Vol. 12, No. 6

MOLECULAR AND CELLULAR BIOLOGY, June 1992, p. 2804-2812

0270-7306/92/062804-09$02.00/0

Copyright C 1992, American Society for Microbiology

Activation of a Mammalian Origin of Replication by Chromosomal Rearrangement TZENG-HORNG LEU AND JOYCE L. HAMLIN* Department of Biochemistry, University of Virginia School of Medicine, Charlottesville, Virginia 22908 Received 30 October 1991/Accepted 11 March 1992

are distributed at very frequent intervals in mammalian chromosomes or (ii) that origins are not fixed genetic ele-

The DNA fiber autoradiographic studies of Huberman and Riggs showed that replication initiates at thousands of sites along each mammalian chromosomal DNA fiber (19). Replication forks move out bidirectionally from most initiation sites, and there is a tendency for clusters of contiguous origins to fire at approximately the same time (19). It has been shown in several studies that there is a temporal order of replication of defined sequences in mammalian genomes (13, 16, 20, 31). Furthermore, the time of replication of different markers in the S period can vary depending upon the transcriptional activity of a particular gene in different tissues (13, 16, 20). Finally, data from a variety of replicon mapping studies suggest that DNA synthesis initiates at preferred loci in mammalian chromosomes (15-17, 20). Therefore, sophisticated controls are exerted over initiation, probably through the agency of defined genetic sequences. However, despite the intensive effort that has gone into identifying replication origins in the complex genomes of mammalian cells, there is still no direct evidence that they are actually genetically fixed elements analogous to the origins of microorganisms. Candidate origins have been rescued from mammalian genomes in assays that depend upon the ability of a sequence to replicate autonomously after transfection into a suitable mammalian cell line (2, 12), but these results so far have not been confirmed in other laboratories, nor have any functional elements within these sequences been implicated in mutagenesis studies. Indeed, there is evidence from a novel long-term pheniotypic assay (in which the vector provides a partition function) that any cloned genomic restriction fragment will replicate after transfection into cultured human cells provided that it is large enough (21). This result suggests either (i) that origins *

ments.

In our laboratory, we have been attempting to identify and characterize initiation sites in the amplified dihydrofolate reductase (DHFR) domains (amplicons) of the methotrexateresistant Chinese hamster cell line CHOC 400. CHOC 400 contains -1,000 DHFR amplicons per cell (28), most of which are 240 kb in length (24). Because of the high copy number of the amplicon, it is possible to monitor replication fork movement through the DHFR domain in synchronized cells by a variety of in vivo labelling approaches (1, 6, 17, 22, 23, 26). In previous studies, we showed that replication initiates preferentially within a small group of restriction fragments that define a 28-kb locus lying downstream from the DHFR gene (17). To increase the resolution of the in vivo labelling approach, we used in-gel renaturation to eliminate background labelling from single-copy sequences and were able to quantitate the relative amount of label in individual amplified fragments (23). These studies suggested that there might actually be two preferred sites or zones of labelling lying within the 28-kb initiation locus (termed ori-1 and ori--y [23, 26]). Supporting data for this proposal were obtained in two independent studies in which the template strand bias of the leading strand of replication in the presence of emetine was used to roughly localize replication start sites (7a, 15). In more recent studies, we have used two-dimensional (2-D) gel electrophoretic methods to map replication intermediates in the amplified DHFR locus and have obtained evidence that, in vivo, replication may actually initiate at multiple, random sites scattered over a broad zone that includes the 28-kb initiation locus, possibly concentrated over the two peaks of early labelling that define ori-1 and ori--y (32). However, a rather different result was recently obtained in

Corresponding author. 2804

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The methotrexate-resistant Chinese hamster cell line DC3F/A3-4K (A3/4K) contains at least two prominent dihydrofolate reductase amplicon types. The type I amplicons, constituting -80%o of the total, are at least 650 kb in length, but the endpoints have not yet been characterized. The type II sequences represent -20% of amplicons, are 450 kb in length, and are arranged as alternating head-to-head and tail-to-tail repeats. In previous studies on the CHOC 400 line, in which the amplicons are much smaller, a replication initiation locus (ori-13/ori-'y) has been shown to reside downstream from the dihydrofolate reductase gene. In a more recent study on the larger amplicons of A3/4K cells, we detected an additional initiation locus (ori-c) lying -240 kb upstream from ori-13/ori--y. Interestingly, in vivo labelling experiments suggested that replication forks diverge from ori-a only in the downstream direction. This finding suggested either that ori-a is a unidirectional origin or that a terminus lies immediately upstream from ori-a However, in this study, we show that ori-a is actually very close to the head-to-head palindromic junction sequence between the minor type II amplicons in A3/4K cells; furthermore, ori-a is active in the early S period in the type II amplicons but not in the larger type I sequences that lack this palindromic junction. This is the first direct demonstration in mammalian cells that a cryptic origin can be activated by chromosomal rearrangement, presumably by deleting negative regulatory elements or by creating a more favorable chromosomal milieu for initiation.

