Vol. 12, No. 10

MOLECULAR AND CELLULAR BioLOGy, Oct. 1992, p. 4733-4741 0270-7306/92/104733-09$02.00/0 Copyright © 1992, American Society for Microbiology

Localization of a DNA Replication Origin and Termination Zone on Chromosome III of Saccharomyces cerevisiae JIGUANG ZHU,lt CAROL S. NEWLON,2 AND JOEL A. HUBERMAN'* Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263,1 and Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School; Newark, New Jersey 071032 Received 16 March 1992/Returned for modification 23 April 1992/Accepted 28 July 1992

Two-dimensional gel electrophoretic replicon mapping techniques were used to identify all functional DNA replication origins and termini in a 26.5-kbp stretch in the left arm of yeast chromosome m. Only one origin was detected; it coincided with an ARS element (ARS306), as have all previously mapped yeast origins. A replication termination region was identified in a 4.3-kbp stretch at the telomere-proximal end of the investigated region, between the origin identified in this paper and the neighboring, previously mapped, ARS305-associated origin (previously called the A6C origin). Termination does not occur at a specific site; instead, it appears to be the consequence of replication forks converging in a stretch of DNA of at least 4.3 kbp.

Replication of eukaryotic chromosomes is accomplished by initiation at multiple replication origins distributed at irregular intervals along DNA molecules. Considerable indirect evidence suggests that these origins correspond to specific nucleotide sequences. However, identification of sequences which serve as origins has been a difficult task. Identification of origins in the yeast Saccharomyces cerevisiae was facilitated by the development of two-dimensional (2D) gel electrophoresis techniques which permit mapping of replication origins and termini (1, 22) and directions of replication (9, 22). These techniques have been used to show that in plasmids (1, 7, 14) and in chromosomes (11, 12, 19), replication origins correspond to ARS elements. ARS elements, or autonomously replicating sequences, were identified by Struhl et al. (28) as segments of yeast DNA capable of promoting the autonomous replication of plasmids into which they were inserted. So far, identifications of only six yeast chromosomal origins have been published. One of these, the ARS305associated origin, is located about 40 kbp from the left end of chromosome III (Fig. 1) (12). Another origin is located in the nontranscribed spacer of the yeast rDNA repeat on chromosome XII (19). The third and fourth origins are in single-copy DNA on chromosomes IV and V (11), the fifth is located in Y' sequences at the ends of many chromosomes (11), and the sixth is in single-copy DNA on chromosome X (31). AH of these origins coincide with previously mapped ARS elements. The number of identified origins, six, is still too small to permit generalization of the correspondence between origins and ARS elements. Characterization of additional origins is essential. In this paper we report identification of an additional origin and show that it, too, coincides with an ARS element. DNA fiber autoradiographic studies have suggested that termination of eukaryotic chromosomal replication occurs by convergence of replication forks emanating from adjacent origins (8). Termination of replication in the simian virus 40 *

(SV40) minichromosome does not depend on specific sequences; it occurs wherever converging replication forks meet, about 1800 around the circular genome from the origin (4, 18). Because of occasional asymmetry in the rate of movement of the two SV40 replication forks, and because of the presence in SV40 DNA of sites where replication forks pause, SV40 termination probably occurs within a zone of about 1 kbp rather than precisely opposite the origin (29, 32). In contrast, replication termination occurs at a specific site in yeast ribosomal DNA because of the existence of a polar barrier to replication forks moving counter to the direction of 37 S pre-rRNA transcription, located about 200 bp downstream of the transcription termination site (2, 19). With the exception of this termination in yeast rDNA, it has not previously been determined for any eukaryotic chromosomal DNA region whether termination occurs at specific nucleotide sequences or nonspecifically. In this paper we show that replication forks emanating from two adjacent origins on S. cerevisiae chromosome III appear to converge and terminate nonspecifically, wherever they happen to meet, primarily within a zone of over 4 kbp. MATERIALS AND METHODS

Cell growth. Unsynchronized S. cerevisiae 4910-3-3 cells (AL4Ta his7 ural cdc7-4 barl; provided by Leland Hartwell) were grown in YPD medium at 23°C to a density of 1.5 x 10 to 2.0 x 107/ml as previously described (25). DNA isolation, purification, and BND-cellulose fractionation. Cells were harvested and chromosomal DNA was purified by CsCl density gradient centrifugation as previously reported (14). The purified DNA was digested to completion with EcoRI, PstI, or BamHI, and then replication fork-containing restriction fragments were enriched by fractionation on benzoylated, naphthoylated DEAE-cellulose (BND-cellulose) as described previously (14). In a typical experiment, about 1 ml of packed BND-cellulose was used for fractionation of 500 1Lg of chromosomal DNA. Most of the DNA (about 480 Fxg) emerged in the flowthrough fraction (which contained fully double-stranded nonreplicating DNA molecules), and about 5 ,ug was recovered in the caffeine wash fraction (which contained partially or fully

Corresponding author.

t Present address: Department of Neurosurgery, Brigham &

Women's Hospital, 221 Longwood Avenue, LMRC Building, room 121, Boston, MA 02115.

