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Mol Cell Biol. Jul 2007; 27(13): 4674–4684.
Published online Apr 23, 2007. doi:  10.1128/MCB.02052-06
PMCID: PMC1951488

Yra1 Is Required for S Phase Entry and Affects Dia2 Binding to Replication Origins[down-pointing small open triangle]


The Saccharomyces cerevisiae F-box protein Dia2 is important for DNA replication and genomic stability. Using an affinity approach, we identified Yra1, a transcription-coupled mRNA export protein, as a Dia2 interaction partner. We find that yra1 mutants are sensitive to DIA2 expression levels. Like Dia2, Yra1 associates with chromatin and binds replication origins, suggesting that they may function together in DNA replication. Consistent with this idea, Yra1 and Dia2 coimmunoprecipitate with Hys2, a subunit of DNA polymerase δ. The C terminus of Yra1 is required to interact with Dia2. A yra1 mutant that lacks this domain is temperature sensitive yet has no apparent defect in RNA export. Remarkably, this mutant also fails to enter S phase at the nonpermissive temperature. Significantly, other mutants in transcription-coupled export do not exhibit S phase entry defects or sensitivity to DIA2 expression levels. Together, these results indicate that Yra1 has a role in DNA replication distinct from its role in mRNA export. Furthermore, Dia2 binding to replication origins is significantly reduced when association with Yra1 is compromised, suggesting that one aspect of the role of Yra1 in DNA replication may involve recruiting Dia2 to chromatin.

The ubiquitin proteasome system plays an important role in mediating a wide variety of cellular processes, including cell division and DNA replication and repair. Members of the highly conserved SCF (Skp1/Cdc53/F-box protein) ubiquitin ligase family are involved in controlling cell proliferation by regulating the ubiquitin-mediated proteolysis of key cell cycle regulators (7, 9, 18, 27, 33, 34, 37, 40, 43). SCF complexes are modular ubiquitin ligases whose specificity is determined by individual F-box proteins, which act as substrate-specific adapters (9, 33). Many F-box proteins have been identified in both humans and model eukaryotic systems, suggesting that SCF pathways are a highly conserved mechanism for controlling protein function.

In Saccharomyces cerevisiae, the F-box protein Dia2 has been linked to DNA replication and genomic stability. The dia2Δ mutant is hypersensitive to DNA damage, exhibits chromosome loss and rearrangement, and accumulates DNA damage foci (6, 17, 28). Two large-scale genomic analyses have identified many synthetic interactions between dia2Δ and mutants involved in DNA replication, replication checkpoint signaling, DNA damage, and DNA repair, suggesting that Dia2 functions in one or more of these pathways (6, 28). The Schizosaccharomyces pombe Pof3 protein is structurally and functionally related to Dia2 (15). The pof3 mutant also exhibits genomic instability, sensitivity to DNA damage, and synthetic interactions with checkpoint genes (15, 25), suggesting that the function performed by Dia2 and Pof3 is evolutionarily conserved.

The mechanistic role that Dia2 performs in DNA replication is not known. We have found that Dia2 associates with origins of replication in a cell cycle-dependent manner, suggesting that Dia2 may regulate DNA replication as a chromatin-bound protein (17). Recent work suggests that Dia2 may play a role in helping replication complexes traverse the replication fork barrier at the ribosomal DNA locus (6). A third possibility is that Dia2 may link DNA replication with sister chromatid cohesion, as Dia2 shows genetic interactions with the anaphase inhibitor Pds1 and with Ctf4, a protein that binds DNA polymerase and is important for sister chromatid cohesion (28, 32). Intriguingly, Pof3 interacts with Mcl1, the S. pombe homolog of Ctf4 (25).

Dia2 is a bona fide F-box protein in that it assembles with Skp1, Cdc53, and Rbx1 into a functional SCF ubiquitin ligase complex (12, 17, 18, 20). Presumably, the role of Dia2 in DNA replication involves targeting a substrate protein for ubiquitination, but no replication-specific targets have been identified. Indeed, little is known about Dia2 interaction partners in general, regardless of whether they might be ubiquitination targets. A large-scale proteomic analysis identified a few replication proteins and a large number of proteins involved in ribosome biogenesis as interaction partners of Dia2 (12), but the relevance of these interactions has recently been called into question (6).

Using an affinity-based approach, we have identified Yra1 as a new Dia2 interaction partner. We demonstrate that endogenous Yra1 and Dia2 coimmunoprecipitate, confirming the validity of the affinity screen. Furthermore, we find that fractions of both Yra1 and Dia2 protein populations behave as classically defined chromatin-bound proteins. Although Yra1 has been extensively studied for its role in transcription-coupled mRNA export (TREX) (11, 13, 21, 22, 35, 38, 44, 45), we present evidence that Yra1 also has a role in DNA replication. Previous studies have found that yra1 mutants are sensitive to the DNA replication inhibitor hydroxyurea (HU) (14) and that Yra1 contributes to the genomic stability of transcribed sequences (31), but it is not clear whether these observations are the result of a direct role in DNA replication or are secondary effects of the function of Yra1 in mRNA export. We find that Yra1 binds replication origins and a subunit of DNA polymerase δ. The association of Yra1 with origins occurs even after treatment with RNase A/T1 and inhibition of transcription. Importantly, we show that a yra1 mutant that lacks 17 amino acids at the C terminus that has been shown to be competent for mRNA export (44) is defective in S phase entry. Significantly, other TREX mutants do not exhibit S phase entry defects or sensitivity to DIA2 overexpression. Together, these results indicate that the role of Yra1 in DNA replication is distinct from its role in mRNA export. In addition, the yra1 C-terminal truncation mutant is defective in binding Dia2. Furthermore, Dia2 association with replication origins is severely compromised in this mutant, suggesting that Yra1 may recruit Dia2 to chromatin.


Generation of strains and constructs.

The Saccharomyces cerevisiae strains used in this study are described in Table Table1.1. Yeast was maintained and cultured according to standard methods (31a). Plasmids yra1-1 (a gift from Ed Hurt, Heidelberg University), HA-YRA1, HA-yra1ΔRBD, and HA-yra1-210 (gifts from Françoise Stutz, University of Geneva) were transformed into yra1Δ (DKY456) and dia2Δyra1Δ (DKY460) strains carrying a CEN URA3 YRA1 plasmid. The transformants were then incubated on 5-fluoroorotic acid (5-FOA) plates to generate DKY457, DKY458, DKY459, DKY479, and DKY480. The strains OAY535 (HA-MCM4) and OAY617 (HA-CDC45) were provided by Anja K. Bielinksy (University of Minnesota). The pRS404-3HA-HYS2 plasmid (a gift from Anja K. Bielinksy, University of Minnesota) was linearized with EcoRI, gel purified, and transformed into DKY456 and DKY408 to generate DKY473 and DKY505, respectively. The HYS2-HA integration and expression were verified by Western blotting. The sub2 alleles were provided by Christine Guthrie (University of California, San Francisco). The CLB2-HA construct was obtained from Mike Tyers (Samuel Lunenfeld Research Institute). The DIA2 overexpression construct pACK123 was generated by PCR amplification using primers LM31 and DK96 (Table (Table2)2) and digested with SalI and BamHI. The resulting fragment was cloned into the SalI and BamHI sites of p1223 (24).