VOL. 12, 1992

MATERIALS AND METHODS Cell culture and synchronizing regimens. The methotrexate-resistant Chinese hamster lung fibroblast cell line A3/4K was maintained as previously described (26). A3/4K is a more resistant derivative of the DC3F/A3 cell line originally developed by Biedler and colleagues (3). Subconfluent cultures of A3/4K cells were arrested in early G1 by isoleucine starvation for 45 h and were then released into complete medium containing aphidicolin (10 ,ug/ml) (17). After 13 h, when cells had collected at the G1/S boundary, the cultures were washed once and incubated in drug-free medium for the times indicated in the figure legends. In the experiments shown in Fig. 2, 3A, 3B, and 3C, bromodeoxyuridine (BrUdR) (10 ,ug/ml) was included during the 13-h aphidicolin block; after release into the S period by washing with BrUdR-containing medium, cells were labelled with BrUdR (10 ,g/ml) and [3H]deoxycytidine (20 ,Ci/ml; 29 Ci/mmol; NEN) for the time periods indicated. The BrUdR and [3H]deoxycytidine were then removed by one wash with complete medium and replacement with complete medium containing thymidine (10 ,ug/ml) and deoxycytidine (2 pg/ ml). All samples were harvested 12 h after initial removal of aphidicolin. In a second BrUdR labelling experiment (Fig. 3D), aphidicolin-blocked A3/4K cells were washed at least twice before labelling with BrUdR and [3H]deoxycytidine for the indicated times. All samples were then cultured in medium containing thymidine (10 ,ug/ml) and deoxycytidine (2 ,g/ml) prior to harvesting 14 h after initial removal of aphidicolin. For the controls, exponentially growing A3/4K cells were labelled with either [3H]thymidine (10 ,Ci/ml; 85.6 Ci/mmol; NEN) or BrUdR (10 ,g/ml) and [3H]deoxycytidine (20 ,uCi/ml) for 12 h before isolating the DNA. The degree of cell synchrony attained in each experiment was monitored by fluorescence-activated cell sorter analysis performed in the University of Virginia Cell Sorter Facility. DNA preparation, restriction enzyme digestion, and Southern blotting. DNA samples were purified by standard meth-

2805

ods as described previously (23). Manipulations with BrUdR-labelled DNA were performed under a 60-W Sears No-bug yellow light at a distance of no closer than 24 in. (ca. 61 cm). DNA was digested with either EcoRI or BamHI before two cycles of fractionation on a neutral CsCl densit gradient. The DNA in the heavy-light (density = 1.74 g/cm) and light-light (1.705 g/cm3) peaks was then recovered by ethanol precipitation, and -30 ng of each was labelled with [32P]dCTP by random priming (10). Each sample was used to probe a slot blot containing 40 ng each of a collection of cosmids spanning -500 kb from the A3/4K DHFR amplicons. Slot blots were prepared on GeneScreen and processed according to the recommendations in NEN brochure NEF-976. Alternatively, each sample from the gradient was further digested with either KpnI (for EcoRI-digested DNA) or HindIlI (for BamHI-digested DNA) before separation on an agarose gel and transfer to GeneScreen by an alkaline blotting procedure (30). The transfers were UV cross-linked (8) and probed with selected labelled cosmids as described previously (25). For reprobing, the previous probe was removed by boiling the membrane for 20 min in 0.1 x SSC (15 mM NaCl, 1.5 mM sodium citrate, pH 7.0) containing 1% sodium dodecyl sulfate. For the restriction mapping of CHO and A3/4K genomic DNA, the DNA of exponentially growing cells was purified by standard procedures (14) and digested with different restriction enzymes according to conditions recommended by the supplier (Bethesda Research Laboratories). Fifty nanograms of each A3/4K digest or 5 ,ug of the CHO digests was loaded onto a 1% agarose gel, and the digests were blotted onto GeneScreen. The transfer was then probed with a 2.1-kb EcoRI-KpnI fragment that was isolated from the cosmid KT27 on a low-melting-point agarose gel (Bethesda Research Laboratories). 2-D mapping of replication intermediates. A3/4K cells were synchronized at the G1/S boundary and were released into the S period for the times indicated in Fig. 6. Replication intermediates were isolated from nuclear matrices by digestion with the appropriate restriction enzymes as described previously (9). Thirty micrograms of each sample was separated on a 2-D gel essentially as described in reference 4. RESULTS Apparent asymmetric fork movement from the ori-a initiation locus in the DHFR amplicon of A3/4K cells. Previous analysis of recombinant cosmids, as well as pulsed-field gradient gel analysis of Sfil fragments, has shown that 80% of the DHFR amplicons in the A3/4K genome are at least 650 kb in length (designated as the type I amplicon in Fig. 1) (25-27). Since the actual endpoints of this amplicon type(s) have not been mapped, it is not known whether they are arranged in head-to-tail or head-to-head arrays in the genome. The minor type II amplicons were shown to be approximately 450 kb in length and to be organized in head-to-head arrays, as determined from cosmid mapping and the presence of a 50-kb Sfi1 fragment in the A3/4K genome that had the properties of a palindromic junction fragment (25). The region shared by these two amplicon types includes the DHFR and 2BE2121 (11) genes as well as the previously identified ori-p/ori--y replication initiation locus (Fig. 1). The arrangements of the type I and II amplicons from CHOC 400 cells are shown for comparison (24, 27). Figure 1A shows a collection of overlapping recombinant

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a study in which labelled Okazaki fragments were synthesized in vitro and were hybridized to the plus and minus template strands of M13 clones from the initiation locus (7). A strong template strand bias was observed on either side of ori-f, and this bias switched abruptly from one template strand to the other within a 500-bp fragment in the center of the ori-O region. Along with several other studies cited above, this result argues for the presence of a strongly preferred initiation site at this locus. These rather disparate findings suggested to us that other initiation loci in the CHO genome should be identified and characterized. Toward this end, we recently localized an additional early-firing origin (ori-a) lying -240 kb upstream from ori-,B/ori-y in the much larger amplicons of the DC3F/ A3-4K (A3/4K) cell line (26). However, replication forks appeared to proceed only in the downstream direction from this locus. This result suggested either that ori-a represents a unidirectional origin or that a strong termination site lies immediately upstream from ori-a. In this study, we have examined the sequence arrangements and patterns of replication in this region of the DHFR amplicon in more detail. We find no evidence that ori-ao is a unidirectional origin or that a terminus lies upstream from ori-ax. Rather, our results provide compelling evidence for the activation of a cryptic origin of replication by chromosome rearrangement.