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ZHU ET AL.

MOL. CELL. BIOL.

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FIG. 1. Restriction maps of a region of the left arm of S. cerevisiae chromosome III. The name assigned to each BamHI fragment (24) is shown above the brackets denoting the fragment. Previously identified ARS elements (24) are indicated by black boxes below the horizontal restriction map lines and by a number (305 or 306). The size scale displays the distance in kilobase pairs from the left telomere. The location of the ARS305-associated origin and the directions of replication fork movement determined in a previous study (12) are shown. The lower portion of this figure presents a more detailed restriction map of the centromere-proximal 26.5 kbp of the region shown in the upper portion. The numbered, highlighted boxes represent the stretches used as hybridization probes in the present study. Restriction sites: B, BamHI; E,

EcoRV, P, PMt; H, HindIII; K, Knl; X, XhoI; G, BgllI.

single-stranded molecules, including replication fork-containing restriction fragments). 2D neutral-alkaline and neutral-neutral gel electrophoresis. About 4 p,g of caffeine wash and flowthrough DNA was subjected to 2D neutral-alkaline gel electrophoresis as previously described (22). For the neutral-neutral 2D gel technique about 1 p,g of DNA was used, and we followed the procedure of Brewer and Fangman (1) with modifications as previously described (12). Preparation of DNA probes. The probes used in this work are numbered and described in Fig. 1 and in the Results section. Each of the two BamHI fragments shown (A4H and C1G) was cloned into the BamHI site of the yeast shuttle vector YIp5 (23, 24). The plasmids containing the cloned DNA were double-digested with BamHI and EcoRI restriction enzymes, and the resulting fragments were separated by agarose gel electrophoresis. Fragments of interest were sliced out and purified by electroelution in dialysis bags. The shorter probes used in this study were obtained by digesting the purified BamHI-EcoRI subfragments with the appropriate restriction enzymes (indicated in Fig. 1), and the desired sub-subfragments were purified by electrophoresis in lowgelling-temperature agarose. The random oligonucleotide labeling procedure (10) was used to label the short DNA fragments to high specific activity with [a-32P]dTlP (800 Ci/mmol; Amersham) for 2 h at 37°C and then at room temperature overnight. Hybridization and autoradiography. The hybridization procedure was the same as previously described (14) with the following modifications. Denaturation of the freshly labeled DNA probes was done by boiling in water for 5 min and then cooling quickly on ice. Prehybridization and hybridization were carried out at 42°C for 16 to 24 h. Exposure of XAR-5 film (Kodak) was carried out at -80°C with an

intensifying screen (DuPont) for 16 h to 2 weeks. Old probes were stripped off the membranes by incubation with alkaline buffer as previously described (12).

RESULTS Replicon mapping techniques. Detailed descriptions of both the neutral-alkaline and neutral-neutral 2D gel electrophoresis techniques have been published previously (1, 22). Since the two techniques are independent and complementary, use of both techniques in combination is helpful in confirming results and minimizing possible misinterpretations (20). Briefly, chromosomal DNA is isolated from unsynchronized, exponentially growing cells, and high-molecular-weight DNA is purified and digested to completion with a restriction enzyme. Replication-fork-containing restriction fragments are enriched by BND-cellulose chromatography and then separated according to extent of replication in a neutral agarose gel (first dimension). The firstdimension gel slices are then cut out, rotated 900, and embedded in gels for the second dimension. For neutralalkaline gels, the second-dimension gel is soaked with alkaline electrophoresis buffer and subjected to alkaline electrophoresis, during which the DNA is denatured so that newly synthesized strands separate from parental stands, and each DNA strand migrates according to its size. The neutralneutral 2D gel technique developed by Brewer and Fangman (1), however, exploits the fact that nonlinear DNA molecules migrate more slowly than linear molecules of the same mass, and this retardation is enhanced by the high voltage, high agarose concentration, low temperature, and high ethidium bromide concentration used for the second dimension. When the second-dimension gel runs are finished, DNA in the gels is transferred to nylon membranes and then hybrid-

