Summary It has been almost twenty-fiveyears since Huberman and Riggs first showed that there are multiple bidirectional origins of replication scattered at -100 kb intervals along mammalian chromosomal fibers. Since that time, every conceivable physical property unique to replicating DNA has been taken advantage of to determine whether origins of replication are defined sequence elements, as they are in microorganisms. The most thoroughly studied mammalian locus to date is the dihydrofolate reductase domain of Chinese hamster cells, which will be used as a model to discuss thc various methods of investigation. While several laboratories agree on the rough location of the ‘initiation locus’ in this large chromosomal domain, different experimental approaches paint different pictures of the mechanism by which initiation occurs. However, a variety of new techniques and synchronizing agents promises to clarify the picture for this particular locus, and to provide the means for identifying and isolating other origins of replication for comparison. Introduction This essay will focus primarily on origins of replication in mammalian cells, the study of which has lagged far behind simpler organisms such as bacteria, plasmids, and viruses. There are several reasons for this, among them: 1) the sheer size and complexity of the typical mammalian genome (-6x109 bp partitioned among 20-50 linear chromosomes, compared to - 4 ~ 1 0for ~ the single circular E. coli chromosome); 2) the lack of workable genetic systems for identifying either the cis-acting elements or the cognate trans-acting protein factors involved in initiating DNA synthesis; and 3) the lack o f a reliable phenotypic ashay for detecting or assessing origin function. Despite these handicaps, significant inroads have been made in the last ten years in localizing mammalian origins. The next decade should yield information about the nature of these elusive genetic elements and the interacting proteins that effect initiation. Early General Studies on Mammalian DNA Replication Historically, studies on DNA replication in mammalian cells have followed many of the same experimental avenues that

were taken in bacterial systems (see ref. 1 for review). When [3H]thymidinebecame commercially available in the 1950’s. Howard and Peld2) used it to show that DNA synthesis in mammalian cells is restricted to a finite interval (the DNA synthetic or S period), just as it is in bacteria. The S period can occupy anywhere from 6 to 9 hr in a cell cycle lasting 1624 hr. [3H]thymidine incorporation was further used to show that there are many replicating chromosomal segments in operation at any moment in the S period, with individual segments replicating at characteristic times in S in a given cell type(3). Thus, sophisticated control mechanisms must be required to guarantee that each sequence in the genome is replicated on schedule, and that each sequence is replicated only once per cell cycle. Based on model prokaryotic systems. it seemed logical to assume that control would be exerted at individual initiation sites, which focussed interest at the molecular level. In 1968, Huberman and Riggd4) crossed the bridge from cytology to molecular biology by answering some of the most basic questions with the elegantly simple DNA fiber autoradiographic technique originally devised to study the replicating E. coli chromosome. In this method, cclls are briefly labelled with [’Hlthymidine, the cells are lysed, and the DNA fibers are stretched out on a microscope slide. After exposure to photographic emulsion, the silver grain tracks that result from replication fork movement during the pulse can be examined by light niicroscopy (the 20 angstrom DNA fibers themselves, of course, are too small to be visualized). The resulting patterns allowed several conclusions to be drawn: 1) replication forks move at about 3 kb per minute (a rate much slower than bacterial forks); 2) each chromosomal DNA fiber contains multiple growing points along its length; 3) most (but not all) replication forks occur as divergent pairs, indicating that they usually arise from bidirectional origins of replication: 4) initiation sites (origins) are spaced, on average, -100 kb apart, with a range of 15-300 kb; thus, there must be 20,000-50,000 origins of replication in a mammalian genome; 5) clusters of 5-10 adjacent origins often appear to fire simultaneously, suggesting that they might be activated coordinately. Thus, a host of information was obtained with one simple experimental technique. In a later fiber autoradiographic study, Hand(5)was able to provoke an increase in the percentage of unidirectional origins by inhibiting protein synthesis for extended time intervals. This result suggested that coordinated bidirectional fork movement from an origin occurs only when there are enough proteins (trans-acting factors) to ensure that both template strands initiate replication synchronously. Ainaldi and coworkerd6) also used the fiber autoradiographic technique to show that loci serving as initiation sites in one cell cycle are often reutilized in the next cell cycle, arguing that origins of replication must be defined sequence elements (although, strictly speaking, the autoradiographic technique cannot distinguish between two sites separated by less than 6-8 kb). These important experiments, as well as many others too numerous to mention, have laid the groundwork for all subsequent studies. However, they do not directly address the question whether origins of replication in mammalian cells

are. indeed, defined sequence elements analogous to those in bacteria and viruses. Necessarily, this question has had to await the development of recombinant DNA techniques that allow thc isolation of hybridization probes for analyzing specific initiation loci in chromosomes. As we will see, however, the tremendous advantage afforded by these techniques has so far opened the window on this question only slightly.