Strains used in this study
Primers used in this study

Protein purification and mass spectrometry.

The glutathione S-transferase (GST)-Dia2 expression vector was generated by moving the NdeI-BamHI insert from pDMK107 (17) into p1205 (24). Protein was expressed in the Escherichia coli strain BL21(DE3). Cells were grown in 4 liters of LB at 37°C to an optical density at 600 nm of 0.7 and then induced with 0.5 mM IPTG (isopropyl-β-d-thiogalactopyranoside) at 25°C overnight. The cells were lysed by sonication in lysis buffer (100 mM NaCl, 50 mM Tris-HCl [pH 8.0], 2.5 mM EDTA, 0.1% Tween 20, 1 mM phenylmethylsulfonyl fluoride [PMSF] with complete protease inhibitors [Roche]). Lysed cells were centrifuged at 10,000 rpm in a Sorvall SS-34 rotor for 30 min at 4°C. Supernatant was incubated for 4 h at 4°C with rotation with 1 ml GT-Sepharose (Amersham Pharmacia) equilibrated with lysis buffer. GT-Sepharose was washed three times with 10 volumes of lysis buffer prior to elution. GST-Dia2 was eluted in three successive incubations with 1 volume each of lysis buffer containing 15 mM glutathione. Eluted GST-Dia2 was desalted on a BioGel-P6 column (3 volumes; Bio-Rad) using 50 mM HEPES-KOH (pH 7.6), 0.5 mM KCl. The GST-Dia2 protein was coupled to Affigel-10 (Bio-Rad) according to the manufacturer's instructions.

To prepare yeast extracts, 2 liters of logarithmically growing yeast cells was incubated overnight at 30°C. The cells were treated with 100 mM Tris-HCl (pH 9.2), 10 mM dithiothreitol (DTT) for 15 min at 30°C prior to spheroplasting. Spheroplasts were generated by incubating cells in 1 M sorbitol, 50 mM Tris-HCl (pH 7.5), 50 mM MgCl2, 50 mM CaCl2, and 1.1 mg Zymolyase 100T per g of cells (wet weight) for 30 min at 30°C. The spheroplasts were washed once in 1 M sorbitol and then centrifuged through a cushion of 2 M sorbitol at 3,500 × g for 15 min at 4°C. The spheroplasts were resuspended in 125 mM potassium acetate, 30 mM HEPES-KOH (pH 7.2), 3 mM EDTA, 3 mM EGTA, 2 mM DTT, 0.5 mM PMSF, and 10 μg each of pepstatin, aprotinin, and leupeptin and then dropped into liquid nitrogen in a precooled mortar. The cells were ground with a pestle while in liquid nitrogen. Lysed cells were thawed and centrifuged at 100,000 × g for 45 min at 4°C. The supernatant was desalted on a Biogel-P6 column (Bio-Rad) using 50 mM Tris-HCl (pH 8.0), 150 mM KCl, 0.5 mM DTT, and 0.5 mM PMSF.

The GST-Dia2 column was equilibrated with 10 volumes of 50 mM Tris-HCl (pH 8.0), 150 mM KCl, and 0.5 mM DTT and then loaded with the yeast extract. After the binding, the column was washed with 10 volumes of 50 mM Tris-HCl (pH 8.0), 150 mM KCl, 0.5 mM DTT. Bound proteins were eluted using a step gradient of KCl, beginning at 0.2 M. Eluted proteins were precipitated with 3% trichloroacetic acid (TCA) and resuspended in loading buffer prior to resolution by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gels were stained with Coomassie blue, and proteins were prepared for mass spectrometry as described previously (41).

Coimmunoprecipitations (co-IPs).

The method for preparation of lysates was adapted from Ricke and Bielinsky (30). Fifty milliliters of logarithmically growing yeast culture was harvested, and cells were lysed in chromatin immunoprecipitation (ChIP) lysis buffer (50 mM HEPES-KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, protease inhibitors [1 mM PMSF and complete protease inhibitors {Roche}]) by vortexing them with glass beads for 40 to 60 min at 4°C. After the addition of 1 mM PMSF, the samples were subjected to sonication three times for 10 seconds each. The lysates were recovered after two centrifugation steps, one at 20,000 × g for 10 min at 4°C followed by one at 20,000 × g for 15 min at 4°C. For DNase treatment of lysates, samples were incubated with 100 kU of DNase I for 45 min on ice. Anti-Myc (9E10) antibodies (Covance Research Products, Inc., Berkeley, CA), antihemagglutinin (anti-HA; HA.11) antibodies (Covance Research Products, Inc., Berkeley, CA), and immunoglobulin G (IgG)-Sepharose beads (Amersham) were used for immunoprecipitation. For experiments with HA-Hys2 and protein A-Yra1 from non-cross-linked samples, lysates were desalted on a Centricon-10 column (Millipore) using NETN (100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 8.0, 0.5% Igepal plus 1 mM PMSF, complete protease inhibitors [Roche], 10 mM NaF, 25 mM β-glycerophosphate). Immunoprecipitates were incubated 4 h to overnight at 4°C with rotation and then washed three times with 10 volumes of NETN. For experiments with cross-linked proteins, samples were treated with formaldehyde and lysed as described for ChIPs. Immunoprecipitates from formaldehyde-treated samples were washed twice in ChIP lysis buffer, once with ChIP lysis buffer containing 500 mM NaCl, and once with 10 mM Tris (pH 8.0), 0.25 M LiCl, 0.5% Igepal, 0.5% sodium deoxycholate. Anti-Myc (9E10) and anti-HA (HA.11) antibodies (Covance Research Products, Inc., Berkeley, CA) and horseradish peroxidase-conjugated anti-mouse antibodies (Jackson Immunoresearch, Inc. West Grove, PA) were used for immunoblotting.

Chromatin fractionation assay.