ORIGIN ACTIVATION

2806

MOL. CELL. BIOL.

LEU AND HAMLIN A

KT27 KD23

K1366

KF115

-500

-400

-300

B A

I

120

N27

KY143

-200

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400

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cosmids from the A3/4K cell line that was isolated in a previous study (26). In the slot blot experiment shown in Fig. 2 (reproduced from reference 26), early-replicating BrUdRlabelled DNA samples from synchronized A3 cells were labelled in vitro with [32P]dCTP and were used as hybridization probes on this cosmid collection to search for a new initiation locus in the large DHFR amplicons of A3/4K cells. In Fig. 2A, the 0.3-h time sample was hybridized to the cosmid collection in the presence of increasing concentrations of unlabelled CHO genomic DNA (as a source of repetitive sequence elements) in order to determine the amount required to eliminate background labelling (>200 ,ug/ml). DNA from an early time point was used in this experiment, since the region between origins would not be replicated in 20 min and would therefore provide an internal baseline. In Fig. 2B, it is clear that early-replicating DNA preferentially hybridizes to the cosmids SE24 and KD23 (compare the 0.3-, 1-, and 3-h samples with the log control). KD23 contains the newly identified ori-t locus (26), while SE24 contains the ori-,B/ori-y locus (1, 6, 7, 15, 17, 23). Note, however, that the signal from KD23 is two- to threefold weaker than that observed for SE24. It is evident that replication forks move outward bidirectionally from ori-13/ori--y and converge with forks from ori-a in the neighborhood of NQ7 -4 h after entry into S phase in this experiment. However, replication forks appear to move only in the 3' direction from ori-a, judging from the low signals emanating from the upstream cosmids, KF115 and KI366, even when probed with samples from later time points (Fig. 2B). This is not the result of unequal loading of cosmids onto the blot, since subsequent hybridization of the same blot to vector (Fig. 2C) shows that all cosmids are present in approximately equal amounts (after corrections for KY143 and Hi, which contain two and three vectors, respectively). It is also apparent, however, that both KF115 and KI366 must contain repeated sequence elements, since even in the blot hybridized to the log-phase DNA sample in the presence of blocking CHO DNA, the signals emanating

y

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re) -n -

y

v

Y

N Z

indicates the early-replicating variant fragment. (B) The membrane was probed with 32P-labelled cosmid KD23. (C) A membrane similar to that in panel B was probed with 32P-labelled KY143. (D) BrUdR-labelled DNA samples from a highly synchronous population of A3/4K cells were digested with EcoRI-KpnI, transferred to GeneScreen, and hybridized with 32P-labelled KT27. (E) Restriction map of cosmid KT27. R, EcoRI; K, KpnI; B, BamHI; H, HindIll. The sizes in kilobases of the fragments in the cosmid inserts are indicated, and the wavy lines indicate vector sequences. Note that the regions mapping between the 4.4- and 3.6-kb HindIII-BamHI fragments and between the 2.6-kb KpnI and 1.5-kb EcoRI fragments contain several restriction sites too closely spaced to be shown on the diagram.

from these two cosmids are very low relative to the others (Fig. 2B). Therefore, the faint signals observed in K1366 and KF115 with the early-replicating hybridization probes could be due either to unidirectional fork movement from ori-a or to large amounts of repetitive sequences that are blocked out by the CHO genomic DNA in the hybridization mixture. An early-replicating variant fragment is detected in the ori-a locus. To distinguish between these two possibilities and to avoid ambiguities arising from the use of genomic DNA samples as hybridization probes, we examined the content of each replicated fraction directly on Southern blots by hybridization to individual cosmids. In the first experiment, BamHI-HindIII or EcoRI-KpnI digests of the BrUdRlabelled samples were electrophoresed, transferred to GeneScreen, and hybridized with selected 32P-labelled cosmids. Equal amounts of DNA were loaded in each lane, except for the experiment in Fig. 3D, in which half the amount was loaded in the first three wells inadvertently. In Fig. 3B and C, it can be seen that all of the EcoRI-I4pnI fragments represented in cosmids KD23 and KY143 (which maps on the downstream side of KD23; Fig. 1) appear to have been replicated by 0.3 and 2 h after removal of aphidicolin, respectively (compare with the log-phase samples in the B and T lanes). This result would be expected if