VOL. 12, 1992

CHROMOSOMAL DNA REPLICATION ORIGIN AND TERMINUS

ized sequentially with probes (Fig. 1) derived from the restriction fragments of interest. Map of a 27.4-kbp portion of yeast chromosome m. Newlon et al. (23, 24) have cloned a -200-kbp stretch of yeast chromosome III that includes sequences from the HML locus near the left telomere to the MA4T locus, which is about halfway out the right arm. They have developed a restriction map of this region and have identified the locations of most of the ARS elements within it (23, 24, 27). Initially, a name was assigned to eachARS element based on the restriction fragment in which it resides. Recently, Campbell and Newlon have proposed a new convention for systematic naming of yeast ARS elements (5) in which each ARS element is assigned a three- or four-digit number. The first digit or pair of digits indicates the chromosome on which the ARS element is located, and the next two digits identify the ARS uniquely. Since most or all of the ARS elements on the left arm of yeast chromosome III have been identified, they are numbered serially from left to right, and the ones relevant to this paper are shown in Fig. 1. We shall use the new convention in this paper; the reader may refer to Fig. 1 and its legend for correlation between the old convention and the new convention. Previously, the B9G, A6C, and AlG restriction fragments (Fig. 1) were investigated for origin activities, and a single origin was detected at ARS305 (12, 20). Replication forks were shown to move through the AlG fragment from left to right (Fig. 1). In this paper we describe an investigation of the two BamHI restriction fragments further to the right, the A4H and ClG fragments

4735

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(Fig. 1).

Unidirectional (towards the telomere) replication fork movement through the 9.7-kbp HIS4-containing PstI fagment. DNA from log-phase yeast cells was digested with PstI and fork-containing restriction fragments were enriched by BND-cellulose fractionation. The enriched DNA fragments were subjected to 2D neutral-alkaline gel electrophoresis and then blotted to a nylon membrane. The membrane was hybridized sequentially with probes 4 and 6 (Fig. 1 and 2). The major spot at the upper left in each autoradiogram in Fig. 2 is due to the intact nonreplicating 9.7-kbp PstI fragment; despite the BND-cellulose enrichment, these nonreplicating fragments are still the most abundant molecules. Because of the overexposure necessary to detect nascent strand signals, the major spot signal in this and the other neutral-alkaline autoradiograms shown in this paper has somewhat merged with the signals from nicked strand fragments, parental strands, and the horizontal streak described below. It is clearly visible as a distinct spot in lighter exposures (data not shown). The vertical streak downwards from the major spot results from occasional randomly located single-strand nicks in the nonreplicating DNA. The nicked strand fragments migrate more rapidly than intact strands in the alkaline second

dimension. The replication fork-containing restriction fragments migrate more slowly in the first dimension than the nonreplicating restriction fragments, according to their extent of replication. Denaturation in the second dimension generates two parental strands of restriction fragment length and two nascent strands. The horizontal line extending rightward from the major spot in each autoradiogram in Fig. 2 is partly due to the parental strands of replicating restriction fragments. The parental strands remain constant in size as the extent of replication increases from left to right. Streaking

FIG. 2. Unidirectional movement of replication forks from right to left through the 9.7-kbp PstI fragment in the ClG BamHI fragment. (Left panel) Hybridization with probe 6; exposure time, 1 day. (Right panel) Hybridization with probe 4; exposure time, 1 day. The numbers in the center show the lengths in nucleotides of marker DNA strands separated in the second (alkaline) dimension. Firstdimension (neutral) electrophoresis was from right to left; seconddimension (alkaline) electrophoresis was from top to bottom.

from the major spot of nonreplicating DNA also contributes to this horizontal line. Nascent strands vary in length from a few nucleotides to full restriction fragment length according to extent of replication. If replication forks move from one end of a fragment to the other, then hybridization with a probe from the end where replication forks enter detects a diagonal arc of nascent strands, and hybridization with a probe from the opposite end does not detect such an arc (14, 22). As shown in Fig. 2, probe 6 detects an arc running upwards and rightward from just to the right of the vertical streak to the parental strand line, while probe 4 does not detect such an arc. Probe 6 produces a detectable signal from nascent strands and from nicked nonreplicating strand fragments (vertical streak) as short as -500 bp (evident on longer exposure). Because probe 6 lies at one end of the 9.7-kbp PstI fragment, it should in theory detect nicked strand fragments of all sizes. The loss of signal below -500 bp is presumably due to diffusion of short strands out of the gel during second-dimension electrophoresis and to the loss of signal which inevitably occurs when the detected strand length is shorter than probe length. The relatively parallel signal intensity from nascent strands and nicked strand fragments suggests that probe 6 would detect even shorter

4736

ZHU ET AL.

