MOLECULAR AND CELLULAR BIOLOGY, Sept. 1992, p. 4056-4066

Vol. 12, No. 9

0270-7306/92/094056-11$02.00/0 Copyright C) 1992, American Society for Microbiology

Replication Forks Pause at Yeast Centromeres SCOTT A. GREENFEDERt AND CAROL S. NEWLON* Department ofMicrobiology and Molecular Genetics, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey 07103 Received 27 April 1992/Returned for modification 26 May 1992/Accepted 29 June 1992

The 120 bp of yeast centromeric DNA is tightly complexed with protein to form a nuclease-resistant core structure 200 to 240 bp in size. We have used two-dimensional agarose gel electrophoresis to analyze the replication of the chromosomal copies of yeast CENI, CEN3, and CEN4 and determine the fate of replication forks that encounter the protein-DNA complex at the centromere. We have shown that replication fork pause sites are coincident with each of these centromeres and therefore probably with all yeast centromeres. We have analyzed the replication of plasmids containing mutant derivatives of CEN3 to determine whether the replication fork pause site is a result of an unusual structure adopted by centromere DNA or a result of the protein-DNA complex formed at the centromere. The mutant centromere derivatives varied in function as well as the ability to form the nuclease-resistant core structure. The data obtained from analysis of these derivatives indicate that the ability to cause replication forks to pause correlates with the ability to form the nuclease-resistant core structure and not with the presence or absence of a particular DNA sequence. Our findings further suggest that the centromere protein-DNA complex is present during S phase when replication forks encounter the centromere and therefore may be present throughout the cell cycle.

In vivo, the DNA linear duplex of eukaryotic chromois packaged with an approximately equal mass of protein into chromatin. The bulk of the DNA is packaged into nucleosomes which consist of approximately 145 bp of DNA wrapped around a histone octamer composed of two molecules each of histones H2A, H2B, H3, and H4 (reviewed in reference 63). The DNA fiber packaged into nucleosomes is further condensed into a 30-nm fiber by histone Hi. Other, much less abundant proteins are also known to interact with specific DNA sequences in chromatin, e.g., transcription factors that bind promoter elements and proteins that interact with centromeric DNA to assemble the kinetochore. Thus, the eukaryotic DNA replication apparatus must be designed to replicate the chromatin complex. It has been shown recently that purified bacteriophage T4 proteins are capable of in vitro replication of a DNA template that is packaged into nucleosomes, and that the replication fork passes through the nucleosomes without the dissociation of the histone octamers from the DNA (5). This finding suggests that nucleosomes can open transiently to allow passage of the replication apparatus. Much less is known about what happens when a replication fork encounters other specific protein-DNA complexes in chromatin. One possibility is that the replication apparatus contains a protein or protein complex capable of disrupting proteinDNA complexes ahead of the replication fork, as has been shown for the bacteriophage T4 dda protein (2, 36). Alternatively, the protein-DNA complexes may be designed to allow passage of a replication fork. Finally, the protein-DNA complex may serve as a replication fork barrier (RFB), as has been demonstrated for a family of repeats present in the Epstein-Barr virus (EBV) origin of replication, which bind

the product of the EBV nuclear antigen 1 (EBNA-1) gene (22, 37). One element at which specific protein-DNA interactions are known to occur is at centromeres of Saccharomyces cerevisiae chromosomes. Fully functional yeast centromeres contain about 120 bp of DNA organized into three elements (reviewed in reference 47). An 8-bp conserved sequence, CDEI (centromere DNA element I), is separated from a 25-bp conserved sequence, CDEIII, by a 78 to 86-bp region of highly A+T-rich DNA, CDEII. Mutational analysis has shown that CDEIII is essential for centromere function (23-25, 44, 49), while CDEI and CDEII contribute to centromere function (11, 14, 23, 25, 53). Centromeric DNA is thought to interact with proteins that mediate attachment of spindle microtubules and provide the motor function(s) that move chromosomes along the spindle (reviewed in reference 12). Yeast centromeres have been shown to interact with proteins by both chromatin digestion studies (4, 57) and in vivo footprinting (17, 45). These protein-DNA interactions are partially or completely eliminated by mutations in centromere DNA that reduce function. A helix-loop-helix protein, identified independently by several laboratories (1, 6, 10, 34) and shown to bind CDEI as well as several promoters in vitro, has recently been shown to bind CDEI in vivo (45). CDEIII is bound by a multisubunit protein complex, CBF3, which has to be phosphorylated for binding (38, 49). In addition, by examining yeast strains in which the synthesis of histone proteins can be controlled, Saunders et al. (58) have shown that histone proteins may also be involved in determining the chromatin structure of yeast centromeres. Interestingly, recent evidence suggests that one component of the mammalian kinetochore (CENP-A) has histonelike properties (51). The roles of the CDEI- and CDEIII-binding proteins in forming the nuclease-resistant centromere structure and the roles of these proteins in centromere function have not been determined. However, the CDEIII-binding protein complex (CBF3) must play a central role because

