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Eukaryotic Cell, April 2005, p. 673-684, Vol. 4, No. 4
1535-9778/05/$08.00+0     doi:10.1128/EC.4.4.673-684.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Contribution of CAF-I to Anaphase-Promoting-Complex-Mediated Mitotic Chromatin Assembly in Saccharomyces cerevisiae

Troy A. A. Harkness,1* Terra G. Arnason,2 Charmaine Legrand,1 Marnie G. Pisclevich,1 Gerald F. Davies,1 and Emma L. Turner1

Department of Anatomy and Cell Biology,1 Department of Medicine, Royal University Hospital, University of Saskatchewan, Saskatoon, Saskatchewan, Canada2

Received 22 June 2004/ Accepted 21 January 2005


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ABSTRACT
 
The anaphase-promoting complex (APC) is required for mitotic progression and genomic stability. Recently, we demonstrated that the APC is also required for mitotic chromatin assembly and longevity. Here, we investigated the role the APC plays in chromatin assembly. We show that apc5CA mutations genetically interact with the CAF-I genes as well as ASF1, HIR1, and HIR2. When present in multiple copies, the individual CAF-I genes, CAC1, CAC2, and MSI1, suppress apc5CA phenotypes in a CAF-1- and Asf1p-independent manner. CAF-I and the APC functionally overlap, as cac1{Delta} cac2{Delta} msi1{Delta} (caf1{Delta}) cells expressing apc5CA exhibit a phenotype more severe than that of apc5CA or caf1{Delta}. The Ts– phenotypes observed in apc5CA and apc5CA caf mutants may be rooted in compromised histone metabolism, as coexpression of histones H3 and H4 suppressed the Ts– defects. Synthetic genetic interactions were also observed in apc5CA asf1{Delta} cells. Furthermore, increased expression of genes encoding Asf1p, Hir1p, and Hir2p suppressed the apc5CA Ts– defect in a CAF-I-dependent manner. Together, these results suggest the existence of a complex molecular mechanism controlling APC-dependent chromatin assembly. Our data suggest the APC functions with the individual CAF-I subunits, Asf1p, and the Hir1p and Hir2p proteins. However, Asf1p and an intact CAF-I complex are dispensable for CAF-I subunit suppression, whereas CAF-I is necessary for ASF1, HIR1, and HIR2 suppression of apc5CA phenotypes. We discuss the implications of our observations.


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INTRODUCTION
 
Chromatin assembly, the deposition of histones onto DNA, is an essential step in chromosome compaction, a process which determines the precision of chromosome segregation during mitosis (12, 24). Imperfections in chromatin assembly and remodeling have been linked to the onset of many disease states, including premature aging and cancer (reviewed in references 4, 6, 10, 29, and 31). Chromatin is composed of a string of nucleosomes, in which 147 bp are wrapped two times around an octameric complex called the nucleosome core particle (54). The nucleosome core particle is made up of two copies of the four histones, H2A, H2B, H3, and H4. Proteins called chromatin assembly factors (CAFs) facilitate nucleosome formation, and they have been isolated from virtually all systems studied (68, 71). However, little is known regarding how CAFs ensure proper chromatin assembly or how they are regulated. Recently, we isolated the apc5CA mutant, which compromised anaphase-promoting complex (APC) activity and impaired a novel mitotic chromatin assembly activity (20, 21). Furthermore, we demonstrated that the APC is required in Saccharomyces cerevisiae for longevity (22). Thus, the possibility exists, at least in S. cerevisiae, that defects in APC-dependent chromatin assembly impact longevity. To understand this potential correlation, it is imperative to explore the molecular mechanisms involved in APC-dependent chromatin assembly.

The APC is a large, evolutionarily conserved complex that is essential in S. cerevisiae. The APC functions as a ubiquitin-protein ligase (E3) in the ubiquitin-dependent targeting pathway (reviewed in references 23 and 51). The E3s mark the endpoint of a molecular cascade which relies on the prior activity of a ubiquitin-activating protein (E1) and one of a family of conserved and homologous ubiquitin-conjugating enzymes (E2s) (27). The E3s are structurally and functionally diverse and share the ability to select proteins for ubiquitination (65). The APC's role is to target proteins that prevent sister chromatid separation (Pds1p) and exit from mitosis (Clb2p) for proteasome-dependent degradation. The segregation of sister chromatids is sensitive to mitotic stress, and this depends in part on proper posttranslational modification of histones (11, 13). Thus, an efficient mechanism maintaining proper chromatin metabolism during mitosis may involve the coupling of sister chromatid separation with a chromatin repair, modification, and/or assembly machinery.

The chromatin assembly machinery potentially targeted by the APC would be expected to be mitosis specific, since the main function of the APC is to promote passage through mitosis. However, of all the CAFs identified, none have been shown to function exclusively during mitosis. The only CAF clearly linked to the cell cycle has been CAF-I, which is critical for S-phase-coupled nucleosome deposition (64). CAF-I is a trimeric complex conserved in virtually all eukaryotic systems studied (37, 39, 55, 64, 67). In S. cerevisiae, the CAF-I subunits are referred to as Cac1p, Cac2p, and Cac3p/Msi1p, whereas in humans the subunits are called p150, p60, and p48. Biochemical studies in vitro have shown that human CAF-I is associated with newly synthesized and acetylated histones H3 and H4 (36, 69). Furthermore, the observation that the p150 subunit of human CAF-I interacts with PCNA, a DNA polymerase clamp, sparked speculation that CAF-I is loaded onto replicating DNA via its interaction with PCNA (61, 73). Recent in vivo studies also support a role for CAF-I in S phase, as a dominant-negative p150 subunit mutation or depletion of CAF-I results in S-phase arrest with concomitant DNA damage and S-phase checkpoint activation (28, 72).

The role that CAF-I plays during S phase is not essential in S. cerevisiae, as S. cerevisiae cells lacking all three CAF-I subunits remain viable (14, 37). This may reflect redundant interactions between different CAFs. For example, yeast CAF-I has been shown to genetically and physically interact with several factors involved in chromatin metabolism, namely, Asf1p and the histone information regulators (Hir) (41, 48, 60, 66, 67). These studies revealed physical interactions between Cac2p/p60 and Asf1p and between Asf1 and the Hir proteins. Taken together, the accumulating data have lead to the suggestion that CAF-I and Asf1p/Hir proteins function in alternative chromatin assembly pathways (41). Moreover, the CAF-I p48 subunit has been isolated from plant and animal cells in CAF-I-independent complexes involved in histone acetylation and deacetylation (26, 57, 70). Although CAF-I is clearly required for S-phase-coupled chromatin assembly, CAF-I function appears to be far more complex than originally anticipated.

CAF-I has been demonstrated to function outside of S phase, as CAF-I is required for DNA repair-coupled chromatin assembly (16, 47, 49), and can assemble bulk chromatin in a replication-independent manner (35), and p60 and p150 colocalize with sites of nucleotide excision repair sites outside of S phase (42, 47). Moreover, yeast CAF-I was recently shown to be required, along with the Hir proteins, for the formation of functional kinetochores (60). Cells lacking both CAC1 and HIR1 exhibited a slow-growth phenotype due to a delay in progression through G2/M (60). Thus, it is becoming apparent that CAF-I can function outside of S phase.

In this report, we present genetic studies that suggest the existence of a complex molecular interaction between the APC and the CAF-I subunits as well as Asf1p, Hir1p, and Hir2p. The individual CAF-I subunits suppress APC defects in a CAF-I- and Asf1p-independent manner, whereas Asf1p, Hir1p, and Hir2p all require CAF-1.