KD23 contains an approximately centered origin and if replication forks move away from it at 2 to 3 kb/min (19). However, when a BamHI-HindIII digest of the same DNA was probed with KT27, which overlaps KD23 on the 5' side (Fig. 1), several fragments in this cosmid are seen to be underrepresented relative to the log-phase controls at all but the latest time point (i.e., the 13-, 4.4-, 3.6-, 3.0-, and 1.7-kb fragments indicated by asterisks in Fig. 3A). Examination of the restriction map for KT27 (Fig. 3E) shows that all of these fragments are contiguous in the genome and map in the upstream two-thirds of KT27. In the experiment presented in Fig. 3A to C, the latereplicating fragments in KT27 do not appear to be synthesized until at least 6 h after removal of aphidicolin. However, cells were moving unusually slowly through the S period in this experiment (>14 h), probably because of inadequate removal of aphidicolin. To gain a more reliable estimate of the time of replication of the region lying upstream from ori-a, the synchrony regimen was therefore repeated under more stringent washing conditions. Fluorescence-activated cell sorter analysis showed that the cultures in this second experiment progressed both into and through the S period in a synchronous wave, the majority completing the S period in -9 h (data not shown). The heavy-light DNA samples recovered from this more

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i-a 1.5-

2808

LEU AND HAMLIN

A. kb 8

--

7-

6-543 --

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R

P

L

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H

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Parental type

16.7kb (38kb) _ 13 kb (5 kb)

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Variant type

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9.5kb; --

7.3kb 22kb 27kb L

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FIG. 4. Identification of the early-replicating variant as one of the type II interamplicon junctions. (A) DNA from exponentially growing A3/4K DNA cells was digested with the indicated enzymes or enzyme combination (B, BamHI; H, HindIll; R, EcoRI; P, PstI; P/H, PstI and HindIII; X, XbaI; X/H, XbaI and HindIII; K, KpnI; K/H, KpnI and HindIII). The DNA was separated on an agarose gel and transferred to GeneScreen. The membrane then was hybridized with the 2.1-kb EcoRI-KpnI fragment. The sizes of parental (major) and variant (minor) fragments, respectively, in each lane are as follows: lane B, 17 and 27 kb; lane H, 13 and 22 kb; lane R, 7.9 and 7.3 kb; lane P, 7.6 and 9.5 kb; lane P/H, 6.6 and 9.5 kb; lane X, 13 and 6.2 kb; lane X/H, 5 and 6.2 kb; lane K, 6.7 and 3.8 kb; and lane K/H, a 3.8-kb doublet. Note that the DNA in lane K/H was largely degraded in this experiment. The positions of the fragments in a 1-kb ladder are shown on the extreme left. (B) DNA isolated from exponentially growing CHO or A3/4K cells (5 ,ug or 50 ng, respectively) was digested with HindIll and separated on a gel. A transfer of the digests was probed with the 2.1-kb EcoRI-KpnI fragment. Two fragments 13 and 22 kb long are detected in A3/4K genomic DNA with this probe, but only a 13-kb fragment is detected in CHO DNA. (C) Restriction maps of the parental (upper) and variant (lower) sequence arrangements are shown with bold lines; relevant restriction fragments are indicated below. The number in parentheses next to each parental restriction fragment denotes the distance in kilobases from the 3' end of each fragment to the 5' end of the 13-kb HindlIl fragment discussed in the text. Note the symmetrical arrangement of restriction sites in the map of the variant sequence.

kb in length and contain no junctions in this region (Fig. 1). The same map could be constructed from CHO genomic DNA, which is homozygous for the allele of the DHFR locus that is amplified in A3/4K cells (data not shown) (25). The second, variant map is symmetrical around a central 3.8-kb KpnI fragment, which itself contains an off-center EcoRI site (Fig. 4C). As can be seen from the map, the 22-kb HindIlI fragment represents the union of two 13-kb HindIII fragments in a head-to-head fashion, with the joint localized somewhat asymmetrically within the variant 3.8-kb KIpnI fragment. Other mapping studies have shown that a 1.7-kb EcoRI-KpnI junction fragment defines the end of the type II amplicons. This fragment comigrates with another 1.7-kb fragment present in both amplicon types (Fig. 3D and data not shown).

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synchronous culture were then digested with EcoRI-KpnI and probed with 32P-labelled KT27. The autoradiogram from this experiment (Fig. 3D) shows that the 4.6-, 2.7-, 2.6-, and 1.9-kb fragments lying upstream from the central 2.1-kb EcoRI-K:pnI fragment did not replicate until sometime after the third hour of the S period (compare the 1-, 2-, and 3-h samples with the log-phase B and T controls). In contrast, sequences in KT27 lying downstream from this fragment (e.g., the 6.0-, 5.6-, and 3.0-kb fragments) had already begun to replicate by the first hour after removal of aphidicolin (Fig. 3D). Note that the 6-, 12-, and 14-h samples are slightly overloaded relative to the other samples. Thus, rather than the ori-a locus representing a unidirectional origin of replication, it appeared that a strong pause site or replication terminus might reside on the upstream end of ori-ao, near the 2.1-kb fragment in KT27. In fact, the latter suggestion was supported by the presence of a variant 22-kb HindIll fragment that was detected in BamHI-HindIII digests of A3/4K genomic DNA (arrowhead, Fig. 3A). This fragment is not present as such in the cosmid KT27 (Fig. 3E) or in CHO DNA (Fig. 4B), and it is highly enriched in early-replicating DNA (compare with the log-phase genomic samples in lanes B and T). Furthermore, the 22- and 13-kb fragments appear to be related, since their levels vary with time in a reciprocal manner (Fig. 3A). It therefore was conceivable that the 22-kb variant fragment represented a long-lived Y-shaped version of the 13-kb fragment resulting from the presence of a replication fork barrier. Indeed, we detected an S1 nuclease-hypersensitive site close to the 2.1-kb EcoRI-KpnI fragment that marks the boundary between the early- and late-replicating fragments in the region of genomic DNA represented by cosmid KT27 (21a). However, the picture became less clear when an Si-sensitive site was also detected in the 13-kb HindIll fragment from the KT27 cosmid itself. This result, along with the observation that the 22-kb fragment was actually a relatively minor species in the DNA of log-phase cells, suggested that it might represent a rearranged variant resulting from the amplification process per se. The early-replicating variant represents one of the type II interamplicon junctions. We therefore mapped the cosmid KT27 and the region of the A3/4K genome circumscribing the novel 22-kb HindIII fragment with several restriction enzymes. The 22-kb variant in A3/4K genomic DNA hybridizes with all sequences in the 13-kb HindIII fragment that map downstream, but not upstream, from the 2.1-kb EcoRIKjpnI fragment (data not shown). Therefore, the 22-kb variant must be derived, at least in part, from the 13-kb HindIll fragment. We previously suggested, on the basis of large-scale mapping of A3/4K genomic DNA with SfiI on pulse-field gradient gels, that an upstream palindromic junction between the type II amplicons might reside somewhere in the neighborhood of KT27 (25). If the 22-kb HindIII variant actually represents a palindromic junction, then it should not be present in CHO DNA, and it should be centered in a symmetrical restriction map in this region. Figure 4B shows that the 22-kb HindIII fragment is not, in fact, present in CHO DNA. Furthermore, when the 2.1-kb EcoRI-KpnI fragment was used as a hybridization probe on selected digests of A3/4K genomic DNA (Fig. 4A), two independent restriction maps could be constructed in the neighborhood of this fragment (Fig. 4C). One of these maps (Fig. 4C) corresponds to the predominant (parental) sequence arrangement in this region of the genome and derives from the major type I amplicons, all of which are at least 650