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FIG. 3. Unidirectional movement of replication forks from left to right through the 8.1-kbp EcoRI fragment in the ClG BamHI fragment. (Left panel) Hybridization with probe 8+; exposure time, 3 days. (Right panel) Hybridization with probe 11; exposure time, 5 days. Other conditions were as in Fig. 2. The spot at about 3,600 bases in the right-hand vertical streak in the right panel was not reproducible.

nascent strands in the absence of diffusion and length problems. Since probe 4 does not detect short nascent strands, replication forks must enter the 9.7-kbp PstI fragment at the probe 6 end (or originate near probe 6) and move unidirectionally through the fragment towards the probe 4 end. These results suggest that the origin responsible for replicating the 9.7-kbp PstI fragment lies near the centromere-proximal end of the fragment or outside the fragment toward the centromere. Replication forks appear to move predominantly in one direction (towards the centromere) through the 8.1-kbp EcoRl fragment. EcoRI-digested DNA was subjected to neutralalkaline 2D gel analysis, the gel was blotted to a nylon membrane, and the membrane was hybridized sequentially with probes 8+ and 11 (Fig. 1 and 3). In Fig. 3, the major spot at the upper left (nonreplicating unit-sized DNA), the vertical streak downwards below the major spot (randomly nicked nonreplicating strands), and the horizontal line extending rightward from the major spot (parental strands) are similar in appearance to those in Fig. 2. However, several features of Fig. 3 are different from Fig. 2. A vertical streak which descends from the right-hand end of the parental strand horizontal line and several faint spots and horizontal smears will be discussed below. The nascent strand signal in Fig. 3 provides another difference from Fig. 2. Whereas the nascent strand diagonal detected by probe 6 in Fig. 2 displays a gradual increase in intensity from short strands to long strands, the nascent strand signal detected by probe 8+

in Fig. 3 is relatively weak for shorter nascent strands and very intense for nascent strands longer than -4,400 bases. The intense portion of the nascent strand signal (from -4,400 to -8,100 bases) displays a steeper slope than the less intense portion because, during electrophoresis in the first dimension, molecules replicated by forks moving through from one end to the other migrate relatively slowly after they are -50% replicated (1, 22). One possible explanation of the dramatic change in intensity along the nascent strand signal detected by probe 8+ is that replication forks entering this fragment at the probe 8+ end may move rapidly through the first -4,400 bp and then much more slowly through the final -3,700 bp of this 8.1-kbp fragment. No nascent strand diagonal signal was detected by probe 11. Therefore replication forks must move predominantly unidirectionally, from left to right towards the centromere, through this fragment. This direction of replication fork movement is opposite to the direction of fork movement through the 9.7-kbp PstI fragment in Fig. 2. Therefore a replication origin must be located within the region which includes the centromere-proximal end of the 9.7-kbp PstI fragment and the telomere-proximal end of the 8.1-kbp EcoRI fragment. The autoradiograms in Fig. 3 reveal two additional types of signal not detected in Fig. 2. The first additional signal is the right-hand vertical streak due to nicks within the parental strands (and possibly the nascent strands) of the mostly replicated restriction fragments which have accumulated, possibly because of the slowing of replication forks, as described in the previous paragraph. The second additional signal-spots connected by faint horizontal lines-is due to the presence of nicks at specific sites within some of the nonreplicating strands and parental strands. Because these nicks were detected after EcoRI digestions but not after digestions with other restriction enzymes, they appear to be artifactual. A replication origin is located at the telomere-proximal end of the 8.1-kbp EcoRI fragment. Because data from the 9.7-kbp PstI fragment and from the 8.1-kbp EcoRI fragment suggested that an origin must be located somewhere near their point of closest approach at 70 to 75 kbp from the telomere (Fig. 1), the 5.5-kbp PstI fragment centered at 74.6 kbp from the telomere (Fig. 1) was chosen for more detailed study. A neutral-alkaline PstI 2D blot, like the one used in Fig. 2, was hybridized sequentially with probes 8, 9, and 10 (Fig. 4). Probe 9 detected the shortest nascent strands (-500 bases), while the minimum nascent strand size detected by probes 8 and 10 was -2 kb (Fig. 4). These data suggest that a replication origin is located within the -600 bp XhoI-BglII fragment (probe 9) and that replication forks proceed bidirectionally from this origin into the fragments used as probes 8 and 10. A striking difference between the neutral-alkaline gels shown in Fig. 4 and those shown in Fig. 2 and 3 is the fact that the vertical streaks due to random nicking do not descend nearly as far in Fig. 4 as they do in the other figures. This difference is a simple consequence of the fact that probes near the ends of the restriction fragment were employed in Fig. 2 and 3 while more centrally located probes were used in Fig. 4. A strand generated by a restriction cut at one end and a randomly located nick at the other end can be detected by a centrally located probe only if the strand is half unit size or longer. Neutral-neutral 2D gel analysis provides confirmation of origin location. Aliquots of the same DNA preparations used for neutral-alkaline gel analysis were also employed for