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MATERIALS AND METHODS
 
Yeast strains. Table 1 lists the S. cerevisiae strains used. All strains were isogenic derivatives of S288c strains. The RMY102 strain (a generous gift from M. Grunstein) was used to purify plasmid pRM102 (45). To construct the cac1{Delta}::LEU2 strain, a plasmid (pPK103) containing cac1{Delta}::LEU2 and the CAC1 flanking region was obtained from P. Kaufman. A cac1{Delta}::LEU2 PCR fragment was generated by using primers designed to amplify 225 bp upstream and 1,063 bp downstream of the CAC1 coding region and pPK103 as the template. This fragment was transformed into YTH3 cells, and correct transformants were confirmed by PCR. YTH1173 was subsequently used in crossing experiments. All other strains were obtained from colleagues or generated through tetrad analyses. Strains containing msi1{Delta}::kanMX6 and cac2{Delta}::kanMX6 (Research Genetics) were selected from tetrads in which the kanMX6 marker segregated 2:2. Triple and quadruple mutants containing msi1{Delta}::kanMX6 and cac2{Delta}::kanMX6 were selected by picking spores from tetrads where the kanMX6 marker segregated 2:2 and the additional desired markers segregated with kanM6X.


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  		TABLE 1. S. cerevisiae strains

Media and methods. YPD (yeast extract, peptone, dextrose), CM (complete medium), SD (synthetic dextrose), SD lacking uracil (SD-ura), and SD lacking leucine (SD-leu) were prepared according to published protocols (56). Glycerol was used at 2% in place of glucose in plates. Standard genetic techniques were performed as described (19). In vitro chromatin assembly assays were performed according to previously published protocols (20, 21). Histone add-back experiments were conducted by including histones isolated from untreated or colcemid-treated HeLa cells, following acid extraction (Upstate), in chromatin assembly reactions according to the nonradioactive protocol (21). Spot dilutions were performed with cells, adjusted to 2 x 107/ml, from a fresh overnight culture. A 10-fold serial dilution series was then prepared, and 5 µl of each dilution was spotted onto the appropriate medium. The plates were placed in the appropriate incubator for 3 to 8 days. The plates were then scanned with an Epson Perfection 1650.

Standard DNA manipulations. Escherichia coli strains JM109 and DH10B were used to propagate DNA plasmids. DNA manipulations such as restriction enzyme digests, DNA minipreps, S. cerevisiae and E. coli transformations, and S. cerevisiae genomic DNA preparation have been described previously (56).

Plasmid construction. All plasmids used in this study are shown in Table 2. pTH130 was constructed by first cloning a CAC1 PCR fragment into pCR2.1-TOPO. The CAC1 PCR fragment contained 226 bp of upstream and 195 bp of downstream sequence. Both Topo-CAC1 and the S. cerevisiae 2µm expression vector, YEplac181 were digested with KpnI and PstI. The appropriate DNA fragments were ligated, resulting in plasmid pTH130 (YEp-CAC1). pRM102, which expresses histones H3 and H4 from the GAL promoter (45), was purified from RMY102.


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  		TABLE 2. Plasmids

Growth curves. Growth curves were generated by inoculating 5-ml YPD cultures with 1 x 104 cells from fresh cultures, followed by incubation at the indicated temperatures. Samples were removed at the indicated times, and the cell concentrations were determined by measurements of the optical density at 600 nm (OD600). Some variation was observed between different growth curves of the same strain. Therefore, the curves shown were all generated at the same time and are a typical representation of the results obtained.

Flow cytometry. Yeast cells were grown overnight in YPD at permissive temperatures. The next morning, cells were diluted to an OD600 of 0.2 and incubated with shaking at 30°C and 37°C till an OD600 of 0.5 was reached. The cells were then harvested and prepared for flow cytometry according to published protocols (15).


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RESULTS
 
Multicopy expression of CAF-I subunits suppresses apc5CA phenotypes. The chromatin assembly deficiency observed in apc5CA cells prompted us to examine whether the APC interacts with a specific CAF. Hence, we asked whether increased levels of CAFs would suppress apc5CA mutant phenotypes. We began our investigation with an analysis of the genes encoding the CAF-I subunits, CAC1, CAC2, and MSI1. Our results demonstrate that multicopy expression of CAC1, CAC2, and MSII could indeed suppress the Ts– growth and chromatin assembly defects associated with apc5CA cells (Fig. 1 and data not shown; a 2µm plasmid expressing MSI1, pJH49, was generously provided by M. Carlson). The finding that multicopy expression of MSI1 suppressed apc5CA defects provides an explanation as to how MSI1 could suppress both snf1{Delta} (30) and Ras2Val19 (58) phenotypes; Snf1p activates the APC (22), while Ras/protein kinase A signaling inhibits it (32, 40). Taken together, these data provide evidence that CAF-I acts downstream of the APC and, when overexpressed, may be capable of repairing damaged chromatin conferred by mutations that reduce APC activity.



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  		FIG. 1. Multicopy expression of individual CAF-I subunits can restore apc5CA phenotypes. (A) Multicopy expression of CAF-I subunit genes CAC1 and MSI1 suppresses the apc5CA growth defect at 37°C. apc5CA cells (YTH1155) were transformed with YEplac181 (2µm-LEU2), YEpCAC1, YEpMSI1, and YCpAPC5. The YCp vector is CEN-ARS based. The transformants were grown for 3 to 5 days on selective medium at 30°C and 37°C. (B) Multicopy expression of CAC1 or MSI1 suppresses the apc5CA in vitro chromatin assembly defect. The transformants described above were assayed for in vitro chromatin assembly according to published protocols (20, 21). The position of uniquely labeled, circular, relaxed pBlueScript plasmid (O, R) is indicated. Efficient chromatin assembly is observed when the amount of highly supercoiled topoisomers (sup.) greatly exceeds that of the intermediate topoisomers (inter.).

apc5CA and CAF-I mutations interact genetically. If increased expression of CAF-I subunits provided a benefit for apc5CA cells, then perhaps decreased expression would be detrimental. This prediction was borne out, as the combination of different caf mutations with apc5CA exacerbated the apc5CA Ts– growth defect (Fig. 2). Previous studies showed that disruption of one CAF-I subunit was as deleterious as deleting all three, indicating that Cac1p, Cac2p, and Msi1p acted together (14, 37). If the intact CAF-I complex was required for APC function, then progressive disruption of CAF-I subunits in apc5CA cells would not be expected to create a more severe phenotype. However, this was not the case, as we observed that when double and triple caf mutants were given apc5CA mutations and grown on glucose-supplemented medium, growth was increasingly impaired (Fig. 2). Furthermore, growth on glycerol illustrated that only a strain lacking all three CAF-I subunits and expressing the apc5CA allele was Ts– at 37°C. We conclude from these observations that individual CAF-I subunits likely support limited viability of apc5CA cells at 37°C, and in the absence of any CAF-I subunit at 37°C, apc5CA cells cannot utilize glycerol as a carbon source.



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  		FIG. 2. CAF-I mutations interact genetically with the apc5CA allele. Progressive disruption of CAF-I subunits in apc5CA cells exacerbates the apc5CA Ts– defect. YTH6 (WT), YTH1155 (apc5CA), YTH1537 (cac1{Delta}), YTH1562 (cac1{Delta} msi1{Delta}), YTH1417 (cac1{Delta} cac2{Delta} msi1{Delta} [caf1{Delta}]), YTH1536 (cac1{Delta} apc5CA), YTH1561 (cac1{Delta} msi1{Delta} apc5CA), and YTH1418 (caf1{Delta} apc5CA) were grown overnight in YPD medium and spot diluted the next day onto the plates shown and grown at the temperatures indicated for 3 to 5 days. The plates were also grown at 16°C, but no differences were observed (data not shown). Following the incubations, the plates were scanned.