MOL. CELL. BIOL.

VOL. 12, 1992

ORIGIN ACTIVATION

2809

Since the distance between the 2.1-kb EcoRI-KpnI frag-

ment and the nearest downstream SfiI site is -25 kb (data not shown), and because the size of a minor palindromic

variant Sfil fragment detected in this region is -50 kb (25), the palindromic joint detected in the 3.8-kb KpnI fragment undoubtedly represents the junction that defines the upstream boundary of the typeII amplicon in the A3/4K cell line. The relative hybridization signals between parental and variant fragments in Fig. 4A suggest that the type II amplicons represent only 20 to 25% of the amplicons in A3/4K cells.

0

0

E

E

uf~ ~ ~ n~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

E~ +

.0 0

variant. By 1 h after entry into the S period, the signal from the variant fragment still predominates (Fig. 3B). By 2 h, the parental fragment begins to be synthesized, although the specific replication activity per fragment is still less than that of the variant (Fig. 3C). By 3 h, very few replication forks are detected in the variant fragment (data not shown), and by 6 h, replication of the variant is basically not detectable (Fig. 6). Note that in this experiment, the degree of synchrony comparable to that in the experiment presented in Fig. 3D, with cells traversing the S period in -9 h. Significantly, no signals indicative of the presence of a replication terminus

was

were the

observed

at any

time point, either in the variant

parental fragment; single-forked

structures

or

in

collected at

a

6.

N, A

...

..

CX

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-I

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]It -D (mass)-~ I It-D (mass) -5. Patterns of typical replication intermediates separated by FIG. 2-D neutral/neutral gel electrophoresis. Each panel shows an idealized autoradiographic image that would be obtained when a restriction digest of replicating DNA is hybridized with probes for fragments that contain different intermediates. (A) A complete simple Y or fork arc (b) resulting from a fragment that is replicated passively from an outside origin. Curve a represents the diagonal of nonreplicating fragments from the genome as a whole. (B) The pattern obtained when a fragment with a centered origin of replication is of probed (curve c). Bubbles migrate more slowly at all extents replication than do forks in a fragment of equal mass (b).rise(C)toThe an presence of an off-center origin in a fragment gives arc when incomplete bubble arc (c), which then reverts to the fork the bubble expands beyond the right-hand restriction site, resulting in a fork arc break. (D) When two forks approach each other in a e or d, fragment either symmetrically or asymmetrically, curve fragment, a in a fixed terminus is obtained. If there is respectively, the collected X-shaped structures would result in a concentrated spot somewhere on curve f. Recombination structures would also

fall along

curve

f (4).

spot single site or within a small zone would result in a strongwould the fork arc (5), whereas double-forked structures result in the curve depicted in Fig. 5 (4). Note that the spike 6A migrating to the left of the variant (larger) fork arc in Fig. not to C is trailing material due to partial digestion and does migrate to the position expected of true double-forked termination structures (Fig. 5D). In addition, no termination signals were observed when we examined EcoRI digests of

on

the

same

DNA (data not shown).