VOL. 12, 1992

CHROMOSOMAL DNA REPLICATION ORIGIN AND TERMINUS 5.5k5

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neutral-neutral analysis. The results of serial hybridizations of the blot of a neutral-neutral gel of EcoRI-digested DNA with probes 3, 4, 5, and 8+ are shown in the lower portion of Fig. 5. Probes 3, 4, and 5 reveal previously described (1) simple Y arcs indicating that the EcoRI fragments detected by these probes are replicated predominantly by replication forks moving from one end of the fragment to the other. The general shape of the arc detected by probe 8+ (sharp rise from the spot of nonreplicating DNA, followed by a segment with slope of about 450, followed by an abrupt vertical downturn) is characteristic of the arcs generated under our standard electrophoresis conditions by restriction fragments larger than about 8 kbp which are replicated as simple Ys (34), and similar distorted arcs have been detected in other laboratories (15). Note that smoother arcs resembling those detected by probes 3, 4, and 5 can also be generated by large restriction fragments when modified electrophoresis conditions are used (15, 16). The presence of simple Y arcs in all these EcoRI autoradiograms indicates that the initiation of replication does not occur in the middle third of any of these EcoRI fragments. However, the presence of simple Y arcs does not rule out the possibility of initiation within the terminal thirds of any of the tested fragments (20). The data presented in Fig. 4 suggested that an origin is located in the region covered by probe 9. This region is in the center of the 5.5-kbp PstI fragment studied for Fig. 4. Therefore, we examined this fragment by neutral-neutral electrophoresis. Hybridization of a PstI neutral-neutral blot with probe 8+ revealed a high-rising "bubble" arc (top portion of Fig. 5), indicative of an origin location at or near the center of this fragment (1). The strong signal below the right-hand end of the bubble arc (near the line of linears) is

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FIG. 4. An origin is located between the XhoI and BglII sites near the center of the 5.5-kbp PstI fragment. (Left panel) Hybridization with probe 8; exposure time, 3 days. (Center panel) Hybridization with probe 9; exposure time, 5 days (lower-specific-activity probe). (Right panel) Hybridization with probe 10; exposure time, 3 days. The effective exposure time of the central panel was less than that of the left and right panels; the background is darker in the central panel because probe 9 generated more background than did probes 8 and 10, not because the exposure was longer. Other conditions were as in Fig. 2. Between the hybridizations with probes 8 and 9, an unknown event (loss of DNA from the membrane? contamination of the membrane?) led to loss of signal in the circular area evident in the middle and right panels. This problem did not affect signal strength from nascent strands shorter than 3.5 kb.

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FIG. 5. A replication origin at or near the center of the 5.5-kbp PstI fragment is detected by 2D neutral-neutral gel electrophoresis. As in Fig. 2 and 3, first-dimension electrophoresis was from right to left and second-dimension electrophoresis was from top to bottom. The four bottom autoradiograms show the results of serial hybridization of a membrane containing DNA from a neutral-neutral 2D gel of EcoRI-digested DNA with the probes indicated in the diagram. First (leftmost) bottom panel: probe 3 was used to detect a 3.6-kbp fragment which overlaps the A4H and ClG BamHI fragments. Exposure time, 4 days. Second bottom panel: probe 4 was used to detect a 4.3-kbp fragment. Exposure time, 1 day. Third bottom panel: probe 5 was used to detect a 3.2-kbp fragment. Exposure time, 1 day. Fourth (rightmost) bottom panel: probe 8+ was used to detect an 8.1-kbp fragment. Exposure time, 3 days. The abnormal arc produced by this large fragment is discussed in the text. For the top panel, a blot of a 2D gel of a preparation of PstI-digested DNA was hybridized with the indicated 5.5-kbp PstI DNA fragment. Exposure time, 1 day.

4738

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ZHU ET AL.

MOL. CELL. BIOL.