Intact CAF-I complex is not required for suppression of apc5CA Ts phenotypes. In order to gather supporting evidence for the results shown in Fig. 2, which suggest that the CAF-I subunits can function independently in apc5CA cells, we asked whether CAF-I subunits can suppress the apc5CA Ts– defect in cells lacking intact CAF-I. Our results indicate that an intact CAF-I complex is not required for apc5CA suppression, as CAC1, CAC2, and MSI1 continued to suppress the apc5CA Ts– defect in apc5CA caf mutants (Fig. 3 and 8C; data not shown). These results clearly show that increased levels of only one CAF-I subunit are sufficient for suppression of the apc5CA Ts– phenotype in the absence of an intact CAF-I complex. These observations confirm that the apc5CA and caf mutations interact genetically and further suggest the possibility that the CAF-I subunits may function individually in apc5CA cells.



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  		FIG. 3. Suppression of apc5CA growth defects by multicopy expression of CAF-I subunits is CAF-I independent. (A) YTH1296 (cac1{Delta} apc5CA), (B) YTH1275 (cac2{Delta} apc5CA), and (C) YTH1298 (msi1{Delta} apc5CA) cells were transformed with YCplac111, YCpAPC5, YEpCAC1, or YEpMSI1. The transformants were then struck out on SD-leu plates and grown at 30°C and 37°C for 3 to 5 days.



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  		FIG. 8. HIR1 and HIR2 suppress apc5CA Ts– defects in a CAF-I-dependent manner. (A) YTH457 (apc5CA) was transformed with YCp50 and YEpHIR1. (B) YTH1637 (apc5CA) cells were transformed with YCp50, YCp50-APC5, and YEpHIR2. The transformants were grown for 3 days at 30°C and 37°C. The plates were then scanned. (C) Suppression of apc5CA Ts– defects by ASF1, HIR1, and HIR2 requires an intact CAF-I. apc5CA caf1{Delta} (YTH1418) cells were transformed with the plasmids shown. The transformants were grown at 30°C and 37°C for 3 days.

Deletion of any of the CAF-I subunits does not exacerbate the apc5CA cell cycle defect. Although CAF-I plays a major role in DNA replication-coupled chromatin assembly (64), it is clear that CAF-I can function in a DNA replication-independent manner, such as in DNA repair (16, 47, 49). To gain insight into the effects of caf mutations on cell cycle progression in apc5CA cells, we prepared the various apc5CA caf mutants for flow cytometry as a means to measure the proportion of cells in each phase of the cell cycle. Cells were grown in YPD to an OD600 of 0.5 at 30°C and 37°C prior to fixation. Our results indicate that deletion of CAF-I subunits does not cause an increase in the accumulation of apc5CA cells in the G2/M phase of the cell cycle (Fig. 4) (2, 20). The caf mutations may in fact mollify the accumulation of apc5CA cells in G2/M. Deletion of CAC1 had previously been shown to have no effect on cell cycle progression (60, 66), and we extended that by showing that caf1{Delta} cells also have little trouble with cell cycle progression. Thus, the increased exacerbation of the apc5CA Ts– defect as CAF-I subunits are progressively deleted (Fig. 2B) is apparently not accompanied by an increased accumulation of apc5CA cells in G2/M.



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  		FIG. 4. CAF-I mutations do not exacerbate apc5CA cell cycle defects. Cells from the strains indicated were analyzed by flow cytometry. Cells were grown overnight, diluted back to an OD600 of 0.2, and then grown at 30°C and 37°C until an OD600 of 0.5 was reached. The strains used were YTH1636 (WT), YTH1637 (apc5CA), YTH1594 (cac1{Delta}), YTH1817 (caf1{Delta}), YTH1589 (apc5CA cac1{Delta}), YTH1561 (apc5CA cac1{Delta} msi1{Delta}), and YTH1588 (apc5CA caf1{Delta}).

Increased expression of histones H3 and H4 suppresses apc5CA and apc5CA caf mutations in vivo. To investigate whether apc5CA and apc5CA caf phenotypes arise from defective chromatin metabolism, implied by the apc5CA chromatin assembly defect (20), we tested whether apc5CA and apc5CA caf Ts– defects could be suppressed by increased coexpression of histones H3 and H4 from a divergent GAL1/10 promoter (pRM102 was kindly provided by M. Grunstein) (45). The results show that coexpression of histones H3 and H4 in apc5CA cells is sufficient to suppress the Ts– defect, whether grown under repressing (glucose) or derepressing (galactose) conditions. Thus, these results are consistent with the notion that histone metabolism is aberrant in apc5CA cells and is exacerbated when CAF-I genes are disrupted.

We next asked whether we could suppress the apc5CA in vitro chromatin assembly defect with the addition of exogenous histones. Acid-extracted histones H2A, H2B, H3, and H4, obtained from untreated or colcemid-treated HeLa cells (Upstate), were added to chromatin assembly reactions with wild-type or apc5CA extracts. Histones isolated from colcemid-treated cells are rich in mitosis-specific histones. However, although addition of histones to wild-type extracts improved chromatin assembly, no effect was observed when histones were added to apc5CA extracts (Fig. 5B). It is likely that apc5CA extracts are "dead end" extracts, as we were previously unable to restore assembly activity when Apc5p protein was added back to apc5CA extracts (data not shown).



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  		FIG. 5. Increased levels of histones H3 and H4 in apc5CA cells suppresses in vivo but not in vitro phenotypes. (A) Suppression of in vivo apc5CA Ts– defects by increased expression of H3 and H4. Wild-type (YTH1636), apc5CA (YTH1637), cac1{Delta} (YTH1594), apc5CA cac1{Delta} (YTH1589), and apc5CA caf1{Delta} (YTH1588) cells were transformed with YCp50 and pRM102, which expresses H3 and H4 under the control of the GAL promoter, and grown on either galactose- or glucose-supplemented medium at 30°C and 37°C. (B) Addition of exogenous histones to apc5CA extracts does not restore chromatin assembly activity. Chromatin assembly reactions using 200 µg of wild-type (YTH5) and apc5CA (YTH1155) extracts were performed with a nonradioactive method (21). Acid-extracted histones obtained from untreated and colcemid-treated HeLa cells (Upstate) were added to the reactions in the amounts shown. Untreated extracts were included as controls; 150 µg of relaxed plasmid was used in each reaction, and the reactions were incubated at 30°C for 90 min. The ethidium bromide-stained gel was photographed and scanned, and the inverse image is shown.

cac1{Delta} interacts genetically with additional APC mutations. Our data thus far indicate that at least apc5CA interacts genetically with the CAF-I subunit genes. Next, we asked whether CAF-I interactions were limited to Apc5p. We extended our analysis by creating apc10{Delta} cac1{Delta} and cac1{Delta} cdc26{Delta} double mutants. The double mutants were grown on glucose-supplemented medium at different temperatures and compared with wild-type and single-mutant cells. The results in Fig. 6 demonstrate that the disruption of CAC1 in apc10{Delta} and cdc26{Delta} cells lowers the restrictive temperature at which these mutants will grow. Thus, our results indicate the APC complex, not just Apc5p, is required for interactions with CAF-I subunits, as the cac1{Delta} mutation exacerbates the Ts– growth phenotype of multiple APC mutants.