somes

* Corresponding author. t Present address: Department of Molecular Genetics, Hoffmann-La Roche, Nutley, NJ 07110.

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point mutations in its binding site that eliminate complex binding also eliminate CEN function and the formation of the nuclease-resistant centromere structure (44, 49, 57). We have examined the replication of S. cerevisiae centromeres by the use of a two-dimensional (2-D) gel electrophoresis method that permits the identification of replication intermediates. We find that replication forks pause at centromeres and that the ability of centromeric DNA to cause replication forks to pause correlates with its ability to form the protein-DNA structure and not with the presence or absence of particular DNA sequences. MATERIALS AND METHODS Strains and enzymes. Eschenichia coli JA226 was used as the host strain for plasmid DNA isolation. S. cerevisiae SG3-47A (MATa his6 trpl-289) (23a) was used for wild-type DNA isolations. In this strain chromosome III is in two parts, a 61-kb ring chromosome and a linear chromosome, stabilized by an insertion of CEN5 near the HIS4 locus, carrying the remainder of chromosome III (61). Therefore, the ring chromosome III sequences are present in only a single copy per cell. Strain SG4-10A (MATa his4-S8 ura3-52 adel) was used to construct the ARS307 deletion chromosome (23a). This strain carries the 61-kb ring chromosome III and a full-length homeologous chromosome III recovered from the Carlsberg brewing strain, S. carlsbergensis (30, 50). The homeologous chromosome III can substitute for S. cerevisiae sequences; however, divergence in its DNA sequence allows the detection of only S. cerevisiae ring chromosome III sequences under high-stringency hybridization conditions. Restriction endonucleases were purchased from New England Biolabs, Inc. [a-32P]ATP was purchased from Amersham Corp. 32P-labeled DNA probes were made using the Amersham Multiprime Random Primer DNA labelling kit. Plasmids. Plasmid DNA was prepared from E. coli by the alkaline lysis procedure (40). The plasmids listed in Fig. 2 were obtained from Molly Fitzgerald-Hayes. pYe(CEN3)-30 carries the 624-bp Sau3A fragment containing CEN3 (20). The 211-bp CEN3-containing fragment carried in 3B14 is a deletion derivative of the 624-bp fragment from which sequences beginning at the CDEIII-proximal Sau3A site and continuing to within 37 bp of CDEIII were removed (44). The CDEI deletion was constructed in the 624-bp Sau3A fragment and removes 101 bp beginning at the CDEI-proximal Sau3A site and continuing through CDEI and 26 bp of CDEII (23). The CDEIII point mutants were constructed in the 211-bp CEN3 derivative (44). Plasmid pYe(CEN3)-30INV was constructed by removing the 624-bp Sau3A CEN3 fragment from pYe(CEN3)-30 (20) and inserting it at the BamHI site of plasmid YRp7' (60) to reverse the orientation of the CEN3 fragment. Plasmids pCEN4 and pCEN4-INV were constructed by inserting the PvuII-to-StuI fragment containing CEN4 (41) at the BamHI site of YRp7' (60). These plasmids differ only in the orientation of the centromere fragments. Determination of plasmid stabilities. The mitotic stabilities of plasmids were determined as the percentage of plasmid bearing cells in a culture grown in selective medium (52, 62). Cells were grown in selective medium to a density of 1 x 106 to 2 x 106 cells per ml and then diluted appropriately and plated onto both selective and nonselective medium at a density of 300 colonies per plate. Cells were plated onto at least five each of selective and nonselective plates, and at least two independent transformants were analyzed for each