Chromatin fractionation was performed as described previously (23), with the following modifications. Strains were grown to ~2 × 107 cells/ml, and a total of 1 × 109 cells were collected. The cells were washed twice with 1× phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) and incubated in 1 ml SNH buffer (400 mM sorbitol, pH 7.5, 150 mM NaCl, 50 mM HEPES) with 10 mM DTT for 10 min. The cells were resuspended in 2 ml SNH plus 10 mM DTT and spheroplasted with 1 mg Zymolyase 20T (ICN Biomedicals, Inc.). Spheroplasting was complete when the optical density at 600 nm in a 1:100 dilution in water was less than 10% of the original value. The spheroplasts were washed with 1 ml SNH plus 2.5 mM MgCl2 and then resuspended in SNH prelysis buffer (SNH plus 50 mM NaF, 1× complete protease inhibitor cocktail [Roche], 1 mM PMSF, 2.5 mM MgCl2). The spheroplasts were lysed by addition of Triton X-100 to a 0.5% final concentration and incubated on ice with gentle agitation for 10 min to generate the whole-cell extract (WCE). The WCE was separated into the supernatant and pellet by microcentrifugation at 12,000 rpm for 10 min at 4°C to generate soluble protein and a crude chromatin pellet. The pellet was washed and resuspended to a volume equal to that of the supernatant with SNH lysis buffer (SNH prelysis plus 0.5% Triton X-100). For micrococcal nuclease (MNase) treatment, the pellet from WCE centrifugation was resuspended to a 1/2 volume with SNH lysis buffer. The suspension was then incubated at 37°C for 2 min, after which 1 unit MNase (Sigma) and 1 mM CaCl2 were added and then incubated at 37°C for an additional 1 min. EGTA was added to a final concentration of 1 mM to halt the reaction, and the reaction mixture was microcentrifuged at 10,000 rpm for 2 min at 4°C. The pellet was digested once more as described above, with 0.2 unit MNase. Supernatants were combined and mixed, and the pellet was resuspended to the starting volume with SNH lysis buffer. Half of the MNase supernatant was centrifuged at 100,000 × g in a Beckman TLA-100.3 rotor for 1 h at 4°C to generate high-speed fractions. The supernatant was removed and the pellet resuspended with SNH lysis buffer. All pellet and supernatant fractions were brought to the same cell equivalents by addition of SNH lysis buffer, and 1 volume of 2× Laemmli loading buffer was added. Samples were boiled for 5 min and then microcentrifuged for 1 min prior to resolution by SDS-PAGE.

Histone association assay.

Extraction and immunoprecipitation (anti-histone H3 [Abcam]) of proteins for the histone association assay were performed as described previously (29). Proteins resolved by SDS-PAGE immunoblotting were probed with anti-HA (HA.11) antibodies (Covance Research Products, Inc., Berkeley, CA) and horseradish peroxidase-conjugated anti-mouse antibodies (Jackson Immunoresearch, Inc., West Grove, PA).


Cultures of 50 ml logarithmically growing cells were harvested after being cross-linked with 1.35 ml of 37% formaldehyde for 15 min. Cross-linking was quenched by the addition of glycine to 125 mM. The WCE was prepared for ChIP in ChIP lysis buffer as described previously for co-IPs (30). Anti-HA (HA.11) antibodies (Covance Research Products, Inc., Berkeley, CA) and IgG-Sepharose beads (Amersham) were used for immunoprecipitation. The samples were washed twice in ChIP lysis buffer, once with ChIP lysis buffer containing 500 mM NaCl, once with 10 mM Tris (pH 8.0), 0.25 M LiCl, 0.5% Igepal, and 0.5% sodium deoxycholate, and finally with 1× Tris-EDTA. Formaldehyde cross-linking was reversed by heating the samples at 65°C for 6 to 12 h, followed by phenol-chloroform extraction of DNA. ARS1, ARS305, and ARS603 primers (2) and Intergenic and PMA1 promoter primers (adapted from reference 45) were used to amplify the immunoprecipitated DNA (Table (Table2).2). RNase (RNase A/T1 cocktail; Ambion) treatment of samples for the ChIP protocol was performed as described previously (1).

Logarithmically growing yeast cells were treated with 1,10-phenanthroline (100-μg/ml final concentration) for 2 h to inhibit transcription before ChIP was performed. Cell cycle arrest was induced by the addition of α-factor, HU (200 mM), and nocodazole (15 μg/ml). Cells were harvested when 90% of the cells exhibited appropriate arrest.

RNA extraction and reverse transcription (RT)-PCR.

Total RNA from exponentially growing cells was isolated using an RNeasy mini kit (QIAGEN Ltd.). Total RNA was incubated with RQ1 RNase-free DNase (Promega) at 37°C for 15 min. DNase-treated total RNA was recovered by acid phenol-chloroform (Ambion) extraction followed by ethanol precipitation. The RNA concentration and purity were calculated by measuring the absorbance values at 260 and 280 nm before and after DNase treatment.

One to 2 μg of total RNA was reverse transcribed using oligonucleotide deoxyribosylthymine primers and Superscript II reverse transcriptase (Invitrogen) by incubation at 42°C for 1 h. The reaction was terminated by incubation at 70°C for 15 min and then used as a template for PCR amplification.

Flow cytometry.

Cells were collected and fixed in 70% ethanol for at least 30 min and then resuspended in PBS (140 mM NaCl, 3 mM KCl, 5 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) plus 0.02% sodium azide. To prepare for flow cytometry, fixed cells were treated with fluorescence-activated cell sorting buffer (200 mM Tris-HCl, 20 mM EDTA plus 0.1% RNase A) overnight at 37°C and then stained in 50 μg/ml propidium iodide in PBS. Prior to analysis, the cells were diluted 10-fold in PBS and sonicated for 5 s at 15% efficiency using a sonic dismembrator (Fisher Scientific, Pittsburgh, PA). Data analysis was performed with FlowJo v6.3.3 software, and graphs were generated using Deltagraph 5.0.


Dia2 interacts with Yra1 physically and genetically.

To identify Dia2-interacting proteins, we used a biochemical affinity column approach. We generated a recombinant GST-Dia2 fusion protein construct for expression in E. coli. The purified GST fusion protein was coupled to an Affigel-10 matrix and used as an affinity column for proteins extracted from a dia2Δ strain (Fig. (Fig.1).1). After a wash in low-salt buffer, proteins were eluted from the column with increasing amounts of KCl. Eluted proteins were TCA precipitated, resuspended in loading buffer, and resolved by SDS-PAGE. At 500 mM KCl, a number of proteins were observed to elute from the column. We used mass spectrometry to identify some of these proteins (41). One of the most prominent bands migrated just above the 25-kDa marker (Fig. (Fig.1)1) and was identified as Yra1 by three peptides, LNLIVDPNQRPVK, EFFASQVGGVQR, and GQSTGMANITFK.

FIG. 1.
Dia2 binds Yra1. (A) Yra1 binds a GST-Dia2 affinity column and elutes at 0.5 M KCl. Recombinant GST-Dia2 was purified from E. coli and coupled to Affigel-10 to generate an affinity column. An extract from dia2Δ cells was passed over the affinity ...

To confirm that Dia2 and Yra1 interact with each other, we performed co-IP experiments using our 18× Myc-tagged version of Dia2 (17) and an HA-tagged allele of YRA1 (44). As shown in Fig. Fig.1B,1B, HA-Yra1 coimmunoprecipitates with Dia2-18xMyc. Likewise, the reciprocal immunoprecipitation indicates that Dia2-18xMyc coimmunoprecipitates with HA-Yra1. The interaction is specific, as no purification is observed when mock immunoprecipitations with normal IgG are performed. The larger Dia2 form, which is close to the predicted size, interacts preferentially with Yra1 in this assay. We have observed a similar preference for interaction with the larger Dia2 form in anti-Skp1 immunoprecipitates (16). To determine whether DNA mediates the interaction between Dia2 and Yra1, we treated protein extracts with DNase I prior to co-IP. As shown in Fig. Fig.1C,1C, HA-tagged Yra1 still coimmunoprecipitates with myc-tagged Dia2 in extracts treated with DNase I. We conclude that Dia2 and Yra1 physically interact, although whether this interaction is direct or is mediated by an intermediary protein remains to be established.

