scholarly journals Physical and Functional Interaction between the Bloom's Syndrome Gene Product and the Largest Subunit of Chromatin Assembly Factor 1

2004 ◽  
Vol 24 (11) ◽  
pp. 4710-4719 ◽  
Author(s):  
Renjie Jiao ◽  
Csanád Z. Bachrati ◽  
Graziella Pedrazzi ◽  
Patrick Kuster ◽  
Maja Petkovic ◽  
...  
Keyword(s):  
Dna Repair ◽  
Cancer Susceptibility ◽  
Chromatin Assembly ◽  
Bloom's Syndrome ◽  
Functional Interaction ◽  
Chromatin Assembly Factor ◽  
Bloom’S Syndrome ◽  
Hydroxyurea Treatment ◽  
Assembly Factor

ABSTRACT Bloom's syndrome (BS) is a genomic instability disorder characterized by cancer susceptibility. The protein defective in BS, BLM, belongs to the RecQ family of DNA helicases. In this study, we found that BLM interacts with hp150, the largest subunit of chromatin assembly factor 1 (CAF-1), in vitro and in vivo. Colocalization of a proportion of the cellular complement of these two proteins is found at specific nuclear foci coinciding with sites of DNA synthesis in the S phase. This colocalization increases in the presence of agents that damage DNA or inhibit DNA replication. In support of a functional interaction between BLM and CAF-1, we show that BLM inhibits CAF-1-mediated chromatin assembly during DNA repair in vitro. Although CAF-1 activity is not altered in BLM-deficient cells, the absence of BLM does impair the ability of CAF-1 to be mobilized within the nucleus in response to hydroxyurea treatment. Our results provide the first link between BLM and chromatin assembly coupled to DNA repair and suggest that BLM and CAF-1 function in a coordinated way to promote survival in response to DNA damage and/or replication blockade.

scholarly journals Essential Role of Chromatin Assembly Factor-1–mediated Rapid Nucleosome Assembly for DNA Replication and Cell Division in Vertebrate Cells

2007 ◽  
Vol 18 (1) ◽  
pp. 129-141 ◽  
Author(s):  
Yasunari Takami ◽  
Tatsuya Ono ◽  
Tatsuo Fukagawa ◽  
Kei-ichi Shibahara ◽  
Tatsuo Nakayama
Keyword(s):  
Dna Replication ◽  
Essential Role ◽  
Chromatin Assembly ◽  
Nuclear Antigen ◽  
Nucleosome Assembly ◽  
Chromatin Assembly Factor ◽  
Assembly Factor

Chromatin assembly factor-1 (CAF-1), a complex consisting of p150, p60, and p48 subunits, is highly conserved from yeast to humans and facilitates nucleosome assembly of newly replicated DNA in vitro. To investigate roles of CAF-1 in vertebrates, we generated two conditional DT40 mutants, respectively, devoid of CAF-1p150 and p60. Depletion of each of these CAF-1 subunits led to delayed S-phase progression concomitant with slow DNA synthesis, followed by accumulation in late S/G2 phase and aberrant mitosis associated with extra centrosomes, and then the final consequence was cell death. We demonstrated that CAF-1 is necessary for rapid nucleosome formation during DNA replication in vivo as well as in vitro. Loss of CAF-1 was not associated with the apparent induction of phosphorylations of S-checkpoint kinases Chk1 and Chk2. To elucidate the precise role of domain(s) in CAF-1p150, functional dissection analyses including rescue assays were preformed. Results showed that the binding abilities of CAF-1p150 with CAF-1p60 and DNA polymerase sliding clamp proliferating cell nuclear antigen (PCNA) but not with heterochromatin protein HP1-γ are required for cell viability. These observations highlighted the essential role of CAF-1–dependent nucleosome assembly in DNA replication and cell proliferation through its interaction with PCNA.

scholarly journals Chromatin assembly factor-1 (CAF-1) chaperone regulates Cse4 deposition into chromatin in budding yeast