REPLICATION OF YEAST CENTROMERES

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plasmid. The mitotic stabilities were determined as the ratio of colonies on the selective plates versus nonselective plates. Transformations. E. coli transformations were performed by the method of Davis et al. (15). Yeast transformations were carried out by using the lithium acetate transformation procedure (33). Isolation of replicating.east DNA. Yeast cells were grown to approximately 2 x 10 cells per ml, and an equal volume of toluene stop solution (95% ethanol, 3% toluene, 20 mM Tris-Cl [pH 7.4]) was added, followed immediately by 0.25 M EDTA to a final concentration of 10 mM (35). The suspension was swirled for 1 min and placed on ice or immediately centrifuged at 4°C. After centrifugation, the cells were washed three times with sterile water, and DNA was prepared by the procedure of Huberman et al. (32). Restriction enzyme digestion. Approximately 10 to 20 ,ug of DNA was digested in a total volume of 400 ,ul in a digestion buffer containing 20 mM Tris (pH 7.4), 50 p,M spermine, 125 ,uM spermidine, 20 mM KCl, 70 mM NaCl, 10 mM MgCl2, 2.5 Kallikrein inhibitor units (KIU) of aprotinin per ml, and 0.1% digitonin (46). Restriction enzymes were used at a concentration of 2 U/Ipg of DNA, and the digest was incubated for 1.5 h at 37°C, followed by a second addition of restriction enzyme and another 1.5-h incubation. 2-D agarose gel electrophoresis. The digested DNA was analyzed by the 2-D electrophoresis procedure of Brewer and Fangman (8). The second-dimension gel was blotted to nitrocellulose membranes by the bidirectional method (59). The blots were hybridized with probe DNA made as described above. Autoradiography was performed by exposing Kodak XAR-5 film with an intensifying screen at -80°C. Exposure times varied between 6 h and 5 days. Quantitation of the centromere pause. Densitometric analysis of autoradiograms was carried out by using a Molecular Dynamics Computing Densitometer. The densitometric results were confirmed by cutting out individual areas of the hybridized blot and determining the radioactive counts by using a scintillation counter. Determination of the direction of replication fork movement. The method of Fangman and Brewer (19) was used to determine the direction of replication fork movement. Replicating DNA was separated in a first-dimension gel as described above. The first-dimension lane was then cut out and digested with a restriction enzyme that cut within the fragment of interest. The gel slice was incubated for 30 min in TE (10/0.1) (10 mM Tris-Cl [pH 8.0], 0.1 mM EDTA) at room temperature (-23°C). The gel slice was then incubated for 1 h in 500 ml of lx restriction enzyme buffer at room temperature. This step was repeated once. The gel slice was then incubated overnight in 7 ml of 1 x restriction buffer with 800 U of restriction enzyme at 37°C, followed by incubation for 30 min in TE (10/1.0) (10 mM Tris-Cl [pH 8.0], 1.0 mM EDTA) at room temperature. The gel slice was then placed in a second-dimension gel and run as described above for normal 2-D gels. In all fork direction analyses, the probe for hybridization was specific for one of the two fragments produced by the restriction enzyme digestion in the gel. RESULTS Yeast centromeres cause replication forks to pause. The replication of genomic DNA fragments containing CEN3 was analyzed by using the 2-D gel electrophoresis replicon mapping method of Brewer and Fangman (8). Figure 1A shows the pattern obtained from analysis of the 5.0-kb

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GREENFEDER AND NEWLON

A

MOL. CELL. BIOL.

D

'I

1.

E.

c

FIG.