A straightforward explanation for the Dia2-Yra1 interaction is that Yra1 might be a substrate of the SCFDia2 complex. To test this possibility, we examined Yra1 protein turnover in wild-type and dia2Δ cells by using a protein stability assay (Fig. (Fig.1D).1D). Cycloheximide was added to the cells expressing HA-tagged Yra1 (35) at time zero to stop protein synthesis. The abundance of the Yra1 protein was determined by immunoblotting protein samples collected at 30-minute intervals for 2 h. Under these conditions, we observed no change in the abundance of the Yra1 protein in either wild-type or dia2Δ cells. As a control, we used the mitotic cyclin Clb2. As expected, HA-tagged Clb2 was unstable in both wild-type and dia2Δ cells. We also examined protein A-tagged Yra1 abundance in cells in G1, S, or M phase by immunoblotting samples from wild-type and dia2Δ cells arrested with α-factor, HU, or nocodazole, respectively (Fig. (Fig.1E).1E). We observed no change in Yra1 abundance under these conditions either, suggesting that Yra1 is not a proteolytic target of the SCFDia2 complex.

As Yra1 has well-documented roles in TREX (13, 21, 22, 35, 38, 44, 45) and Dia2 has been linked to DNA replication (6, 17, 28), we were puzzled as to why they might be in a complex together. To investigate the potential role of the Dia2-Yra1 interaction, we used a number of available yra1 mutants that exhibited different phenotypes (Fig. (Fig.2A).2A). The temperature-sensitive yra1-1 allele exhibits an mRNA export defect as measured by an in situ hybridization assay for poly(A)+ RNA (35). The yra1-1 mutant carries five point mutations, two of which are in the conserved central RNA-binding protein domain (RBD) and two of which are in a C-terminal motif called the C box (35). The RBD motif, also called an RNA recognition motif domain, is found in many RNA-binding proteins (26). The ability of Yra1 to bind RNA in vitro has been mapped to two domains, an N-terminal region following the N box and a C-terminal region upstream of the C box (44). The yra1 ΔRBD mutant has the RBD deleted in frame, whereas the C box is deleted in the temperature-sensitive yra1 1-210 mutant. The yra1 ΔRBD mutant and the yra1 1-210 mutant exhibit binding to RNA in vitro and to an mRNA export partner protein, Mex67, indistinguishable from that exhibited by wild-type Yra1 (44). However, they exhibit striking differences in growth at 37°C and in the export of poly(A)+ RNA from the nucleus (44). The yra1 ΔRBD mutant exhibits an mRNA export defect at 37°C but is viable at this temperature, whereas the yra1 1-210 strain shows a dramatic growth phenotype but does not exhibit an mRNA export defect (44). These observations suggested that Yra1 might have an additional function.

FIG. 2.
Dia2 interacts genetically with Yra1. (A) Diagram of Yra1 domain structure and mutants used in this study. The diagram shows the changes in each mutant and indicates their growth and mRNA export phenotypes (adapted from references 35 and 45). (B) dia2 ...

We crossed the yra1 mutants with a dia2Δ strain covered by a DIA2 URA3 plasmid, induced sporulation of the resulting diploid, dissected tetrads, and recovered the appropriate double-mutant strains. We then tested these strains for growth on media containing 5-FOA to examine what happened to the cells when both DIA2 and YRA1 were mutated. We observed that the dia2Δ yra1-1 and the dia2Δ yra1 1-210 strains were inviable (Fig. (Fig.2B).2B). The dia2Δ yra1 ΔRBD strain was viable but exhibited slower growth than the yra1 ΔRBD strain alone. We also overexpressed DIA2 in each of the yra1 mutants. We found that overexpression of DIA2 induced lethality in the yra1-1 and yra1 1-210 strains at their permissive temperature, whereas the effect of overexpression of DIA2 in the yra1 ΔRBD strain was only moderate (Fig. (Fig.2C).2C). Thus, the strongest genetic interactions were observed between the dia2Δ and the yra1-1 and yra1 1-210 mutants. Both of these mutants are temperature sensitive, but they exhibit different effects on mRNA export, suggesting that the role of Yra1 shared with Dia2 does not involve mRNA export.

Yra1 associates with chromatin and replication origins.

As Dia2 is linked to DNA replication and genomic stability, it is possible that the Dia2-Yra1 interaction is important for these pathways. There are a number of reports suggesting that Yra1 can bind chromatin in ChIP assays (1, 22), and since Dia2 binds replication origins (17), the interaction between these proteins might involve chromatin. To verify that Yra1 is chromatin bound, we used two assays: a histone association assay (30) and a classic chromatin fractionation assay (23). The histone association assay is similar to ChIP, such that cross-linked proteins are immunoprecipitated with anti-histone H3 antibodies. After reversal of the cross-links, immunoprecipitated proteins are resolved by SDS-PAGE and immunoblotted. As shown in Fig. Fig.3A,3A, HA-Yra1 is efficiently immunoprecipitated in this assay, as is myc-tagged Dia2. The results of the chromatin fractionation are shown in Fig. Fig.3B.3B. As expected, substantial amounts of both Yra1 and the positive-control protein Mcm4, a component of the Mcm2-7 replicative helicase complex, are released into the supernatant upon MNase treatment and then found in the chromatin pellet fraction after ultracentrifugation. Similar results were observed with the Dia2 protein. Together, our results confirm that Yra1 and Dia2 associate with chromatin.

FIG. 3.
Yra1 and Dia2 associate with chromatin. (A) HA-Yra1 (top) and Dia2-18xMyc (bottom) coimmunoprecipitate with histone H3 in a histone association assay (30). Mock immunoprecipitations (IP) were used as negative controls (lane 1). (B) Yra1 and Dia2 fractionate ...

We used ChIP to determine whether Yra1 binds replication origins as Dia2 does. We found that Yra1 binds ARS305, ARS1 (early-firing), and ARS603 (late-firing) sequences in a cross-linker-dependent manner (Fig. (Fig.4A).4A). As has been previously shown, Yra1 does not coprecipitate with DNA sequences from the PMA1 promoter or an intergenic region from chromosome IV (45), indicating that the interaction with replication origins is specific.

FIG. 4.
Yra1 associates with replication origins. (A) Yra1 binds origins ARS1, ARS305, and ARS603 but not nonorigin regions (intergenic and PMA1 promoter). ChIP was performed using either the HA-tagged Yra1 strain or the protein A-tagged Yra1 strain. After formaldehyde ...