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  		FIG. 6. CAC1 interacts with additional genes encoding APC subunits. (A) The growth phenotypes of the apc10{Delta} mutant were compared in the presence and absence of the CAC1 gene. YTH1688 (WT), YTH1689 (cac1{Delta}), YTH1690 (apc10{Delta} cac1{Delta}), and YTH1691 (apc10{Delta}) were prepared for spot dilution on glucose-supplemented medium as described for Fig. 4. (B) The growth phenotypes of the apc mutant cdc26{Delta} were compared in the presence and absence of the CAC1 gene. YTH1666 (WT), YTH1668 (cac1{Delta}), YTH 1669 (cdc26{Delta}), and YTH1667 (cac1{Delta} cdc26{Delta}) were prepared for spot dilution on glucose-supplemented medium as described above.

apc5CA interacts genetically with ASF1. The use of a novel APC mutation, apc5CA, allowed us to characterize novel CAF-I-independent functions for each CAF-I subunit. Next, we asked whether other CAFs were capable of genetic interactions with apc5CA. CAF-I is known to physically and genetically interact with the CAF Asf1p (41, 48, 67). To assess whether Asf1p was involved in APC-dependent chromatin assembly, an apc5CA strain was crossed with asf1{Delta} cells and scored for growth at various temperatures. The apc5CA asf1{Delta} mutant grew more slowly at both 30°C and 37°C than either single mutant (Fig. 7A). This broadens the scope of APC-dependent chromatin assembly, which now appears to include Asf1p.



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  		FIG. 7. ASF1 interacts genetically with the apc5CA allele. (A) Disruption of ASF1 in apc5CA cells exacerbates the apc5CA Ts– defect. Wild-type (YTH1156), asf1{Delta} (YTH1149), apc5CA (YTH1155), and asf1{Delta} apc5CA (YTH1159) cells were grown in liquid YPD at the indicated temperatures. At the given times, aliquots were removed and the OD600 was determined. An OD600 of 1 is approximately 2 x 107 cells/ml. (B) Increased expression of ASF1 suppresses the apc5CA Ts– defect. YTH1155 cells were transformed with the plasmids shown, and the transformants were grown at either 30°C or 37°C on glucose-supplemented medium. (C) Overexpression of ASF1 but not APC5 from the CUP1 promoter suppresses asf1{Delta} apc5CA Ts– growth. asf1{Delta} apc5CA cells were transformed with pYEX, pYEX-APC5, and pYEX-ASF1 and grown on SD-ura supplemented with 0.5 mM Cu(SO4)2 at 30°C and 37°C. (D) Single-copy expression of APC5 and multicopy expression of MSI1 suppresses asf1{Delta} apc5CA Ts– growth. asf1{Delta} apc5CA cells were transformed with YCp50, YCp50-APC5, and YEp-MSI1 and grown on SD-ura at 30°C and 37°C. (E) Overexpression of APC5 but not ASF1 suppresses msi1{Delta} apc5CA Ts– growth. msi1{Delta} apc5CA cells were transformed with pYEX, pYEX-APC5, and pYEX-ASF1 and grown on SD-ura supplemented with 0.5 mM Cu(SO4)2 at 30°C and 37°C.

To complement the investigation of asf1{Delta}-apc5CA genetic interactions, we tested whether increased expression of ASF1 could influence the apc5CA Ts– growth defect. As expected, expression of ASF1 from the 2µm plasmid suppressed apc5CA Ts– growth (Fig. 7B). We also show that overexpression of ASF1, resulting from growth of CUP1prom-ASF1-expressing cells in 0.5 mM Cu(SO4)2, was capable of suppressing apc5CA asf1{Delta} Ts– growth (Fig. 7C). As in apc5CA cells (data not shown), CUP1prom-dependent overexpression of APC5 could not restore the Ts– defect in apc5CA asf1{Delta} cells (Fig. 7C). This is not due to an inability of APC5 to suppress asf1{Delta} mutations, as single-copy expression of APC5 suppressed apc5CA asf1{Delta} cells (Fig. 7D). Although overexpression of APC5 in wild-type cells had no apparent effect on growth rate at 37°C (data not shown), it did reduce the replicative life span of wild-type cells (22). We interpret our observations regarding overexpression of APC5 to reflect that APC5 expression is tightly controlled in cells. Indeed, Northern analysis of APC genes demonstrated that APC4, APC5, and APC9 transcripts are extremely rare (22).

Multicopy expression of MSI1 restored the growth of the apc5CA asf1{Delta} mutant at 37°C (Fig. 7D), indicating that suppression of apc5CA Ts– growth defects by MSI1 did not require Asf1p or CAF-I. ASF1 could not, however, restore the Ts– defect in apc5CA msi1{Delta} cells (Fig. 7E). This observation suggests that Asf1p requires at least Msi1p to suppress apc5CA Ts– defects. It is also worthy of note that overexpression of APC5 could restore the Ts– growth defect of apc5CA cells when MSI1 is deleted (compare Fig. 7C with Fig. 7E). Together, these observations add strength to our hypothesis that the APC controls mitotic chromatin assembly, perhaps by recruiting multiple CAFs to mitotic chromatin templates.

HIR1 and HIR2 suppress apc5CA in a CAF-I-dependent manner. Lastly, we asked if the interaction of APC5 with the CAF-I subunit genes and ASF1 involves an additional pair of CAF-I- and Asf1p-interacting proteins, Hir1p and Hir2p. As found for the CAF-I subunits and ASF1, the expression of HIR1 and HIR2 in multicopy suppressed the apc5CA Ts– defect (Fig. 8A and 8B; HIR1 and HIR2 multicopy plasmids were kindly provided by M. Osley). Asf1p and Hir1p have been linked to a chromatin assembly pathway that functions in parallel with CAF-I (41, 59, 68). Our results agree with this hypothesis, as increased expression of ASF1, HIR1, and HIR2 could not suppress apc5CA when CAF-I subunits were deleted (Fig. 8C). We also observed that while CUP1prom-dependent overexpression of APC5 could suppress the Ts– growth of an apc5CA msi1{Delta} mutant (Fig. 7E), it could not suppress the Ts– growth of the apc5CA caf1{Delta} strain (Fig. 8C). Taken together, our results suggest that the APC requires only one CAF-I subunit for mitotic chromatin assembly activity, supporting a model in which the individual CAF-I subunits assemble mitotic chromatin in a CAF-I-independent manner.

The observation that all CAF-I subunits must be deleted in apc5CA cells to confer Ts– growth on glycerol (Fig. 2B) provides supporting evidence for this model. The genetic complexity of this model becomes apparent when one considers that Asf1p, Hir1p, and Hir2p likely require an intact CAF-I complex to suppress apc5CA Ts– defects. Thus, it remains possible that an intact CAF-I complex also contributes to APC-dependent chromatin assembly.


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DISCUSSION
 
The results reported here suggest a molecular regulatory mechanism for mitotic chromatin assembly. Our genetic and biochemical experiments support a model in which the APC plays a crucial role in mitotic chromatin assembly regulation, a function mediated by interactions with subunits of the chromatin assembly factor CAF-I. In apc5CA cells, the CAF-I subunits can function independently in an apparently redundant manner; the apc5CA Ts– phenotype becomes increasingly impaired as additional CAF-I subunits are deleted (Fig. 2B). Asf1p as well as the Hir proteins, which interact physically and genetically with CAF-I and with each other (41, 48, 59, 60, 66, 67), also play a role in APC-dependent chromatin assembly but require an intact CAF-I complex. Thus, our results suggest that CAF-I may play a dual role in mitotic chromatin assembly, both as an intact complex in conjunction with Asf1p and the Hir proteins and as individual subunits that act independently of Asf1p and the Hir proteins.