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FIG. 6. Predicted results of neutral-alkaline 2D gel analyses restriction fragments in which termination of replication specific sites or randomly. (A) A restriction fragment (heavy zontal line) and probe (grey box) for the left end of the fragment. Diagrams representing the configurations of parental (heavy and nascent (light lines) strands during termination events occurring 25, 50, or 75% of fragment length from the left end of the

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probably the final portion of a simple Y arc. The presence this signal suggests that the origin is not precisely in the center of the PstI fragment, and that one of the forks reaches the end of the fragment before the other, leading to an abrupt switch from a bubble arc to a simple Y arc (1). We have explanation for the faint signals which are also visible in the top portion of Fig. 5. The location of an origin near the center of the 5.5-kbp PstI fragment is consistent, within experimental error of several hundred nucleotides, both with replication of all EcoRI fragments within the ClG BamHI fragment as dominantly simple Y structures and with the origin location determined by neutral-alkaline 2D gel analysis. of

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Mechanism of termination during chromosomal DNA replication. Two models for termination of DNA replication eukaryotic chromosomes have been proposed (13). According to the specific termination model, there are specific sites on DNA molecules where replication forks coming from either direction are stopped. According to the nonspecific model, converging replication forks meet at random sites. The neutral-alkaline 2D gel patterns predicted by the two different models are diagrammed in Fig. 6. Figure 6A shows a restriction fragment (heavy horizontal line) and a hybridization probe specific for the left end of the fragment (gray in

box). Figure 6B illustrates the configurations of parental and nascent strands anticipated if replication were to terminate specifically at positions 25, 50, or 75% of fragment length from the left end of the fragment. Note that the maximum nascent strand length detectable by the left end probe would correspond to 25, 50, or 75% of full fragment length, respectively. Consequently, as shown in Fig. 6C, the diagonal signal produced by the nascent strands should terminate, at its upper right extreme, at a strand length corresponding to 25, 50, or 75% of full fragment length, respectively (14, 22). Notice, in Fig. 6C, that the nascent strand arcs produced by specific termination at 50 or 75% partially overlap each other at their lower left-hand ends. In fact, all nascent strand arcs produced by specific termination events occurring 50% or more from the probed end of the restriction fragment should overlap in this region, with diminishing extents of overlap at longer nascent strand lengths. Consequently, if replication were to terminate nonspecifically at random locations throughout the diagrammed restriction fragment, then the overlapping portions of the nascent strand arcs should produce a signal running from lower left to upper right, with intensity diminishing continuously from bottom to top, as in Fig. 6D. In addition, the nonoverlapping portions of the nascent strand arcs should produce a diffuse elevated background in the region below the overlapping signal (lightly shaded region in Fig. 6D). If nonspecific termination occurs throughout a restriction fragment, then probes from the left end (illustrated in Fig. 6A and B) and probes from the right end should both produce the type of result diagrammed in Fig. 6D. By combining the information about directions of DNA replication provided by the current study with previous information about directions of replication flanking the ARS305-associated origin (Fig. 1) (12), we could deduce that termination between these two origins was likely to occur within the 4.3-kbpA4H BamHI fragment. Hybridization of a BamHI neutral-alkaline 2D blot with probes 1 and 2 revealed (Fig. 7) that both probes detect signals corresponding to nascent strands of all sizes, although the signal from nascent strands appears to be stronger for probe 1 than for probe 2. Because both probes yielded similar patterns, we confirmed by Southern blot hybridization of appropriately restricted genomic DNA that each probe hybridized specifically to the expected fragments and that the two probes did not crosshybridize (33). Consequently, we can conclude from Fig. 7 that replication forks must enter and pass through theA4H BamHI fragment in both directions. Although the results in Fig. 7 are clearly inconsistent with the specific termination model (Fig. 6C), they are at least partially consistent with the nonspecific model (Fig. 6D). As predicted in Fig. 6D, nascent strands of all sizes are detected by both probes 1 and 2 (Fig. 7). However, although the model (Fig. 6D) predicts decreasing signal intensity from longer nascent strands, the detected signal (Fig. 7) appears to be about the same for nascent strands of all sizes. This could be due to the fact that longer nascent strands are concentrated in a smaller area of the gel. Consequently, the signal from longer nascent strands is ordinarily expected to be (and is) stronger than the signal from shorter nascent strands (for example, Fig. 2 to 4 in this paper and similar figures in reference 12). The fact that the signal from nascent strands ofall sizes appears approximately constant in Fig. 7 may therefore indicate that the number of short nascent strands detected by both probes is greater than the number of longer nascent strands, as predicted for nonspecific termination.

CHROMOSOMAL DNA REPLICATION ORIGIN AND TERMINUS

VOL. 12, 1992

4.3 kbp

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FIG. 7. Replication forks enter the 4.3-kbp A4H BamHI fragment from both ends, and some of these forks pass all the way through the fragment. (Left panel) Hybridization with probe 1. Exposure time, 8 days. (Right panel) Hybridization with probe 2. Exposure time, 9 days. Other conditions were as in Fig. 2.