1. 2-D

gel replication patterns

of chromosomal

copies of diagram of the fragment analyzed, showing the position of the centromere (black boxes) in each fragment. In this and all other figures, the first dimnension was run from left to right and the second dimension was run from top to bottom. The spots along the diagonal line of linear molecules of higher molecular weight than the unreplicated monomer spot result from partial digestion of the DNA. In all figures, open arrows point to spots of accumulated Y-shaped replication intermediates. (A) The 2-D gel pattern arising from analysis of the 5.0-kb EcoRI fragment containing CEN3. (B) The 2-D pattern arising from a second experiment analyzing the same fragment as in panel A, clearly showing the hybridization sigual in the termination region of the pattern. The shape of the Y arc in this pattern differs slightly in shape from that in panel A because the second dimension was run for a shorter time, and the paused replication intermediates are difficult to discern because of the long exposure. (C) Diagram of the patterns in panels A and B. The shapes of replication intermediates present in various regions of the patterns are indicated by the stick figures. Although we have indicated specific arcs of double-Y termination and X-shaped replication intermediates for clarity, the CEN3, CEN4, and

CENI.

Below each pattern is

termination intermediates in the patterns that

we

a

obtained arise from

termination

throughout the fragments analyzed and produce a diffuse triangular hybridization signal in the termination region. (D) The 2-D gel patten arising from the 5.1-kb EcoRI fragment containfrom the 4.6-kb ing CEN4. (E) The 2-D gel patter fragment containing CEN4. (F) The 2-D gel patter arising from the

arising

4.5-kb EcoRI

fragment containing CENI.

Hinddll

EcoRI fragment, containing CEN3, from a strain in which CEN3 is located on a 61-kb circular derivative of chromosome III (strain SG3-47A; see Materials and Methods). When the same restriction fragment was analyzed by using DNA isolated from a yeast strain that carries the wild-type linear chromosome III, the results were indistinguishable from those shown in Fig. 1A. The presence of a complete simple-Y arc indicates that the fragment is replicated primarily by a single replication fork moving completely through it. On the simple-Y arc is a prominent spot of accumulated replication intermediates that are approximately 8 kb in mass. The presence of this spot suggests that replication forks pause in this fragment (9), and its position on the Y arc in Fig. 1A and on that of the overlapping BamHI-XbaI restriction fragment (23a) indicates that the replication fork pause site is coincident with the centromere. The presence of a complete simple-Y pattern shows that, despite pausing at the centromere, replication forks must be able to pass through the centromere and move all the way through the fragment. If the centromere caused an absolute block to replication fork movement, then one fork would halt and a second fork would enter the fragment from the other side, giving rise to double-Y replication intermediates. This would result in early replication intermediates (less than 50% replicated) that are Y-shaped, late replication intermediates (greater than 50% replicated) that are double-Y shaped, and no Y-shaped late replication intermediates. The pattern visualized would begin as a Y arc and change to a double-Y arc at the spot of accumulated intermediates (42). Since this pattern is not prominent, we suggest that CEN3 causes replication forks to pause and not stop. However, in darker exposures of the autoradiogram shown in Fig. 1A and in the independent DNA preparation shown in Fig. 1B, a region of hybridization containing double-Y termination intermediates can be seen. This signal is triangular in shape, extends leftward from the nearly vertical late Y arc, and is bounded by the arc of X-shaped intermediates (Fig. 1C). The presence of this signal demonstrates that some replication forks pause long enough at CEN3 to allow a fork to enter the fragment from the other end; however, the diffuse shape of this signal suggests that termination occurs throughout this entire fragment and is not limited to a single site. We determined the fraction of replication forks. that are paused at CEN3 in our DNA preparations by densitometric analysis of autoradiograms and by direct scintillation counting of radioactively labeled filters. We estimate that 20% ± 5% of the replication forks are paused at the centromere. Since the DNA used for these analyses was isolated from asynchronously grown cells, we cannot estimate the actual fraction of replication forks that pause at the centromere. To ascertain whether the ability to cause replication forks to pause is a general property of yeast centromeres, we examined the replication of genomic DNA fragments containing CEN4 and CEN1. All of these fragments yielded patterns of complete simple-Y arcs with spots of accumulated replication intermediates. CEN4 is positioned asymmetrically in the fragment examined in Fig. 1D. If replication forks move through this fragment from left to right, then pausing at the centromere would lead to the accumulation of the late Y-shaped replication intermediates indicated by the open arrow. Pausing of forks traversing the fragment from right to left would lead to the accumulation of early Y-shaped replication intermediates, and a possible signal from these intermediates is indicated by the filled arrow. The termination intermediates that are apparent in Fig. 1D demonstrate that forks must enter this fragment from both ends.