CAF-I subunits play a role in APC-dependent mitotic chromatin assembly. Evidence exists that potentially ties CAF-I to APC activity. For example, the smallest S. cerevisiae CAF-I subunit, Msi1p, has been found to suppress phenotypes resulting from overactive protein kinase A when expressed in multicopy (58). As protein kinase A inhibits APC activity (40), Msi1p may either play a role in activating the APC by downregulating protein kinase A, or act downstream of the APC by directly compensating for chromatin defects resulting from APC impairment. Furthermore, multicopy expression of MSI1-suppressed phenotypes associated with an SNF1 disruption (30). Snf1p is activated upon glucose limitation and acts to ensure that genes required to utilize alternate carbon sources are transcribed (9).

We recently characterized a role for Snf1p in APC activation (22). We demonstrated that Snf1p, a known aging determinant in S. cerevisiae (3, 43), was required to promote APC-dependent longevity (22). We also have evidence to suggest that protein kinase A induced life span reduction (reviewed in references 33, 44, and 63) is mediated through inhibition of the APC, as blocking Ras2p function extends apc5CA reduced life span (C. R. Geyer and T. A. A. Harkness, unpublished data). Thus, the complex MSI1 genetic interactions could be explained if Msi1p were acting downstream of the APC and was capable of repairing damaged chromatin resulting from APC inhibition through overactive protein kinase A or reduced Snf1p signaling.

The suppression of apc5CA defects by increased expression of CAF-I subunits could also be explained as an indirect effect, as each subunit of CAF-I can separately bind histones (36, 62, 69). Thus, suppression of apc5CA phenotypes could result from the compensatory action of histone binding, followed by either deposition into chromatin or sequestration. This would occur if histones were stabilized or accumulating in a free form in apc5CA cells, as excess free histones are deleterious to cell health (18). However, this is clearly not the case, as increased expression of histones H3 and H4 in apc5CA caf mutants suppressed the Ts– growth defects (Fig. 5A). Even low levels of H3 and H4 expression, resulting from growth of the GAL1/10prom driven plasmid in repressing conditions (glucose), are sufficient to suppress the Ts– growth defects. Our attempts to suppress the in vitro chromatin assembly defect in apc5CA extracts with the addition of purified histones demonstrated that apc5CA extracts could not be rescued by the addition of exogenous histones (Fig. 5B). We were also unable to rescue apc5CA extracts with purified Apc5p protein (data not shown). It is therefore likely that the defects conferred by the apc5CA allele are permanent once an extract is generated. We conclude from these experiments that in apc5CA and apc5CA caf cells, defects in histone metabolism can be overcome by increased expression of histones H3 and H4 in vivo.

CAF-I subunits play separate yet partially redundant functions in apc5CA cells. Our conclusion that the CAF-I subunits can function independently of an intact CAF-I complex in apc5CA cells is supported by two observations: (i) increased temperature sensitivity of apc5CA cells as additional CAF-I subunits were deleted, indicating that the individual subunits support limited viability of apc5CA cells at 37°C (Fig. 2B); and (ii) the ability of individual CAF-I subunits to suppress apc5CA Ts– defects when a different CAF-I subunit was deleted (Fig. 3). Basically, if an intact CAF-I complex was interacting with the APC, then double and triple caf mutations would create the same phenotype as single caf mutations in apc5CA cells. Msi1p had previously been shown to function independently of CAF-I (34, 74). A recent study also ascribed a CAF-I-independent function for Cac1p in histone nuclear import (17). This report demonstrates additional CAF-I-independent functions for Cac1p and Msi1p and a novel CAF-I-independent function for Cac2p.

The notion that the individual subunits are at least partially redundant in apc5CA cells is based on the following observations: (i) only apc5CA cells lacking all three CAF-I subunits were Ts– on glycerol, which also suggests that the CAF-I subunits provide some function that is redundant with at least Apc5p (Fig. 2B); (ii) low-level expression of histones H3 and H4 had a greater suppressive effect on apc5CA cac1{Delta} cells than on apc5CA caf1{Delta} cells (Fig. 5A); and (iii) overexpression of APC5 could suppress apc5CA msi1{Delta} cells but not apc5CA caf1{Delta} cells (compare Fig. 7E with 8C). Therefore, our results are consistent with a model in which the individual CAF-I subunits act independently and most likely redundantly with each other.

We obtained additional evidence supporting the hypothesis that the CAF-I subunits can function in the absence of an intact CAF-I complex. We performed in vitro chromatin assembly assays on extracts prepared from the mutants shown in Fig. 2B. Mild chromatin assembly defects were observed in extracts prepared from single CAF-I mutants (C. Legrand and T. A. A. Harkness, unpublished data), implying a role for intact CAF-I in mitosis-specific chromatin assembly. However, the defects were increased marginally when additional subunits were mutated, implying that CAF-I subunits may function outside of an intact CAF-I complex. Combining each CAF-I mutation with the apc5CA allele also slightly decreased assembly efficiency. The marginal defects that we observed in chromatin assembly efficiency were reproducible and consistent with the involvement of single CAF-I subunits in mitotic chromatin assembly. However, the marginal results clearly show that other factors are involved.

ASF1, HIR1, and HIR2 interact genetically with the apc5CA allele in a CAF-I-dependent manner. Work in several laboratories has demonstrated physical and genetic interactions between CAF-I, Asf1p, and the Hir proteins (38, 41, 59, 67). Asf1p has been shown to stimulate the assembly of newly replicated chromatin by CAF-I in several systems (41, 48, 59, 67) and may act by delivering histones to CAF-I in vivo (48). Consistent with Asf1p functioning as a CAF, disruption of ASF1 was recently shown to cause in vitro chromatin assembly defects (52). On the other hand, an in vivo study in S. cerevisiae suggests that Asf1p may mediate global chromatin disassembly (1). Disruption of ASF1 was shown to increase the in vivo chromatin assembly capacity of the 2µm plasmid, as opposed to disruption of CAC1, which partially reduced assembly efficiency (1).

We have been unable to observe in vitro chromatin assembly defects with asf1{Delta} extracts (data not shown). The differences observed with asf1{Delta} strains might reflect the different methods employed to measure in vitro chromatin assembly. Nonetheless, our in vivo observations are consistent with models proposing that Asf1p acts as a CAF, perhaps by facilitating the role that CAF-I plays in mitotic chromatin assembly. In the absence of intact CAF-I, however, increased ASF1 cannot suppress apc5CA defects. Perhaps under these circumstances (the absence of CAF-I), Asf1p is free to act as a chromatin disassembly factor.

Our experiments suggest that an intact CAF-I complex may play a role in mitotic chromatin assembly that is facilitated by Asf1p and the Hir proteins (Fig. 7 and 8). We have recently shown that the mitotic chromatin assembly pathway may be regulated as early as G1, as SCF (Skp1/Cdc53/F-box protein) and RSP5 ubiquitin-ligase mutants, which arrest or accumulate in G1 at restrictive temperatures, respectively, are defective in our in vitro mitotic chromatin assembly assay (2). That work also provided evidence that RSP5 interacts genetically with SCF and the APC to promote mitotic chromatin assembly.