A second apparent difference between Fig. 7 and the prediction in Fig. 6D is the lack of diffuse signal below and to the right of the line of nascent strands. It is possible that the high background obtained with these probes obscures the

4739

expected diffuse signal. It is also possible that the predicted diffuse signal is not present, in which case replication must terminate in this region by a mechanism somewhat different from the mechanisms considered in Fig. 6 (and Fig. 8; see below). One can also predict the type of signal which specific or nonspecific termination would produce after neutral-neutral 2D gel electrophoresis. As first described by Brewer and Fangman (1), a restriction fragment replicated as a double Y by forks entering simultaneously from both ends and meeting in the middle (specific termination at 50% fragment length) would generate a nearly linear signal (Fig. 8, left panel, darkly shaded line). In addition, Brewer, Sena, and Fangman (3) have demonstrated that recombining restriction fragments with crossover points located throughout the fragment form a family of nonlinear structures (X structures) which, in neutral/neutral gels, yield a nearly vertical line signal, just above the 2n position in the arc of linear structures (Fig. 8, left panel). The position of a recombining molecule in this line depends on the position of the crossover point relative to the ends of the fragment. Molecules with crossover points at 50% are maximally nonlinear and therefore most retarded in the gel, forming the top portion of the line. Molecules with crossover points closer to one of the ends (for example, at 25 or 75%) are more linear and migrate more rapidly, as illustrated (3). Molecules with two converged replication forks should be identical or nearly identical to X structures and thus should migrate at the same positions as the corresponding X structures. In contrast, molecules with two converging forks should migrate in various positions, depending on the extent of replication of each of the two forks. Several examples are illustrated in Fig. 8, left panel. In each case, the molecules should migrate like simple Y structures until the second replication fork enters at the opposite end; then they should deviate from the simple Y pattern, forming a signal which should extend rightward to the appropriate position on the line of X structures. Similar predictions have recently been

33qr,167q

"SimpleY" Structures IlOS'd90% tructures

Liner Structres

In

FIG. 8. Neutral-neutral 2D gel electrophoresis suggests that termination occurs nonspecifically in the A4H BamHI fragment. (Left panel) Diagram illustrating the autoradiographic signals predicted after neutral-neutral 2D gel electrophoresis of DNA molecules containing converging replication forks. The large round spot at lower left represents the strong signal produced by nonreplicating monomeric DNA. The lower black line rising upwards and rightward from this spot represents the arc produced by double-stranded linear molecules. The upper black line which rises to a peak and then falls to the line of linear molecules represents the signal from simple Y structures. The other lines represent the signals which would be produced by molecules containing two converging replication forks; the positions and lengths of the lines produced depend on the points in the restriction fragment where the two forks finally meet. Examples are provided for forks meeting at 10% (or 90%) of fragment length from one end, 25% (or 75%) from one end, 33% (or 67%) from one end, or 50% from one end. The line diagrams represent the following structures: a partially replicated molecule with forks converging symmetrically toward the center (lower center), a Y-shaped molecule which has been half replicated (upper center), a fully replicated terminating molecule with forks meeting at 50% of unit length (upper right), and a terminating molecule with forks meeting at 75% (or 25%) of unit length (far right). If nonspecific termination were to occur, then the signals produced by the different types of double Y molecules would form a continuous smear in the roughly triangular area bounded by the last portion of the simple Y arc, the line of X structures, and the 50% line (grey area in diagram). (Center and right panels) Blots of neutral-neutral 2D gels of two independent preparations of BamHI-digested DNA were hybridized with probe 2 (Fig. 1). Exposure times were 10 days. Other conditions were as in Fig. 5.

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ZHU ET AL.

made both for random termination of converging forks (15) and for replication termination in unidirectionally replicating molecules (21). Therefore, if replication were to terminate at a specific location, the signal produced in a neutral-neutral 2D gel would be a single line which would end at a defined position along the line of X structures. The signal actually obtained for the A4H BamHI fragment (Fig. 8, right panels) is not a single line. Instead, it is a composite of a simple Y arc, a termination line, and a continuous smear of signals. The simple Y arc fades out as it descends to the line of linears at the 2n position, consistent with gradual conversion of simple Y structures to double-Y, terminating structures. The continuous smear is similar to the smear predicted for molecules terminating at all possible positions (Fig. 8, left panel), including a triangular section bounded by the simple Y arc, the 50% line, and the line of X structures. The relatively strong line projecting upward and rightward from the simple Y arc, in the position expected for the final portion of the 50% termination line, would appear to suggest that termination occurs most frequently at about 50% of fragment length. However, that interpretation is inconsistent with the neutralalkaline results (Fig. 7), in which the type of arc predicted for termination at 50% is not visible. In addition, Hyrien and Mechali (15) detect a similarly strong signal from the 50% line (as well as the type of triangular signal seen in Fig. 8) in neutral-neutral 2D gel analyses of plasmids replicating in Xenopus eggs and egg extracts. The fact that such signals are generated, in their case, by multiple overlapping restriction fragments proves that the presence of a strong 50% signal does not always mean that termination occurs at 50% of fragment length. In contrast, the strong 50% signal, in combination with a triangular signal, appears to be indicative of termination taking place randomly throughout the studied restriction fragment (15). Therefore, as we concluded from the neutral-alkaline analysis of the A4H fragment, the neutral-neutral data are not consistent with termination at a specific site but are largely consistent with a simple nonspecific termination model.