2018 ◽  
Vol 46 (9) ◽  
pp. 4440-4455 ◽  
Author(s):  
Geetha S Hewawasam ◽  
Karthik Dhatchinamoorthy ◽  
Mark Mattingly ◽  
Chris Seidel ◽  
Jennifer L Gerton
Keyword(s):  
Gene Expression ◽  
Chromosome Segregation ◽  
Budding Yeast ◽  
Chromatin Assembly ◽  
Growth Defects ◽  
Chromatin Assembly Factor ◽  
Genome Wide ◽  
Assembly Factor ◽  
Underlying Mechanisms

Abstract Correct localization of the centromeric histone variant CenH3/CENP-A/Cse4 is an important part of faithful chromosome segregation. Mislocalization of CenH3 could affect chromosome segregation, DNA replication and transcription. CENP-A is often overexpressed and mislocalized in cancer genomes, but the underlying mechanisms are not understood. One major regulator of Cse4 deposition is Psh1, an E3 ubiquitin ligase that controls levels of Cse4 to prevent deposition into non-centromeric regions. We present evidence that Chromatin assembly factor-1 (CAF-1), an evolutionarily conserved histone H3/H4 chaperone with subunits shown previously to interact with CenH3 in flies and human cells, regulates Cse4 deposition in budding yeast. yCAF-1 interacts with Cse4 and can assemble Cse4 nucleosomes in vitro. Loss of yCAF-1 dramatically reduces the amount of Cse4 deposited into chromatin genome-wide when Cse4 is overexpressed. The incorporation of Cse4 genome-wide may have multifactorial effects on growth and gene expression. Loss of yCAF-1 can rescue growth defects and some changes in gene expression associated with Cse4 deposition that occur in the absence of Psh1-mediated proteolysis. Incorporation of Cse4 into promoter nucleosomes at transcriptionally active genes depends on yCAF-1. Overall our findings suggest CAF-1 can act as a CenH3 chaperone, regulating levels and incorporation of CenH3 in chromatin.

scholarly journals The p55 subunit of Drosophila chromatin assembly factor 1 is homologous to a histone deacetylase-associated protein.

1996 ◽  
Vol 16 (11) ◽  
pp. 6149-6159 ◽  
Author(s):  
J K Tyler ◽  
M Bulger ◽  
R T Kamakaka ◽  
R Kobayashi ◽  
J T Kadonaga
Keyword(s):  
Drosophila Melanogaster ◽  
Dna Replication ◽  
Histone Modification ◽  
Molecular Mass ◽  
Histone Deacetylase ◽  
Nuclear Extract ◽  
Chromatin Assembly ◽  
Chromatin Assembly Factor ◽  
Assembly Factor

To gain a better understanding of DNA replication-coupled chromatin assembly, we have isolated the cDNA encoding the smallest (apparent molecular mass, 55 kDa; termed p55) subunit of Drosophila melanogaster chromatin assembly factor 1 (dCAF-1), a multisubunit protein that is required for the assembly of nucleosomes onto newly replicated DNA in vitro. The p55 polypeptide comprises seven WD repeat motifs and is homologous to the mammalian RbAp48 protein, which is associated with the HD1 histone deacetylase. dCAF-1 was immunopurified by using affinity-purified antibodies against p55; the resulting dCAF-1 preparation possessed the four putative subunits of dCAF-1 (p180, p105, p75, and p55) and was active for DNA replication-coupled chromatin assembly. Moreover, dCAF-1 activity was specifically depleted with antibodies against p55. Thus, p55 is an integral component of dCAF-1. p55 is localized to the nucleus and is present throughout Drosophila development. Consistent with the homology between p55 and the HD1-associated RbAp48 protein, histone deacetylase activity was observed to coimmunoprecipitate specifically with p55 from a Drosophila nuclear extract. Furthermore, a fraction of the p55 protein becomes associated with the newly assembled chromatin following DNA replication. These findings collectively suggest that p55 may function as a link between DNA replication-coupled chromatin assembly and histone modification.