DISCUSSION Because the sequences lying upstream from the newly locus are greatly underrepresented in the identified cells (26) (Fig. 2), we early-replicating DNA of unidioriginally proposed either (i) that ori-o represents a immerectional origin or (ii) that a replication terminus lies diately upstream from this origin. However, the data presented here argue that both of these to explanations are incorrect. Although we were unable se, per movement fork for unidirectional biased) (or assay

ori-ot

A3/4K

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To confirm this suggestion and to rule out the presence of a terminus, we examined the replication intermediates in this region by a 2-D gel electrophoretic method that is capable of determining whether a given fragment contains an origin or a terminus or is replicated through by forks from distant origins (4). In this method (diagrammed in Fig. 5), restriction digests of genomic DNA from actively dividing cells (either log or synchronized in the S period) are separated in the first dimension of an agarose gel largely according to molecular mass and in the second dimension by a combination of both mass and shape. Because of their nonlinear configurations, restriction fragments containing single forks, replication bubbles, or double-forked structures trace characteristic arcs above the linear diagonal of nonreplicating fragments (Fig. 5). The intermediates that characterize a fragment of interest can then be identified by using a cognate hybridization probe. Replicating DNA was partially purified from synchronized A3/4K cells at various times after entry into S phase by a matrix isolation procedure developed in our laboratory (9), using a combination of XbaI and HindIll to digest the DNA. The digests were separated on a 2-D gel and probed with the 2.1-kb EcoRI-KpnI fragment. In this digest, the parental and variant fragments detected with the probe are 5 and 6.2 kb in length, respectively (Fig. 4C). By 0.3 h after entry into S phase, the larger variant fragment (V in Fig. 6A) is the predominant replicating species, displaying an intense fork arc (Fig. 6A) and, after a longer exposure, a faint bubble arc (Fig. 6E). In contrast, only a very faint fork arc is observed in the parental position on the same autoradiogram (P in Fig. 6A), even though the parental fragment is four times more prevalent than the

c

1

0

.0

The junction fragment, but not its wild-type counterpart, is early replicating. The mapping data presented above showed

quite convincingly that the prominent 22-kb variant HindIII fragment in early-replicating DNA represents a palindromic junction between the typeII amplicons. Since only 20 to 25% of amplicons in A3/4K cells are in the type II arrangement, this result suggested that the 22-kb variant was preferentially replicated in the early S period.

~~

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0.3 hr.

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FIG. 6. Determination that the junction fragment, but not the wild-type fragment, is early replicating. A3/4K cells were synchronized as described in Materials and Methods. Thirty micrograms of an XbaI-HindIII digest of DNA from cells harvested at the times indicated was separated on a 2-D gel, transferred to GeneScreen, and hybridized with a probe for the 2.1-kb EcoRI-KpnI fragment. V, the variant 6.2-kb fragment in the type II amplicon; P, the parental 5-kb fragment. The films were exposed for 24 h (A) and 16 h (B to D). (E) A 60-h exposure of the transfer shown in panel A.

to effectively rule out the existence of a strong termination signal in the neighborhood of the 2.1-kb EcoRIKpnI fragment that defines the start of the late-replicating we were able

region (Fig. 6). Most important was the finding that the variant earlyreplicating 22-kb HindIII fragment actually represents the upstream palindromic junction between the type II amplicons, lying only 25 kb upstream from the Sfil site in KD23 (Fig. 3E). Therefore, the late-replicating sequences upstream from ori-a can be derived only from the larger type I amplicons (Fig. 1). In addition, the data from the BrUdR labelling experiment (Fig. 3A) and from 2-D gel analysis (Fig. 6A and B) showed clearly that the palindromic type II junction fragment replicates much earlier than the parental nonrearranged fragment in the larger type I amplicons. In total, these data show that the ori-a locus is active in

It is formally possible that any genomic rearrangement that results in the formation of a palindromic junction can serve as a replication origin itself. However, we have examined fragments containing the downstream palindromic junction of the type II amplicons in both A3/4K and CHOC 400 cells (Fig. 1B) and did not detect preferential early replication of either of these junctions (data not shown). Therefore, the special rearrangement that activated ori-a in the A3/4K type II amplicon either (i) has activated a region that was never an origin in the first place or (ii) has stimulated a cryptic origin, either by the removal of a negative constraint or by the formation of a more permissive environment for origin function. In fact, in BrUdR labelling studies on the ori-a locus in MK42 cells (29), in which all of the amplicons are larger than the A3/4K type II and do not contain a palindromic junction in this region (25), we have shown that ori-ao is actually active, albeit only 2 to 3 h after entry into the S period (21a). We have not yet confirmed this suggestion in parental CHO cells, nor were we able to detect a bubble arc over the parental-sized fragment in longer exposures of the 2- or 3-h A3/4K genomic samples from the experiment shown in Fig. 6 (probably because not enough DNA was loaded on the gel in this experiment, in combination with the decay of synchrony that occurs as cells traverse the S period and the consequent diminution in the strength of the bubble arc). However, we tentatively conclude that the palindromic rearrangement somehow has affected the time of activation rather than the ability to function as an origin per se. In this regard, there appear to be some inconsistencies in the time of replication suggested by the two different methods of analysis used here. In the BrUdR labelling experiment in which the cell populations entered and traversed the S period in a highly synchronous wave (Fig. 3D), sequences upstream from the palindromic junction (e.g., the 4.6-kb EcoRI-KpnI fragment) do not appear to replicate until about 3 h after entry into the S period. This result contrasts with the 2-D gel data shown in Fig. 6, in which the population entered and traversed the S period with the same kinetics. In these cells, replication of the parental-size fragment from the

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the early S period in the type II amplicon but not in the larger type I. A necessary consequence is that sequences lying upstream from ori-a in the type I arrangement must wait to be replicated by forks from a more distant upstream or downstream origin or, if ori-a normally fires later in the S period, from ori-a itself. After long film exposures of 2-D gels, a faint and complete bubble arc, in addition to a prominent and complete fork arc, could be detected in the palindromic junction fragment (Fig. 6E). We have shown previously that this composite pattern results from initiation occurring at many random sites both within and outside the respective fragment being analyzed. Thus, at least some initiation actually appears to occur near the junction itself in the early S period. Although we have not yet examined fragments flanking the variant, other 2-D gel studies on the ori-a locus have so far shown that initiation also occurs at multiple random sites within at least a 15-kb zone in the center of KD23 (8a). It is therefore possible that the initiation zone extends all the way from the 3' boundary of ori-a (yet to be defined) to the palindromic junction in KT27. This would be very analogous to the situation in the ori-13/ori--y locus, in which extensive analysis by two independent 2-D gel techniques has shown that, in vivo, replication in this locus begins at multiple random sites scattered over a zone -55 kb in length