DISCUSSION The ARS306-associated origin. The results obtained with both the neutral-alkaline 2D gel technique (Fig. 4) and with the independent and complementary neutral-neutral technique (Fig. 5) demonstrate the existence of a bidirectional replication origin near the middle of the 5.5-kbp PstI subfragment of the ClG BamHI fragment. The data in Fig. 4 suggest that this origin is contained within a 650-bp XhoIBglII restriction fragment which also contains an ARS element, ARS306 (24, 27). Therefore, the newly identified origin coincides (within experimental error) with an ARS element, as have the six previously mapped yeast chromosomal origins (11, 12, 19, 31). We shall refer to this origin as the ARS306-associated origin. Another ARS element, different from ARS306, has recently been reported to lie within the ClG BamHI fragment between probes 3 and 4 (26). This region has been reexamined for possible ARS function by analyzing the transformation efficiency of plasmids containing the 4.3-kbp EcoRI fragment as well as plasmids containing the 2.0-kbp BglIIPvuII subclone of the 4.3-kbp EcoRI fragment reported to have ARS activity (6). These fragments were cloned in both orientations at the EcoRI site of a URA43/CEN4 vector, pVHA (30). None of the plasmids gave rise to transformants visible after 4 days of growth, while transformants from the

MOL. CELL. BIOL.

control ARSJ-containing plasmid were visible after 2 days. Thus we have been unable to confirm the presence of an ARS element in this region. Furthermore, the data reported here show that there is no chromosomal origin within this region. The fact that probe 4 detects only very long nascent strands (Fig. 2) while probe 6 detects nascent strands of all sizes shows that replication forks enter the 9.7-kbp PstI fragment (Fig. 1) at the probe 6 end, not the probe 4 end. If the putative new ARS element (which is close to probe 4) were an active chromosomal origin, then one would expect some replication forks to enter this restriction fragment at the probe 4 end. The conclusion that this region does not contain an origin is strengthened by our observation that the EcoRI-PstI fragment containing the putative new ARS element detects only long strands in the blot of a neutralalkaline 2D gel of EcoRI-digested genomic DNA (33). Termination between chromosomal origins. Measurements of replication fork direction by neutral-alkaline 2D gel electrophoresis in previous studies (12, 20) and this one (Fig. 2) suggested that replication forks initiated at the adjacent ARS305-associated and ARS306-associated origins must converge and terminate in or near the A4H BamHI fragment (Fig. 1). The data obtained in this study by the neutralalkaline (Fig. 6 and 7) and neutral-neutral (Fig. 8) 2D gel techniques are consistent with that expectation. Furthermore, these studies suggest that termination does not occur at a specific site within the A4H fragment but at multiple, probably random, sites throughout it. The A4H fragment is located halfway between the ARS305-associated and ARS306-associated origins (Fig. 1). However, replication timing studies (27) suggest that the ARS306-associated origin fires earlier in S phase than does the ARS305-associated origin. It is possible that replication forks moving leftward from the early ARS306-associated origin travel somewhat more slowly than forks moving rightward from the later ARS305-associated origin, resulting in termination approximately halfway between the two ori-

gins. Although the data in Fig. 7 and 8 suggest that most termination events take place within the A4H fragment, the data in both figures also suggest that a significant minority of replication forks move all the way through this fragment. These forks must terminate outside the A4H fragment. We have not been able to detect termination signals outside this fragment, presumably because termination outside the fragment, like termination within it, occurs at variable sites, leading to signals which are too diffuse to be detected. The results presented in this paper do not exclude the possibility that all termination events may take place within a broad zone defined at its extremes by specific polar termination sites. This type of termination zone is found in the Escherichia coli chromosome, where polar termination sites serve to confine random termination events to a region of -270 kbp (reviewed in reference 17). ACKNOWLEDGMENTS We are grateful to Leslie R. Davis for plasmid preparation and to Leland Hartwell for the yeast strain. For encouragement and helpful discussions we thank our colleagues in the Huberman and Newlon laboratories. An anonymous referee also provided helpful suggestions. This work was supported by grants to J.A.H. from the National Science Foundation and the American Cancer Society.

VOL. 12, 1992

CHROMOSOMAL DNA REPLICATION ORIGIN AND TERMINUS

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