As Yra1 has been previously shown to immunoprecipitate with the 3′ ends of actively transcribed genes in preparation for RNA processing and export (22) and since many origins of replication are in highly transcribed areas of the genome, we sought to determine whether Yra1 association with origins was dependent on transcription. Using an assay described by Abruzzi et al. (1), we determined that the binding of Yra1 to ARS305 and ARS603 is resistant to RNase A/T1 treatment (Fig. (Fig.4B).4B). We examined Yra1 association with origins in the presence of 1,10-phenanthroline, a metal ion chelator that inhibits RNA polymerases in S. cerevisiae (10). As shown in Fig. Fig.4C,4C, treatment of cells with 1,10-phenanthroline has no effect on the binding of Yra1 to ARS1 and ARS603 sequences (Fig. (Fig.4C,4C, top). To verify that 1,10-phenanthroline inhibited transcription as reported previously (10), we assayed the abundance of the unstable RPL8B transcript by RT-PCR, using the ACT1 transcript (actin) as a control (Fig. (Fig.4C,4C, bar graph). We quantified the results of these experiments, expressing the abundance of the RPL8B transcript as a percentage, with the value for the untreated sample arbitrarily set to 100%. By this analysis, we observed a fourfold decrease in the abundance of the RPL8B transcript after treatment with 1,10-phenanthroline, which is similar to the decrease previously described (10). Together, these results suggest that the Yra1 association with origins is not simply a mere coincidence due to its recruitment to transcriptionally active genes.

We examined Yra1 association with origins during G1, S, or M phase using ChIP from cells arrested with α-factor, HU, or nocodazole, respectively. Yra1 showed association with early-origin ARS305 and late-origin ARS603 in cells arrested in all three phases, although binding during G1 may be reduced (Fig. (Fig.4D).4D). Similar results were observed with early-origin ARS607 and late-origin ARS501 (data not shown). Interestingly, the association of Dia2 with origins is strongest in M phase (17), indicating that Dia2 and Yra1 may interact on chromatin at this time.

A role for Yra1 in DNA replication.

Given the association of Yra1 with Dia2 and replication origins, we asked whether Yra1 might bind other origin-binding proteins involved in DNA replication. We generated strains coexpressing protein A-tagged Yra1 with either HA-tagged Cdc45, required for DNA replication initiation, or HA-tagged Hys2, a component of DNA polymerase δ, for use in co-IP experiments (3). We performed these co-IP experiments using protein samples from either untreated (Fig. (Fig.5A)5A) or formaldehyde-cross-linked (Fig. (Fig.5B)5B) cells. We observed immunoprecipitation of HA-Hys2 with protein A-Yra1 purified with IgG-Sepharose in both cross-linked and non-cross-linked samples (Fig. 5A and B). HA-Hys2 did not independently bind to the IgG-Sepharose in either situation, indicating that this reaction was dependent on Yra1. Furthermore, we observed no interaction between Cdc45 and Yra1 in either situation (Fig. (Fig.5A;5A; results from cross-linked samples not shown), suggesting that Yra1 does not promiscuously interact with HA-tagged proteins. We also examined whether Dia2 might interact with Hys2 using co-IP (Fig. (Fig.5C).5C). Intriguingly, a fraction of the Dia2 protein population also copurified with HA-Hys2, suggesting that Yra1, Dia2, and Hys2 may function together in a complex.

FIG. 5.
Yra1 interacts with Hys2. (A) HA-Hys2 copurifies with protein A-tagged Yra1 from non-cross-linked protein extracts. Protein extracts were prepared from the indicated strains and incubated with IgG-Sepharose. Precipitates were immunoblotted with anti-HA ...

To determine if yra1 mutants exhibited defects in DNA replication, we performed two types of synchronization experiments. In the first, we arrested cells in late G1 phase by α-factor treatment, released the cells from the arrest at 36°C, and examined samples at 30-minute intervals by flow cytometry to assay entry into S phase. As shown in Fig. Fig.6A,6A, both yra1-1 and yra1 1-210 mutants exhibit defects in S phase entry, whereas the yra1 ΔRBD mutant enters S phase indistinguishably from wild-type cells. No obvious defects in S phase entry were observed at the permissive temperature in any of the mutants (data not shown). To determine if the yra1 mutants were also defective for S phase progression, we released α-factor-arrested cells into media containing HU at the permissive temperature to allow them to enter early S phase. After 2 h, the cells were released from HU arrest at 36°C and samples collected at 30-minute intervals were prepared for flow cytometry. In this case, all four strains renewed progression through S phase at approximately the same rate (Fig. (Fig.6B).6B). As a comparison, we performed the same experiments with the dia2Δ strain and observed no defects in S phase entry or progression. These results suggest that Yra1 has a role in S phase initiation but not in S phase progression.

FIG. 6.
yra1 mutants are defective for S phase entry. (A) The indicated strains were arrested in G1 using alpha factor at 25°C and then released at 36°C. Samples were taken at the indicated time points and prepared for flow cytometry. (B) The ...

To determine if the role of Yra1 in DNA replication was linked to the function of the TREX complex, we examined conditional alleles of another TREX subunit, Sub2 (13, 16, 36, 45). The sub2-1 allele is temperature sensitive and cold sensitive, whereas the sub2-5 allele is cold sensitive (16). Unlike yra1 mutants, neither sub2 allele was sensitive to the overexpression of DIA2 (Fig. (Fig.7A).7A). Furthermore, neither sub2 mutant exhibited S phase entry (Fig. (Fig.7B)7B) or S phase progression (Fig. (Fig.7C)7C) defects. These results strongly suggest that the role of Yra1 in DNA replication is independent of the role of the TREX complex.

FIG. 7.
sub2 mutants are competent in S phase entry. (A) The indicated strains were transformed with either an empty vector or a DIA2 overexpression construct. Serial dilutions of cultures at equal densities were spotted on media containing glucose (as a control) ...

Yra1 is important for Dia2 origin association.

Intriguingly, the two yra1 mutants that are defective in S phase entry have mutations at the C terminus of the Yra1 protein, in the C box. In addition, these mutants exhibit the most sensitivity to DIA2 expression levels. We thus sought to determine if the C box is important for binding to Dia2. We generated strains expressing Dia2-18xMyc and the HA-tagged yra1 mutant alleles and used them in co-IP experiments. The protein levels of the yra1 1-210 and yra1 ΔRBD mutants are indistinguishable from those of wild-type HA-Yra1 (44) (Fig. (Fig.8A).8A). The yra1 1-210 mutant is severely compromised in binding myc-tagged Dia2, whereas the yra1 ΔRBD mutant still binds Dia2, although perhaps not quite as well as wild-type Yra1 (Fig. (Fig.8B).8B). Similar results were observed in both asynchronous and nocodazole-arrested cultures. We observed no difference in the ability of the HA-tagged proteins to be immunoprecipitated with anti-HA antibodies, indicating that the failure to copurify Dia2 is a result of impaired binding to the Yra1 1-210 protein. These results indicate that the C box is important in Yra1 association with Dia2.

FIG. 8.
Binding to Yra1 is important for Dia2 origin association. (A) Protein expression in the double-tagged Dia2-18xMyc HA-Yra1 strains is equivalent. Protein extracts from the indicated strains were blotted with anti-Myc (top) or anti-HA (bottom) antibodies. ...

Finally, we examined whether Dia2 could still bind replication origins when Yra1 association is compromised. We performed ChIP of myc-tagged Dia2 in the yra1 1-210 mutant using both asynchronous and nocodazole-arrested cultures. As shown in Fig. Fig.8C,8C, Dia2 origin association is compromised in the yra1 1-210 mutant background in both asynchronous and nocodazole-arrested cells, although it is competent to bind in the yra1 ΔRBD mutant background under these conditions. Together, these results suggest that Yra1 is required to recruit Dia2 to replication origins.