As CAF-I is critical for replication-dependent chromatin assembly (64) and passage through S phase (28, 50), it is possible that the role that an intact CAF-I complex plays in APC-dependent chromatin assembly is limited to ensuring that cells progress through S phase. Cells must complete DNA replication and chromosome compaction in order to progress through mitosis, and this is regulated by cell cycle checkpoints that inhibit APC activity (5). Thus, our results are consistent with a model in which CAF-I assembly and disassembly are regulated at mitosis, allowing CAF-I subunits to contribute to both S-phase- and mitosis-specific chromatin assembly. There is evidence to support CAF-I disassembly in mammalian cells, as p60 is hyperphosphorylated as cells enter mitosis, and this coincides with release of CAF-I from chromatin, export of monomeric hyperphosphorylated p60 to the cytosol, and an inability of purified CAF-I to assemble chromatin in a replication-dependent manner (46).

In conclusion, the experiments presented in this report provide evidence that a novel molecular mechanism controls chromatin assembly during mitosis. It is feasible that this mechanism is in place to maintain functionally intact chromosomes during the segregation of sister chromatids or to establish a transcriptionally primed genome as cells exit mitosis. The possibility that this is a conserved mechanism raises several interesting questions. For example, what would be the consequences to a mammalian cell that harbored mutations in this pathway? We have recently demonstrated that S. cerevisiae APC is required for longevity (22). Premature aging in mammalian cells is often accompanied by cancer and genomic instability (7, 8). Strikingly, several reports have indicated that the APC plays a pivotal role in resistance to cancer (5, 53, 65). In addition, CAF-I mutations can lead to S-phase arrest and genomic instability (50, 72), while p48 and the protein that directs CAF-I to replication forks, PCNA, are deregulated in tumors found in patients with tuberous sclerosis (25). Future studies aimed at fully characterizing the mechanisms employed by the APC to control chromatin assembly in S. cerevisiae and higher eukaryotic cells will provide valuable insights into disease onset and progression.


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ACKNOWLEDGMENTS
 
We thank Marian Carlson, Mike Ellison, Mike Grunstein, Paul Kaufman, Walter Neupert, Mary Ann Osley, and Rick Wozniak for the generous contributions of strains and plasmids. Mark Boyd assisted with the flow cytometry analysis. Kyla Shea is acknowledged for valuable technical assistance.

This work was supported by grants to T.A.A.H. from the Canadian Institutes for Health Research Regional Partnership Program and the Canadian Foundation for Innovation. T.G.A. was supported by a part-time fellowship from the Alberta Heritage Foundation for Medical Research. The initial experiments were supported by grants to Mike Schultz from the Alberta Heritage Foundation for Medical Research and the Canadian Institutes for Health Research.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Anatomy and Cell Biology, College of Medicine, University of Saskatchewan, B313 Health Sciences Building, 107 Wiggins Road, Saskatoon, Saskatchewan S7N 5E5, Canada. Phone: (306) 966-1995. Fax: (306) 966-4298. E-mail: troy.harkness{at}usask.ca. Back