VOL. 12, 1992

ACKNOWLEDGMENTS We thank June L. Biedler (Sloan-Kettering Institute) for kindly providing the DC3F/A3 cell line. We thank all members of our laboratory for many helpful and critical discussions, particularly W. Carlton White and Kevin Cox for dedicated and valuable technical assistance in all aspects of this project. This work was supported by NIH grants GM26108 and CA52559 to J.L.H. REFERENCES 1. Anachkova, B., and J. L. Hamlin. 1989. Replication in the amplified dihydrofolate reductase domain in CHO cells may initiate at two distinct sites, one of which is a repetitive sequence element. Mol. Cell. Biol. 9:532-540.

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2. Ariga, H., T. Itani, and S. M. M. Iguchi-Ariga. 1987. Autonomous replicating sequences from mouse cells which can replicate in mouse cells in vivo and in vitro. Mol. Cell. Biol. 7:1-6. 3. Biedler, J. L., and B. A. Spengler. 1976. A novel chromosome abnormality in human neuroblastoma and antifolate-resistant Chinese hamster cells in culture. J. Natl. Cancer Inst. 57:683695. 4. Brewer, B. J., and W. L. Fangman. 1987. The localization of replication origins in ARS plasmids in S. cerevisiae. Cell 51: 463-471. 5. Brewer, B. J., and W. L. Fangman. 1988. A replication fork barrier at the 3' end of yeast ribosomal RNA genes. Cell 55:637-643. 6. Burhans, W. C., J. E. Selegue, and N. H. Heintz. 1986. Isolation of the origin of replication associated with the amplified Chinese hamster dihydrofolate reductase domain. Proc. Natl. Acad. Sci. USA 83:7790-7794. 7. Burhans, W. C., L. T. Vassilev, M. S. Caddle, N. H. Heintz, and M. L. DePamphilis. 1990. Identification of an origin of bidirectional replication in mammalian chromosomes. Cell 62:955-965. 7a.Burhans, W. C., L. T. Vassilev, J. Wu, J. M. Sogo, F. S. Nallaseth, and M. L. DePamphilis. 1991. Emetine allows identification of origins of mammalian DNA replication by imbalanced DNA synthesis, not through conservative nucleosome segregation. EMBO J. 10:4351-4360. 8. Church, G. M., and W. Gilbert. 1984. Genomic sequencing. Proc. Natl. Acad. Sci. USA 81:1991-1995. 8a.Dijkwel, P. A. Unpublished data. 9. Dijkwel, P. A., J. P. Vaughn, and J. L. Hamlin. 1991. Mapping of replication initiation sites in mammalian genomes by twodimensional gel analysis: stabilization and enrichment of replication intermediates by isolation on the nuclear matrix. Mol. Cell. Biol. 11:3850-3859. 10. Feinberg, A. P., and B. Vogelstein. 1983. High specific activity labeling of DNA restriction endonuclease fragments. Anal. Biochem. 132:6-13. 11. Foreman, P. K., and J. L. Hamlin. 1989. Identification and characterization of a gene that is coamplified with dihydrofolate reductase in a methotrexate-resistant CHO cell line. Mol. Cell. Biol. 9:1137-1147. 12. Frappier, L., and M. Zannis-Hadjopoulos. 1987. Autonomous replication of plasmids bearing monkey DNA origin-enriched sequences. Proc. Natl. Acad. Sci. USA 84:6668-6672. 13. Goldman, M. A., G. P. Holmquist, L. A. Gray, L. A. Caston, and A. Nage. 1984. Replication timing of genes and middle repetitive sequences. Science 224:686-692. 14. Gross-Bellard, M., P. Oudet, and P. Chambon. 1978. Isolation of high molecular weight DNA from mammalian cells. Eur. J. Biochem. 36:32-38. 15. Handeli, S., A. Klar, M. Meuth, and H. Cedar. 1989. Mapping replication units in animal cells. Cell 57:909-918. 16. Hatton, K. S., V. Dhar, E. H. Brown, M. A. Iqbal, S. Stuart, V. T. Didamo, and C. L. Schildkraut. 1988. Replication program of active and inactive multigene families in mammalian cells. Mol. Cell. Biol. 8:2149-2158. 17. Heintz, N. H., and J. L. Hamlin. 1982. An amplified chromosomal sequence that includes the gene for dihydrofolate reductase initiates replication within specific restriction fragments. Proc. Natl. Acad. Sci. USA 79:4083-4087. 18. Hohn, B., and J. Collins. 1980. A small cosmid for efficient cloning of large DNA fragments. Gene 11:291-298. 19. Huberman, J. A., and A. D. Riggs. 1968. On the mechanism of DNA replication in mammalian chromosomes. J. Mol. Biol. 32:327-337. 20. James, D. C., and M. Leffak. 1986. Polarity of DNA replication through the avian alpha-globin locus. Mol. Cell. Biol. 6:976-984. 21. Krysan, P. J., S. B. Haase, and M. P. Calos. 1989. Isolation of human sequences that replicate autonomously in human cells. Mol. Cell. Biol. 9:1026-1033. 21a.Leu, T.-H. Unpublished data. 22. Leu, T.-H., B. B. Anachkova, and J. L. Hamlin. 1990. Repetitive sequence elements in an initiation locus of the amplified dihydrofolate reductase domain in CHO cells. Genomics 7:428-433.