The results of our study support two main conclusions: (i) Yra1 has a role in DNA replication that is distinct from its role in mRNA export and (ii) Yra1 binding to Dia2 is important for Dia2 association with replication origins.

Yra1 functions in DNA replication.

Previous work has suggested the possibility of a role for Yra1 in S phase, but it has been difficult to determine whether the results observed were an indirect result of the role of Yra1 in mRNA export. For example, the yra1-2 mutant has been shown to be sensitive to HU (14). Yra1 is part of the TREX complex, which bridges transcription and mRNA export (36). TREX subunits have been shown to be important for maintenance of the genomic stability of transcribed sequences (31). Recent work indicates that mutants in the TREX subunit called Hpr1 lead to disrupted transcription and mRNP biogenesis that inhibit the movement of replication forks through areas of the genome undergoing transcription during S phase (42). As Yra1 is a member of the TREX complex, it is possible that yra1 mutants could also lead to defective mRNP biogenesis that disrupts DNA replication. This might explain the replication defects that we observe in yra1 mutants. However, we show that mutants in another TREX subunit, Sub2, do not exhibit S phase entry defects. Moreover, in such a role, we predict that Yra1 would be important for S phase progression and not necessarily S phase entry. Rather, our results suggest that the opposite is the case, i.e., that Yra1 is important for S phase entry but is not essential for S phase progression. Therefore, we think that this possibility is unlikely.

All evidence to date suggests that the yra1 1-210 mutant is competent for mRNA export, yet we observe an S phase entry phenotype with this mutant. The yra1 1-210 mutant does not accumulate poly(A)+ RNA in the nucleus at the nonpermissive temperature (44). In addition, the yra1 1-210 mutant behaves indistinguishably from wild-type Yra1 in its abilities to bind RNA in vitro and to associate with the Mex67 mRNA export protein (44). In principle, it is possible that the yra1 1-210 mutant is defective in the export of a subset of mRNAs at sufficiently low levels that they cannot be detected by in situ hybridization and that this defect causes the S phase entry phenotype that we observe. However, in this situation we would expect the C box to be important for binding such RNAs and no RNA-binding activity has been identified for the C-box domain (44).

A simpler interpretation of our results is that the functions of Yra1 in mRNA export and DNA replication are genetically separable. We demonstrate that two yra1 temperature-sensitive mutants with distinct mRNA export phenotypes, yra1-1 and yra1 1-210, exhibit defects in S phase entry from an α-factor arrest. Intriguingly, both mutants have mutations in the C box. The yra1 1-210 mutant has a complete deletion of the C box, and the yra1-1 mutant has two point mutations in this domain. Interestingly, these mutations in yra1-1 appear to be largely responsible for the temperature-sensitive phenotype (35), which is consistent with the severe growth defect of the yra1 1-210 mutant (44). Indeed, these results suggest that the essential function of Yra1 may be in DNA replication.

Other RNA-binding proteins have been suggested to play roles in DNA synthesis and S phase. In humans, the DNA and RNA helicase UPF1 has been shown to function in maintaining genomic stability by contributing to DNA replication and cell cycle progression (4). In addition, transcriptional complexes are suggested to be critical determinants in proper initiation and progression of DNA replication (39).

The results presented here likely provide clues to the specific role that Yra1 plays in DNA replication. Our results suggest that Yra1 functions on chromatin at replication origins. The observation that Yra1 associates with Hys2, a component of DNA polymerase δ, coupled with the requirement for Yra1 for S phase entry, suggests that Yra1 may function at the transition from initiation to DNA synthesis. Since Yra1 still binds origins after treatment of lysates with RNase A/T1, it is unlikely that RNA serves as an intermediate for the association with chromatin, although we cannot rule out the possibility that an RNA transcript is protected from digestion by blocking proteins. As Yra1 does not appear to associate with Cdc45, a helicase cofactor important for loading DNA polymerases α and epsilon at the origin (5), it is possible that Yra1 interacts with only a subset of proteins found at the origin during the transition to DNA synthesis.

Yra1 is important for Dia2 origin association.

Our data imply that Dia2 and Yra1 function in the same pathway. Mutations in YRA1 and DIA2 exhibit synthetic defects, which is typically associated with function in the same or parallel pathways. Since Yra1 and Dia2 physically interact, they likely function together in the same pathway. As both proteins associate with chromatin and with replication origins, it is probable that they function together on chromatin. We demonstrate that the C box of Yra1 is required to interact efficiently with Dia2 and that when Dia2 binding to Yra1 is compromised, so is its ability to bind replication origins. Thus, Yra1 may have a role in recruiting Dia2 to replication origins.

One possibility is that Yra1 might recruit SCFDia2 to replication origins to target an unknown substrate for ubiquitination. Yra1 itself is unlikely to be a substrate of SCFDia2, as we observe no changes in Yra1 protein stability in dia2Δ mutants, nor do we observe any obvious modified forms of Yra1. Finding common interaction partners of Dia2 and Yra1 may be key to determining how they function together. Large-scale proteomic analyses have not yet identified any common interaction partners of Dia2 and Yra1, but we find that both can immunoprecipitate with Hys2 (12, 19). We look forward to future studies examining further the composition of Dia2 and Yra1 complexes.

The roles that Dia2 and Yra1 play in DNA replication do not appear to be equivalent, as the yra1 temperature-sensitive mutants and the dia2Δ strain exhibit different S phase entry phenotypes. We show here that yra1 mutants are defective for S phase entry. In contrast, dia2Δ mutants have been suggested to prematurely enter S phase (17) or to be defective in replication fork progression through the replication fork barrier at the ribosomal DNA locus (6). Thus, the role that Yra1 plays in DNA replication encompasses functions in addition to recruiting Dia2 to replication origins.

Chromatin structure is likely to have a significant impact on DNA replication, and a number of chromatin-remodeling complexes have been suggested to play a role in DNA replication (reviewed in reference 8). We show here that fractions of both the Yra1 and the Dia2 protein populations behave as classically defined chromatin proteins. Furthermore, Yra1 has been shown to associate with chromatin at locations other than replication origins (1, 22, 45). In addition, a large-scale proteomic screen has identified a number of proteins involved either in chromatin modification or in DNA replication as interaction partners of Yra1 (19). Perhaps Yra1 has a general role in the assembly of complexes on chromatin that may explain its function in DNA replication. Future work will be necessary to mechanistically define the role of Yra1 in DNA replication.

In conclusion, we have identified Yra1 as a protein that interacts both physically and genetically with the F-box protein Dia2. We demonstrate that both Dia2 and Yra1 are chromatin-associated proteins that bind replication origins. Importantly, we find that Yra1 has a novel role in DNA replication distinct from its role in mRNA export. Yra1 and Dia2 bind to a DNA polymerase δ subunit, and yra1 mutants are defective for S phase entry, including a temperature-sensitive allele that is competent for mRNA export. Significantly, other TREX mutants do not exhibit S phase entry defects or sensitivity to DIA2 overexpression. Yra1 may recruit Dia2 to chromatin, as the yra1 C-terminal truncation mutant exhibits reduced binding to Dia2 and Dia2 origin association is compromised in this mutant. Together, these results broaden our understanding of S phase entry and the dynamics of chromatin-associated proteins.