scholarly journals Role of Saccharomyces cerevisiae Chromatin Assembly Factor-I in Repair of Ultraviolet Radiation Damage in Vivo

Genetics ◽  
1999 ◽  
Vol 151 (2) ◽  
pp. 485-497 ◽  
Author(s):  
John C Game ◽  
Paul D Kaufman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Repair ◽  
Gene Silencing ◽  
Chromatin Assembly ◽  
Yeast Cells ◽  
Repair Gene ◽  
Uv Sensitivity ◽  
Factor I ◽  
Chromatin Assembly Factor ◽  
Assembly Factor

Abstract In vitro, the protein complex Chromatin Assembly Factor-I (CAF-I) from human or yeast cells deposits histones onto DNA templates after replication. In Saccharomyces cerevisiae, the CAC1, CAC2, and CAC3 genes encode the three CAF-I subunits. Deletion of any of the three CAC genes reduces telomeric gene silencing and confers an increase in sensitivity to killing by ultraviolet (UV) radiation. We used double and triple mutants involving cac1Δ and yeast repair gene mutations to show that deletion of the CAC1 gene increases the UV sensitivity of cells mutant in genes from each of the known DNA repair epistasis groups. For example, double mutants involving cac1Δ and excision repair gene deletions rad1Δ or rad14Δ showed increased UV sensitivity, as did double mutants involving cac1Δ and deletions of members of the RAD51 recombinational repair group. cac1Δ also increased the UV sensitivity of strains with defects in either the error-prone (rev3Δ) or error-free (pol30-46) branches of RAD6-mediated postreplicative DNA repair but did not substantially increase the sensitivity of strains carrying null mutations in the RAD6 or RAD18 genes. Deletion of CAC1 also increased the UV sensitivity and rate of UV-induced mutagenesis in rad5Δ mutants, as has been observed for mutants defective in error-free postreplicative repair. Together, these data suggest that CAF-I has a role in error-free postreplicative damage repair and may also have an auxiliary role in other repair mechanisms. Like the CAC genes, RAD6 is also required for gene silencing at telomeres. We find an increased loss of telomeric gene silencing in rad6Δ cac1Δ and rad18Δ cac1Δ double mutants, suggesting that CAF-I and multiple factors in the postreplicative repair pathway influence chromosome structure.

scholarly journals Insights into the molecular architecture and histone H3-H4 deposition mechanism of yeast Chromatin assembly factor 1

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Paul Victor Sauer ◽  
Jennifer Timm ◽  
Danni Liu ◽  
David Sitbon ◽  
Elisabetta Boeri-Erba ◽  
...  
Keyword(s):  
Dna Binding ◽  
Histone H3 ◽  
Chromatin Assembly ◽  
Molecular Architecture ◽  
Dna Molecules ◽  
Nucleosome Assembly ◽  
Chromatin Assembly Factor ◽  
Assembly Factor ◽  
Chaperone Complex

How the very first step in nucleosome assembly, deposition of histone H3-H4 as tetramers or dimers on DNA, is accomplished remains largely unclear. Here, we report that yeast chromatin assembly factor 1 (CAF1), a conserved histone chaperone complex that deposits H3-H4 during DNA replication, binds a single H3-H4 heterodimer in solution. We identify a new DNA-binding domain in the large Cac1 subunit of CAF1, which is required for high-affinity DNA binding by the CAF1 three-subunit complex, and which is distinct from the previously described C-terminal winged-helix domain. CAF1 binds preferentially to DNA molecules longer than 40 bp, and two CAF1-H3-H4 complexes concertedly associate with DNA molecules of this size, resulting in deposition of H3-H4 tetramers. While DNA binding is not essential for H3–H4 tetrasome deposition in vitro, it is required for efficient DNA synthesis-coupled nucleosome assembly. Mutant histones with impaired H3-H4 tetramerization interactions fail to release from CAF1, indicating that DNA deposition of H3-H4 tetramers by CAF1 requires a hierarchical cooperation between DNA binding, H3-H4 deposition and histone tetramerization.

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