the cell cycle due to activation of the DNA damage checkpoint. Furthermore, yeast lacking Asf1p are highly sensitive to mutations in DNA polymerase alpha and to DNA replicational stresses. Although yeast lacking Asf1p do complete DNA replication, they have greatly elevated rates of DNA damage occurring during DNA replication, as indicated by spontaneous Ddc2p-green fluorescent protein foci. The presence of elevated levels of spontaneous DNA damage in asf1 mutants is due to increased DNA damage, rather than the failure to repair double-strand DNA breaks, because asf1 mutants are fully functional for double-strand DNA repair. Our data indicate that the altered chromatin structure in asf1 mutants leads to elevated rates of spontaneous recombination, mutation, and DNA damage foci formation arising during DNA replication, which in turn activates cell cycle checkpoints that respond to DNA damage.

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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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    • "In yeast, it was demonstrated that Asf1 shields H3/H4 from unfavorable DNA interactions and aids the formation of favorable histone-DNA interactions through the formation of disomes (Donham et al., 2011). In addition, yeast cells lacking Asf1 display an increased frequency of genome instability and spontaneous genome rearrangement (Myung et al., 2003; Prado et al., 2004; Ramey et al., 2004). ASF1 is also required to efficiently complete DNA replication in the presence of DNA-damaging agents or compromised replication machinery (Franco et al., 2005). "
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    ABSTRACT: ASF1 is a key histone H3/H4 chaperone that participates in a variety of DNA and chromatin-related processes, including DNA repair, where chromatin assembly and disassembly is of primary relevance. Information concerning the role of ASF1 proteins in post-UV response in higher plants is currently limited. In Arabidopsis thaliana, an initial analysis of in vivo localization of ASF1A and ASF1B indicates that both proteins are mainly expressed in proliferative tissues. In silico promoter analysis identified ASF1A and ASF1B as potential targets of E2F transcription factors. These observations were experimentally validated, both in vitro by electrophoretic mobility shift assays, and in vivo by chromatin immunoprecipitation assays and expression analysis using transgenic plants with altered levels of different E2F transcription factors. These data suggest that ASF1A and ASF1B are regulated during cell cycle progression through E2F transcription factors. In addition, we found that ASF1A and ASF1B are associated with the UV-B induced DNA damage response in Arabidopsis thaliana. Transcript levels of ASF1A and ASF1B were increased following a UV-B-treatment. Consistent with a potential role in UV-B response, RNAi silenced plants of both genes showed increased sensitivity to UV-B compared to wild type plants. Finally, by coimmunoprecipitation analysis we found that ASF1, physically interacts with N-terminal acetylated histones H3 and H4, and with acetyltransferases of the HAM subfamily, which are known to be involved in cell cycle control and DNA repair, among other functions. Together, here we provide evidence that ASF1A and ASF1B are regulated by cell cycle progression and DNA repair after UV-B irradiation.
    Plant physiology 04/2013; 162(2). DOI:10.1104/pp.112.212837 · 6.84 Impact Factor
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    • "The chaperones we identify are orthologues of ASF1 and CAF-1. The genes encoding these proteins are dispensable in the yeast, Saccharomyces cerevisiae (32), possibly due to redundancy with other related functions, but essential in mammalian or other vertebrate cells. Depletion of either vertebrate ASF1 (33) or CAF-1 (34,35) leads to accumulation in S-phase, and these histone chaperones have also been linked to chromatin-based silencing in yeast (36–39) and mammals (40), similar to the situation we now observe in trypanosomes. "
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    ABSTRACT: Antigenic variation in African trypanosomes involves monoallelic expression and reversible silencing of variant surface glycoprotein (VSG) genes found adjacent to telomeres in polycistronic expression sites (ESs). We assessed the impact on ES silencing of five candidate essential chromatin-associated factors that emerged from a genome-wide RNA interference viability screen. Using this approach, we demonstrate roles in VSG ES silencing for two histone chaperones. Defects in S-phase progression in cells depleted for histone H3, or either chaperone, highlight in particular the link between chromatin assembly and DNA replication control. S-phase checkpoint arrest was incomplete, however, allowing G(2)/M-specific VSG ES derepression following knockdown of histone H3. In striking contrast, knockdown of anti-silencing factor 1A (ASF1A) allowed for derepression at all cell cycle stages, whereas knockdown of chromatin assembly factor 1b (CAF-1b) revealed derepression predominantly in S-phase and G(2)/M. Our results support a central role for chromatin in maintaining VSG ES silencing. ASF1A and CAF-1b appear to play constitutive and DNA replication-dependent roles, respectively, in the recycling and assembly of chromatin. Defects in these functions typically lead to arrest in S-phase but defective cells can also progress through the cell cycle leading to nucleosome depletion and derepression of telomeric VSG ESs.
    Nucleic Acids Research 08/2012; 40(20). DOI:10.1093/nar/gks813 · 9.11 Impact Factor
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