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Examination of the overlapping CEN4-containing fragment (Fig. 1E) confirms that the pause site maps to CEN4. In the CENJ-containing fragment (Fig. 1F), both the centromere and the replication pause site are in the middle of the fragment. Taken together, the results for CEN3, CENI, and CEN4 suggest that all yeast centromeres are able to cause replication forks to pause. The nature of the replication fork pause site. Two models can explain the replication fork pause at yeast centromeres. First, the pause could result from the formation of an unusual DNA structure by the conserved centromere DNA elements. Alternatively, the pause could result from the specific protein-DNA interactions at the centromere. To distinguish between these two possibilities, we analyzed the replication of plasmids containing either wild-type or mutant derivatives of CEN3 (Fig. 2). ARSI has been previously shown to function as the plasmid replication origin (8), and these centromere derivatives have been previously analyzed for centromere function (21, 23, 44) and their ability to form the nuclease-resistant core structure (57). Our determination of the relative function of each of these centromere derivatives by mitotic stability measurements is consistent with previous observations (Fig. 2B). Figure 3 shows the patterns obtained from analysis of the BglII-NdeI fragment (Fig. 2A) of plasmids containing the CEN3 derivatives shown in Fig. 2B. The four plasmids that contain functional or partially functional CEN3 derivatives, pYe(CEN3)-30, pCEN3-3B14, pCEN3-B58A1, and pCEN3BCT1, show complete simple-Y arcs along with spots of accumulated replication intermediates (open arrows) and faint triangles of termination intermediates extending leftward from the portion of the Y arc containing late replication intermediates (Fig. 3A to D). The position of the spot in each of these patterns correlates with the position of the centromere in each plasmid. The position of the pause site was confirmed to be at CEN3 by analysis of the overlapping 5.0-kb PstI-PstI fragment (Fig. 2A) of all of the plasmids. The presence of termination intermediates indicates that the fork initiating fromARSI and moving toward the centromere pauses long enough at the centromere to allow the fork moving in the other direction around the plasmid to enter the fragment. Without a pause at the centromere, this fragment is replicated by a single fork and yields a simple-Y pattern (Fig. 3E). These results indicate that the 624- and 211-bp wild-type CEN3 fragments, the CDEI deletion derivative, and the partially functional CDEIII mutant derivative all cause replication forks to pause. In contrast, the pattern that arises from analysis of the BglII-NdeI fragment of the fifth plasmid, pCEN3-BCT2, shows only a simple-Y arc with little or no spot of accumulated replication intermediates and little or no termination signal (Fig. 3E). This result demonstrates that the nonfunctional CDEIII mutant is unable to cause replication forks to pause. These results suggest that formation of the CDEIII protein complex is required for the replication fork pause. A point mutation in CDEIII (BCT2) that eliminates CBF3 binding in vitro, the nuclease-resistant centromere core structure in vivo, and centromere function also eliminates the replication fork pause. In contrast, partially functional CEN3 derivatives carrying either a deletion of CDEI and part of CDEII (B58A1) or a point mutation in CDEIII (BCT1) maintained the ability to cause replication forks to pause. Orientation dependence of the centromere pause. The second feature of the patterns arising from the genomic fragments containing CEN3 is that only a single spot of accumulated replication intermediates is observed even though the

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A

B CENTRONERE

WILD-TYPE (CEN3-30)

NITOTIC STABILITY

DNA SEQUENCE

CDEIII COEI CDEII GTCACATG -- - -84bp ---- TGTA MTGATMTCCGAAAGTTAAAA >90% A+T

100%

-58bp ---- TGTATTTGATMTCCGAAAGTTAAAA

64%

GTCACATG --- -84bp-- -- TGTATMTGATMfCCGAAAGTTAAAA I >90% A+T

94%

(3B14)

CDEI DELETION (B58A1) COEIII POINT INIUTATION (BCT1)

>90% AdT

t

CDEIII POINT NUTATION (BCT2)

GTCACATG ----84bp----TGTATGAMCCGAGTTAA I >90% A+T t

Replication forks pause at yeast centromeres.

The 120 bp of yeast centromeric DNA is tightly complexed with protein to form a nuclease-resistant core structure 200 to 240 bp in size. We have used ...
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