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same region of the type I amplicon already can be detected by the second hour of the S period (although it constitutes a very minor amount of replication relative to the larger variant fragment, since there are four times as many parental-size fragments in the A3/4K genome). These disparate replication times probably result, at least in part, from the fact that the BrUdR-labelled samples analyzed in Fig. 3 were pooled from the center of the heavy-light peaks on CsCl gradients before being run on the gels for detection, which would require that most fragments be fully replicated in the presence of BrUdR. However, 2-D gels analyze only those fragments that are in the act of replicating and would necessarily detect replication of a fragment earlier than would the BrUdR approach. It is also possible that the introduction of BrUdR into the DNA of replicating cells slows progression through the S period somewhat. A possible explanation for early activation of the ori-a locus in the type II amplicon is that the palindromic arrangement and/or the close juxtaposition of two ori-a loci might have affected chromosome condensation or higher-order architectural features of the chromatin in this region. Consequently, the ori-a loci in the type II amplicons might become a more accessible target for the protein or complex that activates replication initiation at origins of replication. This proposal could explain the early activation of ori-a if it is further assumed that initiation factors accumulate during the S period and normally build up to a level high enough to activate ori-a only after 2 or 3 h. Finally, it is interesting to note that during preparation of the matrix-attached and loop DNA fractions that were analyzed on 2-D gels in this study, we observed that a larger percentage of the nonreplicating (in) type II junction fragment partitions with the matrix fraction than does the parental fragment (21a). We have no idea why this should be so, since there is no detectable matrix attachment region (MAR) in the parental fragment that gave rise to the junction fragment (data not shown). It is therefore conceivable that a MAR was accidentally created during the formation of the type II junction. The preferential usage of ori-ao in the type II amplicons in the early S period may therefore be facilitated by the presence of this newly created MAR in the region. (Note, however, that replicating variant fragments are not artifactually enriched over the parental fragment by isolation on the nuclear matrix, since the variant is observed to be replicated preferentially in the early S period even when the DNA is isolated by other means, such as standard proteinase K treatment followed by organic extraction [21a]. Furthermore, in the very minor quantities of replication intermediates that partition with the loop fraction in the matrix isolation procedure, the variant is also observed to replicate preferentially in the early S period [21a].)

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30. 31. 32.

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and J. L. Hamlin. 1981. Methotrexate-resistant Chinese hamster ovary cells have amplified a 135 kilobase pair region that includes the gene for dihydrofolate reductase. Proc. Natl. Acad. Sci. USA 78:6042-6047. Nunberg, J. H., R. J. Kaufman, R. T. Schimke, G. Urlaub, and L. A. Chasin. 1978. Amplified dihydrofolate reductase genes are localized to a homogeneously staining region of a single chromosome in a methotrexate-resistant Chinese hamster ovary cell line. Proc. Natl. Acad. Sci. USA 75:5553-5556. Reed, K. C., and D. A. Mann. 1985. Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 13:72077221. Taljanidisz, J., J. Popowski, and N. Sarkar. 1989. Temporal order of gene replication in Chinese hamster ovary cells. Mol. Cell. Biol. 9:2881-2889. Vaughn, J. P., P. A. Djkwel, and J. L. Hamlin. 1990. Replication initiates in a broad zone in the amplified CHO dihydrofolate reductase domain. Cell 61:1075-1087. Wahl, G. M., K. A. Lewis, J. C. Ruiz, B. Rothenberg, J. Zhao, and G. A. Evans. 1987. Cosmid vectors for rapid genomic walking, restriction mapping, and gene transfer. Proc. Natl. Acad. Sci. USA 84:2160-2164.

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23. Leu, T.-H., and J. L. Hamlin. 1989. High resolution mapping of replication fork movement through the amplified dihydrofolate reductase domain in CHO cells by in-gel renaturation. Mol. Cell. Biol. 9:523-531. 24. Looney, J. E., and J. L. Hamlin. 1987. Isolation of the amplified dihydrofolate reductase domain from methotrexate-resistant Chinese hamster ovary cells. Mol. Cell. Biol. 7:569-577. 25. Looney, J. E., C. Ma, T.-H. Leu, W. F. Flintoff, W. B. Troutman, and J. L. Hamlin. 1988. The dihydrofolate reductase amplicons in different methotrexate-resistant Chinese hamster cell lines share at least a 273-kb core sequence, but the amplicons in some cell lines are much larger and are remarkably uniform in structure. Mol. Cell. Biol. 8:5268-5279. 26. Ma, C., T.-H. Leu, and J. L. Hamlin. 1990. Multiple origins of replication in the dihydrofolate reductase amplicons of a methotrexate-resistant Chinese hamster cell line. Mol. Cell. Biol. 10:1338-1346. 27. Ma, C., J. E. Looney, T.-H. Leu, and J. L. Hamlin. 1988. Organization and genesis of dihydrofolate reductase amplicons in the genome of a methotrexate-resistant Chinese hamster ovary cell line. Mol. Cell. Biol. 8:2316-2327. 28. Milbrandt, J. D., N. H. Heintz, W. C. White, S. M. Rothman,