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REFERENCES
 
    1
  1. Adkins, M. W., and J. K. Tyler. 2004. The histone chaperone Asf1p mediates global chromatin disassembly in vivo. J. Biol. Chem. 279:52069-52074.[Abstract/Free Full Text]
    2. 2
  2. Arnason, T. G. M. G. Pisclevich, M. D. Dash, G. F. Davies, and T. A. A. Harkness. 2004. Novel interaction between Apc5p and Rsp5p in an intracellular signaling pathway in yeast. Eukaryot. Cell 4:134-146.[CrossRef]
    3. 3
  3. Ashrafi, K., S. S. Lin, J. K. Manchester, and J. I. Gordon. 2000. Sip2p and its partner snf1p kinase affect aging in S. cerevisiae. Genes Dev. 14:1872-1885.[Abstract/Free Full Text]
    4. 4
  4. Ausio, J., D. B. Levin, G. V. De Amorim, S. Bakker, and P. M. Macleod. 2003. Syndromes of disordered chromatin remodeling. Clin. Genet. 64:83-95.[CrossRef][Medline]
    5. 5
  5. Bharadwaj, R., and H. Yu. 2004. The spindle checkpoint, aneuploidy, and cancer. Oncogene 23:2016-2027.[CrossRef][Medline]
    6. 6
  6. Bitterman, K. J., O. Medvedik, and D. A. Sinclair. 2003. Longevity regulation in Saccharomyces cerevisiae: linking metabolism, genome stability, and heterochromatin. Microbiol. Mol. Biol. Rev. 67:376-399.[Abstract/Free Full Text]
    7. 7
  7. Busuttil, R. A., M. Dolle, J. Campisi, and J. Vijga. 2004. Genomic instability, aging, and cellular senescence. Ann. N. Y. Acad. Sci. 1019:245-255.[CrossRef][Medline]
    8. 8
  8. Campisi, J. 2003. Cancer and ageing: rival demons? Nat. Rev. Cancer 3:339-349.[CrossRef][Medline]
    9. 9
  9. Carlson, M. 1998. Regulation of glucose utilization in yeast. Curr. Opin. Genet. Dev. 8:560-564.[CrossRef][Medline]
    10. 10
  10. Chen, J. S., D. V. Faller, and R. A. Spanjaard. 2003. Short-chain fatty acid inhibitors of histone deacetylases: promising anticancer therapeutics? Curr. Cancer Drug Targets 3:219-236.[CrossRef][Medline]
    11. 11
  11. Cimini, D., M. Mattiuzzo, L. Torosantucci, and F. Degrassi. 2003. Histone hyperacetylation in mitosis prevents sister chromatid separation and produces chromosome segregation defects. Mol. Biol. Cell 14:3821-3823.[Abstract/Free Full Text]
    12. 12
  12. Cohen-Fix, O. 2001. The making and breaking of sister chromatid cohesion. Cell 106:137-140.[CrossRef][Medline]
    13. 13
  13. Elledge, S. J. 1998. Mitotic arrest: Mad2 prevents sleepy from waking up the APC. Science 279:999-1000.[Abstract/Free Full Text]
    14. 14
  14. Enomoto, S., and J. Berman. 1998. Chromatin assembly factor I contributes to the maintenance, but not the re-establishment, of silencing at the yeast silent mating loci. Genes Dev. 12:219-232.[Abstract/Free Full Text]
    15. 15
  15. Epstein, C. B., and F. R. Cross. 1992. CLB5: a novel B cyclin from budding yeast with a role in S phase. Genes Dev. 6:1695-1706.[Abstract/Free Full Text]
    16. 16
  16. Gaillard, P.-H. L, E. M.-D. Martini, P. D. Kaufman, B. Stillman, E. Moustacchi, and G. Almouzni. 1996. Chromatin assembly coupled to DNA repair: a new role for chromatin assembly factor I. Cell 86:887-896.[CrossRef][Medline]
    17. 17
  17. Glowczewski, L., J. H. Waterborg, and J. G. Berman. 2004. Yeast chromatin assembly complex 1 protein excludes nonacetylatable forms of histone H4 from chromatin and the nucleus. Mol. Cell. Biol. 24:10180-10192.[Abstract/Free Full Text]
    18. 18
  18. Gunjan, A., and A. Verreault. 2003. A Rad53 kinase-dependent surveillance mechanism that regulates histone protein levels in S. cerevisiae. Cell 115:537-549.[CrossRef][Medline]
    19. 19
  19. Guthrie, C., and G. R. Fink (ed.). 1991. Methods in enzymology, vol. 194. Guide to yeast genetics and molecular biology. Academic Press, Inc., San Diego, Calif.
    20. 20
  20. Harkness, T. A. A., G. F. Davies, V. Ramaswamy, and T. G. Arnason. 2002. The ubiquitin-dependent targeting pathway in Saccharomyces cerevisiae plays a critical role in multiple chromatin assembly regulatory steps. Genetics 162:615-632.[Abstract/Free Full Text]
    21. 21
  21. Harkness, T. A. A., T. G. Arnason, C. Legrand, and A. Lone. 2003. Methods designed for the identification and characterization of in vitro and in vivo chromatin assembly mutants in Saccharomyces cerevisiae. Biol. Proced. Online. 5:162-169.[CrossRef][Medline]
    22. 22
  22. Harkness, T. A. A., K. A. Shea, C. Legrand, M. Brahmania, and G. F. Davies. 2004. A functional analysis reveals dependence on the anaphase promoting complex for prolonged lifespan in yeast. Genetics 168:759-774.[Abstract/Free Full Text]
    23. 23
  23. Harper, J. W., J. L. Burton, and M. J. Solomon. 2002. The anaphase-promoting complex: it's not just for mitosis any more. Genes Dev. 16:2179-2206.[Free Full Text]
    24. 24
  24. Haushalter, K. A., and J. T. Kadonaga. 2003. Chromatin assembly by DNA-translocating motors. Nat. Rev. Mol. Cell. Biol. 4:613-620.[CrossRef][Medline]
    25. 25
  25. Hengstschlager, M., M. Rosner, M. Fountoulakis, and G. Lubec. 2003. Regulation of PCNA and CAF-1 expression by the two tuberous sclerosis gene products. Biochem. Biophys. Res. Commun. 307:737-742.[CrossRef][Medline]
    26. 26
  26. Hennig, L., P. Taranto, M. Walser, N. Schonrock, and W. Gruissem. 2003. Arabidopsis MSI1 is required for epigenetic maintenance of reproductive development. Development 130:2555-2565.[Abstract/Free Full Text]
    27. 27
  27. Hochstrasser, M., P. R. Johnson, C. S. Arendt, A. Y. Amerik, S. Swaminathan, R. Swanson, S. J. Li, J. Laney, R. Pals-Rylaarsdam, J. Nowak, and P. L. Connerly. 1999. The Saccharomyces cerevisiae ubiquitin-proteasome system. Phil. Trans. R. Soc. Lond. B Biol. Sci. 354:1513-1522.[Abstract/Free Full Text]
    28. 28
  28. Hoek, M., and B. Stillman. 2003. Chromatin assembly factor 1 is essential and couples chromatin assembly to DNA replication in vivo. Proc. Natl. Acad. Sci. USA 100:12183-12188.[Abstract/Free Full Text]
    29. 29
  29. Huang, C., E. A. Sloan, and C. F. Boerkoel. 2003. Chromatin remodeling and human disease. Curr. Opin. Genet. Dev. 13:246-252.[CrossRef][Medline]
    30. 30
  30. Hubbard, E. J. A., X. Yang, and M. Carlson. 1992. Relationship of the cAMP-dependent protein kinase pathway to the SNF1 protein kinase and invertase expression in Saccharomyces cerevisiae. Genetics 130:71-80.[Abstract]
    31. 31
  31. Imai, S., F. B. Johnson, R. A. Marciniak, M. McVey, P. U. Park, L. Guarente. 2000. Sir2: an NAD-dependent histone deacetylase that connects chromatin silencing, metabolism, and aging. Cold Spring Harb. Symp. Quant. Biol. 65:297-302.[CrossRef][Medline]
    32. 32
  32. Irniger, S., M. Baumer, and G. H. Braus. 2000. Glucose and ras activity influence the ubiquitin ligases APC/C and SCF in Saccharomyces cerevisiae. Genetics 154:1509-1521.[Abstract/Free Full Text]
    33. 33
  33. Jazwinski, S. M. 1999. The RAS genes: a homeostatic device in Saccharomyces cerevisiae longevity. Neurobiol. Aging 20:471-478.[CrossRef][Medline]
    34. 34
  34. Johnston, S. D., S. Enomoto, L. Schneper, M. C. McClellan, F. Twu, N. D. Montgomery, S. A. Haney, J. R. Broach, and J. Berman. 2001. CAC3(MSI1) suppression of RAS2(G19V) is independent of chromatin assembly factor I and mediated by NPR1. Mol. Cell. Biol. 21:1784-1794.[Abstract/Free Full Text]
    35. 35
  35. Kamakaka, R. T., M. Bulger, P. D. Kaufman, B. Stillman, and J. T. Kadonaga. 1996. Postreplicative chromatin assembly by Drosophila and human chromatin assembly factor 1. Mol. Cell. Biol. 16:810-817.[Abstract/Free Full Text]
    36. 36
  36. Kaufman, P. D., R. Kobayashi, N. Kessler, and B. Stillman. 1995. The p150 and p60 subunits of chromatin assembly factor I: a molecular link between newly synthesized histones and DNA replication. Cell 81:1105-1114.[CrossRef][Medline]
    37. 37
  37. Kaufman, P. D., R. Kobayashi, and B. Stillman. 1997. Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I. Genes Dev. 11:345-357.[Abstract/Free Full Text]
    38. 38
  38. Kaufman, P. D., J. L. Cohen, and M. A. Osley. 1998. Hir proteins are required for position-dependent gene silencing in Saccharomyces cerevisiae in the absence of chromatin assembly factor I. Mol. Cell. Biol. 18:4793-4806.[Abstract/Free Full Text]