We thank Ed Hurt (University of Heidelberg) for yra1 strains and plasmids, Françoise Stutz (Geneva University) for HA-tagged Yra1 constructs, Christine Guthrie (University of California, San Francisco) for the sub2 alleles, Katherine Kragtorp for advice and reagents for the RT-PCR experiments, and Anja-Katrin Bielinsky (University of Minnesota) for strains, advice, and critical reading of the manuscript. We are grateful for the help of Jun Qin with the mass spectrometry analysis (Baylor College of Medicine), and we are indebted to Stephen J. Elledge (Harvard Medical School) for insightful discussions early in this study.

This work was funded in part by a Minnesota Medical Foundation grant and an Academic Health Center Faculty Seed Grant to D.M.K.


[down-pointing small open triangle]Published ahead of print on 23 April 2007.


1. Abruzzi, K. C., S. Lacadie, and M. Rosbash. 2004. Biochemical analysis of TREX complex recruitment to intronless and intron-containing yeast genes. EMBO J. 23:2620-2631. [PMC free article] [PubMed]
2. Aparicio, O. M., A. M. Stout, and S. P. Bell. 1999. Differential assembly of Cdc45p and DNA polymerases at early and late origins of DNA replication. Proc. Natl. Acad. Sci. USA 96:9130-9135. [PMC free article] [PubMed]
3. Aparicio, O. M., D. M. Weinstein, and S. P. Bell. 1997. Components and dynamics of DNA replication complexes in S. cerevisiae: redistribution of MCM proteins and Cdc45p during S phase. Cell 91:59-69. [PubMed]
4. Azzalin, C. M., and J. Lingner. 2006. The human RNA surveillance factor UPF1 is required for S phase progression and genome stability. Curr. Biol. 16:433-439. [PubMed]
5. Bell, S. P., and A. Dutta. 2002. DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71:333-374. [PubMed]
6. Blake, D., B. Luke, P. Kanellis, P. Jorgensen, T. Goh, S. Penfold, B. J. Breitkreutz, D. Durocher, M. Peter, and M. Tyers. 2006. The F-box protein Dia2 overcomes replication impedance to promote genome stability in Saccharomyces cerevisiae. Genetics 174:1709-1727. [PMC free article] [PubMed]
7. Carrano, A. C., E. Eytan, A. Hershko, and M. Pagano. 1999. SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27. Nat. Cell Biol. 1:193-199. [PubMed]
8. Falbo, K. B., and X. Shen. 2006. Chromatin remodeling in DNA replication. J. Cell. Biochem. 97:684-689. [PubMed]
9. Feldman, R. M., C. C. Correll, K. B. Kaplan, and R. J. Deshaies. 1997. A complex of Cdc4p, Skp1p, and Cdc53p/cullin catalyzes ubiquitination of the phosphorylated CDK inhibitor Sic1p. Cell 91:221-230. [PubMed]
10. Grigull, J., S. Mnaimneh, J. Pootoolal, M. D. Robinson, and T. R. Hughes. 2004. Genome-wide analysis of mRNA stability using transcription inhibitors and microarrays reveals posttranscriptional control of ribosome biogenesis factors. Mol. Cell. Biol. 24:5534-5547. [PMC free article] [PubMed]
11. Hieronymus, H., and P. A. Silver. 2003. Genome-wide analysis of RNA-protein interactions illustrates specificity of the mRNA export machinery. Nat Genet. 33:155-161. [PubMed]
12. Ho, Y., A. Gruhler, A. Heilbut, G. D. Bader, L. Moore, S. L. Adams, A. Millar, P. Taylor, K. Bennett, K. Boutilier, L. Yang, C. Wolting, I. Donaldson, S. Schandorff, J. Shewnarane, M. Vo, J. Taggart, M. Goudreault, B. Muskat, C. Alfarano, D. Dewar, Z. Lin, K. Michalickova, A. R. Willems, H. Sassi, P. A. Nielsen, K. J. Rasmussen, J. R. Andersen, L. E. Johansen, L. H. Hansen, H. Jespersen, A. Podtelejnikov, E. Nielsen, J. Crawford, V. Poulsen, B. D. Sorensen, J. Matthiesen, R. C. Hendrickson, F. Gleeson, T. Pawson, M. F. Moran, D. Durocher, M. Mann, C. W. Hogue, D. Figeys, and M. Tyers. 2002. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415:180-183. [PubMed]
13. Jimeno, S., A. G. Rondon, R. Luna, and A. Aguilera. 2002. The yeast THO complex and mRNA export factors link RNA metabolism with transcription and genome instability. EMBO J. 21:3526-3535. [PMC free article] [PubMed]
14. Kashyap, A. K., D. Schieltz, J. Yates, III, and D. R. Kellogg. 2005. Biochemical and genetic characterization of Yra1p in budding yeast. Yeast 22:43-56. [PubMed]
15. Katayama, S., K. Kitamura, A. Lehmann, O. Nikaido, and T. Toda. 2002. Fission yeast F-box protein Pof3 is required for genome integrity and telomere function. Mol. Biol. Cell 13:211-224. [PMC free article] [PubMed]
16. Kistler, A. L., and C. Guthrie. 2001. Deletion of MUD2, the yeast homolog of U2AF65, can bypass the requirement for sub2, an essential spliceosomal ATPase. Genes Dev. 15:42-49. [PMC free article] [PubMed]
17. Koepp, D. M., A. C. Kile, S. Swaminathan, and V. Rodriguez-Rivera. 2006. The F-box protein Dia2 regulates DNA replication. Mol. Biol. Cell 17:1540-1548. [PMC free article] [PubMed]
18. Koepp, D. M., L. K. Schaefer, X. Ye, K. Keyomarsi, C. Chu, J. W. Harper, and S. J. Elledge. 2001. Phosphorylation-dependent ubiquitination of cyclin E by the SCFFbw7 ubiquitin ligase. Science 294:173-177. [PubMed]
19. Krogan, N. J., G. Cagney, H. Yu, G. Zhong, X. Guo, A. Ignatchenko, J. Li, S. Pu, N. Datta, A. P. Tikuisis, T. Punna, J. M. Peregrin-Alvarez, M. Shales, X. Zhang, M. Davey, M. D. Robinson, A. Paccanaro, J. E. Bray, A. Sheung, B. Beattie, D. P. Richards, V. Canadien, A. Lalev, F. Mena, P. Wong, A. Starostine, M. M. Canete, J. Vlasblom, S. Wu, C. Orsi, S. R. Collins, S. Chandran, R. Haw, J. J. Rilstone, K. Gandi, N. J. Thompson, G. Musso, P. St. Onge, S. Ghanny, M. H. Lam, G. Butland, A. M. Altaf-Ul, S. Kanaya, A. Shilatifard, E. O'Shea, J. S. Weissman, C. J. Ingles, T. R. Hughes, J. Parkinson, M. Gerstein, S. J. Wodak, A. Emili, and J. F. Greenblatt. 2006. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature 440:637-643. [PubMed]