    39. 39
  39. Kaya, H., K.-i. Shibahara, K.-i. Taoka, M. Iwabuchi, B. Stillman, and T. Araki. 2001. FASCIATA genes for chromatin assembly factor-1 in Arabidopsis maintain the cellular organization of apical meristems. Cell 104:131-142.[CrossRef][Medline]
    40. 40
  40. Kotani, S., S. Tugendreich, M. Fujii, P. M. Jorgensen, N. Watanabe, C. Hoog, P. Hieter, and K. Todokoro. 1998. PKA and MPF-activated polo-like kinase regulate anaphase-promoting complex activity and mitosis progression. Mol. Cell 1:371-380.[CrossRef][Medline]
    41. 41
  41. Krawitz, D. C., T. Kama, and P. D. Kaufman. 2002. Chromatin assembly factor I mutants defective for PCNA binding require Asf1/Hir proteins for silencing. Mol. Cell. Biol. 22:614-625.[Abstract/Free Full Text]
    42. 42
  42. Krude, T. 1995. Chromatin assembly factor 1 (CAF-1) colocalizes with replication foci in HeLa cell nuclei. Exp. Cell Res. 220:304-311.[CrossRef][Medline]
    43. 43
  43. Lin, S. S., J. K. Manchester, and J. I. Gordon. 2003. Sip2, an N-myristoylated beta subunit of Snf1 kinase, regulates aging in Saccharomyces cerevisiae by affecting cellular histone kinase activity, recombination at rDNA loci, and silencing. J. Biol. Chem. 278:13390-13397.[Abstract/Free Full Text]
    44. 44
  44. Longo, V. D. 2003. The Ras and Sch9 pathways regulate stress resistance and longevity. Exp. Gerontol. 38:807-811.[CrossRef][Medline]
    45. 45
  45. Mann, R. K., and M. Grunstein. 1992. Histone H3 N-terminal mutations allow hyperactivation of the yeast GAL1 gene in vivo. EMBO J. 11:3297-3306.[Medline]
    46. 46
  46. Marheineke, K., and T. Krude. 1998. Nucleosome assembly activity and intracellular localization of human CAF-1 changes during the cell division cycle. J. Biol. Chem. 273:15279-15286.[Abstract/Free Full Text]
    47. 47
  47. Martini, E., D. M. Roche, K. Marheineke, A. Verreault, and G. Almouzni. 1998. Recruitment of phosphorylated chromatin assembly factor 1 to chromatin after UV irradiation of human cells. J. Cell Biol. 143:563-575.[Abstract/Free Full Text]
    48. 48
  48. Mello, J. A., H. H. Sillje, D. M. Roche, D. B. Kirschner, E. A. Nigg, and G. Almouzni. 2002. Hum. Asf1 and CAF-1 interact and synergize in a repair-coupled nucleosome assembly pathway. EMBO Rep. 3:329-334.[CrossRef][Medline]
    49. 49
  49. Moggs, J. G., P. Grandi, J. P. Quivy, Z. O. Jonsson, U. Hubscher, P. B. Becker, and G. Almouzni. 2000. A CAF-1-PCNA-mediated chromatin assembly pathway triggered by sensing DNA damage. Mol. Cell. Biol. 20:1206-1218.[Abstract/Free Full Text]
    50. 50
  50. Myung, K., V. Pennaneach, E. S. Kats, and R. D. Kolodner. 2003. Saccharomyces cerevisiae chromatin-assembly factors that act during DNA replication function in the maintenance of genome stability. Proc. Natl. Acad. Sci. USA 100:6640-6645.[Abstract/Free Full Text]
    51. 51
  51. Peters, J. M. 2002. The anaphase-promoting complex: proteolysis in mitosis and beyond. Mol. Cell 9:931-943.[CrossRef][Medline]
    52. 52
  52. Prado, F., F. Cortes-Ledesma, and A. Aguilera. 2004. The absence of the yeast chromatin assembly factor Asf1 increases genomic instability and sister chromatid exchange. EMBO Rep. 5:497-502.[CrossRef][Medline]
    53. 53
  53. Pray, T. R., F. Parlati, J. Huang, B. R. Wong, D. G. Payan, M. K. Bennett, S. D. Issakani, S. Molineaux, and S. D. Demo. 2002. Cell cycle regulatory E3 ubiquitin ligases as anticancer targets. Drug Resist. Updat. 5:249-258.[CrossRef][Medline]
    54. 54
  54. Richmond, T. J., and C. A. Davey. 2003. The structure of DNA in the nucleosome core. Nature 423:145-150.[CrossRef][Medline]
    55. 55
  55. Ridgway, P., and G. Almouzni. 2000. CAF-1 and the inheritance of chromatin states: at the crossroads of DNA replication and repair. J. Cell Sci. 113:2647-2658.[Abstract]
    56. 56
  56. Rose, M. D., F. Winston, and P. Hieter. 1990. Methods in yeast genetics: a laboratory course manual. Cold Spring Harbor Laboratory Press, Plainview, N.Y.
    57. 57
  57. Roth, S. Y., and C. D. Allis. 1996. Histone acetylation and chromatin assembly: a single escort, multiple dances? Cell 87:5-8.[CrossRef][Medline]
    58. 58
  58. Ruggieri, R., K. Tanaka, M. Nakafuku, Y. Kaziro, A. Toh-E, and K. Matsumoto. 1989. MSI1, a negative regulator of the RAS-cAMP pathway in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 86:8778-8782.[Abstract/Free Full Text]
    59. 59
  59. Sharp, J. A., E. T. Fouts, D. C. Krawitz, and P. D. Kaufman. 2001. Yeast histone deposition protein Asf1p requires Hir proteins and PCNA for heterochromatic silencing. Curr. Biol. 11:463-473.[CrossRef][Medline]
    60. 60
  60. Sharp, J. A., A. A. Franco, M. A. Osley, and P. D. Kaufman. 2002. Chromatin assembly factor I and Hir proteins contribute to building functional kinetochores in S. cerevisiae. Genes Dev. 16:85-100.[Abstract/Free Full Text]
    61. 61
  61. Shibahara, K.-i., and B. Stillman. 1999. Replication-dependent marking of DNA by PCNA facilitates CAF-1-coupled inheritance of chromatin. Cell 96:575-585.[CrossRef][Medline]
    62. 62
  62. Shibahara, K., A. Verreault, and B. Stillman. 2000. The N-terminal domains of histones H3 and H4 are not necessary for chromatin assembly factor-1-mediated nucleosome assembly onto replicated DNA in vitro. Pro. Natl. Acad. Sci. USA 97:7766-7771.[Abstract/Free Full Text]
    63. 63
  63. Sinclair, D., K. Mills, and L. Guarente. 1998. Aging in Saccharomyces cerevisiae. Annu. Rev. Microbiol. 52:533-560.[CrossRef][Medline]
    64. 64
  64. Smith, S., and B. Stillman. 1989. Purification and characterization of CAF-I, a human cell factor required for chromatin assembly during DNA replication in vitro. Cell 58:15-25.[CrossRef][Medline]
    65. 65
  65. Sun, Y. 2003. Targeting E3 ubiquitin ligases for cancer therapy. Cancer Biol. Ther. 2:623-629.[Medline]
    66. 66
  66. Tyler, J. K., C. R. Adams, S. R. Chen, R. Kobayashi, R. T. Kamakaka, and J. T. Kadonaga. 1999. The RCAF complex mediates chromatin assembly during DNA replication and repair. Nature 402:555-560.[CrossRef][Medline]
    67. 67
  67. Tyler, J. K., K. A. Collins, J. Prasad-Sinha, E. Amiott, M. Bulger, P. J. Harte, R. Kobayashi, and J. T. Kadonaga. 2001. Interaction between the Drosophila CAF-1 and ASF1 chromatin assembly factors. Mol. Cell. Biol. 21:6574-6584.[Abstract/Free Full Text]
    68. 68
  68. Tyler, J. K. 2002. Chromatin assembly. Cooperation between histone chaperones and ATP-dependent nucleosome remodeling machines. Eur. J. Biochem. 269:2268-2274.[Medline]
    69. 69
  69. Verreault, A., P. D. Kaufman, R. Kobayashi, and B.Stillman. 1996. Nucleosome assembly by a complex of CAF-1 and acetylated histones H3/H4. Cell 87:94-104.
    70. 70
  70. Verreault, A., P. D. Kaufman, R. Kobayashi, and B. Stillman. 1998. Nucleosomal DNA regulates the core-histone-binding subunit of the human Hat1 acetyltransferase. Curr. Biol. 8:96-108.[CrossRef][Medline]
    71. 71
  71. Verreault, A. 2000. De novo nucleosome assembly: new pieces in an old puzzle. Genes Dev. 14:1430-1438.[Free Full Text]
    72. 72
  72. Ye, X., A. A. Franco, H. Santos, D. M. Nelson, P. D. Kaufman, and P. D. Adams. 2003. Defective S phase chromatin assembly causes DNA damage, activation of the S phase checkpoint, and S phase arrest. Mol. Cell 11:341-351.[CrossRef][Medline]
    73. 73
  73. Zhang, Z., K.-i. Shibahara, and B. Stillman. 2000. PCNA connects DNA replication to epigenetic inheritance in yeast. Nature 408:221-225.[CrossRef][Medline]
    74. 74
  74. Zhu, X., N. Demolis, M. Jacquet, and T. Michaeli. 2000. MSI1 suppresses hyperactive RAS via the cAMP-dependent protein kinase and independently of chromatin assembly factor-1. Curr. Genet. 38:60-70.[CrossRef][Medline]

Eukaryotic Cell, April 2005, p. 673-684, Vol. 4, No. 4
1535-9778/05/$08.00+0     doi:10.1128/EC.4.4.673-684.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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