20. Kus, B. M., C. E. Caldon, R. Andorn-Broza, and A. M. Edwards. 2004. Functional interaction of 13 yeast SCF complexes with a set of yeast E2 enzymes in vitro. Proteins 54:455-467. [PubMed]
21. Lei, E. P., H. Krebber, and P. A. Silver. 2001. Messenger RNAs are recruited for nuclear export during transcription. Genes Dev. 15:1771-1782. [PMC free article] [PubMed]
22. Lei, E. P., and P. A. Silver. 2002. Intron status and 3′-end formation control cotranscriptional export of mRNA. Genes Dev. 16:2761-2766. [PMC free article] [PubMed]
23. Liang, C., and B. Stillman. 1997. Persistent initiation of DNA replication and chromatin-bound MCM proteins during the cell cycle in cdc6 mutants. Genes Dev. 11:3375-3386. [PMC free article] [PubMed]
24. Liu, Q., M. Z. Li, D. Leibham, D. Cortez, and S. J. Elledge. 1998. The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes. Curr. Biol. 8:1300-1309. [PubMed]
25. Mamnun, Y. M., S. Katayama, and T. Toda. 2006. Fission yeast Mcl1 interacts with SCF(Pof3) and is required for centromere formation. Biochem. Biophys. Res. Commun. 350:125-130. [PubMed]
26. Maris, C., C. Dominguez, and F. H. Allain. 2005. The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression. FEBS J. 272:2118-2131. [PubMed]
27. Mathias, N., S. L. Johnson, M. Winey, A. E. Adams, L. Goetsch, J. R. Pringle, B. Byers, and M. G. Goebl. 1996. Cdc53p acts in concert with Cdc4p and Cdc34p to control the G1-to-S-phase transition and identifies a conserved family of proteins. Mol. Cell. Biol. 16:6634-6643. [PMC free article] [PubMed]
28. Pan, X., P. Ye, D. S. Yuan, X. Wang, J. S. Bader, and J. D. Boeke. 2006. A DNA integrity network in the yeast Saccharomyces cerevisiae. Cell 124:1069-1081. [PubMed]
29. Ricke, R. M., and A. K. Bielinsky. 2005. Easy detection of chromatin binding proteins by the histone association assay. Biol. Proced. Online 7:60-69. [PMC free article] [PubMed]
30. Ricke, R. M., and A. K. Bielinsky. 2004. Mcm10 regulates the stability and chromatin association of DNA polymerase-alpha. Mol. Cell 16:173-185. [PubMed]
31. Rondon, A. G., S. Jimeno, M. Garcia-Rubio, and A. Aguilera. 2003. Molecular evidence that the eukaryotic THO/TREX complex is required for efficient transcription elongation. J. Biol. Chem. 278:39037-39043. [PubMed]
31a. Rose, M. D., F. Winston, and P. Hieter. 1990. Methods in yeast genetics: a laboratory course manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
32. Sarin, S., K. E. Ross, L. Boucher, Y. Green, M. Tyers, and O. Cohen-Fix. 2004. Uncovering novel cell cycle players through the inactivation of securin in budding yeast. Genetics 168:1763-1771. [PMC free article] [PubMed]
33. Skowyra, D., K. L. Craig, M. Tyers, S. J. Elledge, and J. W. Harper. 1997. F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex. Cell 91:209-219. [PubMed]
34. Skowyra, D., D. M. Koepp, T. Kamura, M. N. Conrad, R. C. Conaway, J. W. Conaway, S. J. Elledge, and J. W. Harper. 1999. Reconstitution of G1 cyclin ubiquitination with complexes containing SCFGrr1 and Rbx1. Science 284:662-665. [PubMed]
35. Strasser, K., and E. Hurt. 2000. Yra1p, a conserved nuclear RNA-binding protein, interacts directly with Mex67p and is required for mRNA export. EMBO J. 19:410-420. [PMC free article] [PubMed]
36. Strasser, K., S. Masuda, P. Mason, J. Pfannstiel, M. Oppizzi, S. Rodriguez-Navarro, A. G. Rondon, A. Aguilera, K. Struhl, R. Reed, and E. Hurt. 2002. TREX is a conserved complex coupling transcription with messenger RNA export. Nature 417:304-308. [PubMed]
37. Strohmaier, H., C. H. Spruck, P. Kaiser, K. A. Won, O. Sangfelt, and S. I. Reed. 2001. Human F-box protein hCdc4 targets cyclin E for proteolysis and is mutated in a breast cancer cell line. Nature 413:316-322. [PubMed]
38. Stutz, F., A. Bachi, T. Doerks, I. C. Braun, B. Seraphin, M. Wilm, P. Bork, and E. Izaurralde. 2000. REF, an evolutionary conserved family of hnRNP-like proteins, interacts with TAP/Mex67p and participates in mRNA nuclear export. RNA 6:638-650. [PMC free article] [PubMed]
39. Tan, B. C., C. T. Chien, S. Hirose, and S. C. Lee. 2006. Functional cooperation between FACT and MCM helicase facilitates initiation of chromatin DNA replication. EMBO J. 25:3975-3985. [PMC free article] [PubMed]
40. Tsvetkov, L. M., K. H. Yeh, S. J. Lee, H. Sun, and H. Zhang. 1999. p27(Kip1) ubiquitination and degradation is regulated by the SCF(Skp2) complex through phosphorylated Thr187 in p27. Curr. Biol. 9:661-664. [PubMed]
41. Wang, Y., D. Cortez, P. Yazdi, N. Neff, S. J. Elledge, and J. Qin. 2000. BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures. Genes Dev. 14:927-939. [PMC free article] [PubMed]
42. Wellinger, R. E., F. Prado, and A. Aguilera. 2006. Replication fork progression is impaired by transcription in hyperrecombinant yeast cells lacking a functional THO complex. Mol. Cell. Biol. 26:3327-3334. [PMC free article] [PubMed]
43. Willems, A. R., S. Lanker, E. E. Patton, K. L. Craig, T. F. Nason, N. Mathias, R. Kobayashi, C. Wittenberg, and M. Tyers. 1996. Cdc53 targets phosphorylated G1 cyclins for degradation by the ubiquitin proteolytic pathway. Cell 86:453-463. [PubMed]
44. Zenklusen, D., P. Vinciguerra, Y. Strahm, and F. Stutz. 2001. The yeast hnRNP-like proteins Yra1p and Yra2p participate in mRNA export through interaction with Mex67p. Mol. Cell. Biol. 21:4219-4232. [PMC free article] [PubMed]
45. Zenklusen, D., P. Vinciguerra, J. C. Wyss, and F. Stutz. 2002. Stable mRNP formation and export require cotranscriptional recruitment of the mRNA export factors Yra1p and Sub2p by Hpr1p. Mol. Cell. Biol. 22:8241-8253. [PMC free article] [PubMed]

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