Studies on Defined Origins of Replication In vivo and in vitro labelling protocols Careful analysis of the replicating stretch of chromosomal DNA diagrammed in Fig. IA suggests several unique properties that can be taken advantage of to roughly localize origins of replication. For example, the bold lines in the figure are meant to indicate that cells can be labelled with a variety of specific precursors that aid the identification and/or isolation of nascent or recently replicated DNA. Sarkar and c~lleagues(~)labelled DNA synthesized in vitro at different times in the S period with mercurated dCTP and isolated the labelled DNA on sulfhydryl columns. The order of synthesis of more than 30 Chinese hamster genes was then determined by utilizing cloned cDNAs as molecular probes on the isolated fractions. This comprehensive study confirmed earlier suggestions(8’that each gene is synthesized at a characteristic time in the S pcriod in a particular cell type. The dense thymidine analogue, bromodeoxyuridine (BrUdR), has been used in several different approaches to isolate and identify nascent DNA. Schildkraut and coworke r ~ (labelled ~) exponentially growing mammalian cultured cells very briefly with BrUdR, and fractionated them by centrifugal elutriation on the basis of DNA content into different stages of the S period. Using cloned hybridization probes scattered at intervals over large. defined chromosomal loci A

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Fig. 1. Properties of replicating DNA. Panel A. Two bidirectional origins (0)are shown that have already initiated replication. The nascent DNA is shown as bold lines, with the arrowheads indicating the direction of nascent chain growth. The short Okazah fragments on the lagging side of each fork have not yet been ligated together in this diagram but would normally have a half life of only a few minutcs. Panel B . The DNA in this cell has been cross-linked with psoralen in vivo prior to entry into the S period. The vertical arrows show the position of replication forks at the time of release from an aphidicolinblock into rnediutn containing BrUdR. Replication fork progression is impeded by the first cross-links encountered. If origins initiation during the BrUdR labelling period, nascent DNA (bold lines) will be small and centered around origins. However, replication forks at distant locations will incorporate BrUdR only into high molecular weight DNA.

(e.g., the immunoglobulin heavy chain genes), they were able to determine the time interval in the S period when each cognate DNA sequence was synthesized and, hence, to determine the direction of replication fork movement through the locus in question. Although the resolution of this method is not great enough to identify the position of origins of replication with high precision, it did allow these investigators to show that the direction of fork movement through this locus changes after a chromosomal rearrangement that juxtaposes the c-myc gene to the immunoglobulin locus, presumably by bringing an activc origin into the region(I1). BrUdR was also used in a run-off replication assay lo determine the direction of fork movement through the globin and c-myc Exponentially growing avian cells were briefly labelled in vitro with BrUdR and the DNA was purified and digested with a restriction enzyme. The distribution of BrUdR in appropriate fragments was then determined. Since only completed fragments are analyLed in this method, more BrUdR will be concentrated in the origin-distal end than in the origin-proximal end of a given fragment. By analyzing several fragments from the relevant loci, it was possible to show that the direction of fork movement can change in a locus (e.g., globin) depending upon whether or not the gene is expressed in a particular cell type(I2). The method was also applied to the c-myc locus in human cells and suggested the presence of an initiation site upstream from the promoter(l3). Radioactive analogues of thymidine or thymine were first used to localize bacterial origins of replication by synchronizing temperature-sensi tive initiation mutants and then radiolabelling the first sequences replicated after release from the block (e.g.. ref. 12). However, a similar protocol would not be informative in mammalian cells because, even in a single cell, several thousand different origins of replication probably fire at the beginning of S. This would result in an uninterpretdbk smear of thousands of labelled fragments on a gel. To focus on only one defined origin, a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) was developed that has amplified one allele of the dihydrofolate reductase (DHFR) gene and flanking scquenccs more than 1,000 times(I3). The multiple amplicons occur as tandem repeats in homogeneously-staining chromosome regions (HSRs). Since the unit of amplification (amplicon) was deduced to be at least 150 kb in length(’”),it was reasonable to assume that it would contain at least one origin (subsequent molecular cloning of the ainplicon showed it to be -240 kb in length(l4!). This suggestion was confirmed by showing that when synchronized cells (see below) are briefly labelled with [3H]thymidine at the very beginning of the S period, labelling commences synchronously at multiple positions scattered all over the length of the HSRs(13). The advantages provided by the high copy number of thc DHFR amplicon have rendered this particular model replicon the best studied of any mammalian locus. Indeed, a minor cottage industry has been spawned in which several laboratories are attempting to determine the precise location and properties of the origin(s) of replication in the Chinese hamster DHFR locus by a variety or approaches. Each of these methods exemplifies a different aspect outlined in Fig.

1 A. However, it will become obvious that what each method sees depends upon the approach. Most interestingly, when genomic DNA from CHOC 400 cells is digested with a restriction enzyme and separated on an agarose gel containing ethidium bromide, the vcry high copy number of the DHFR amplicon in the CHOC 400 cell line produces a distinct pattern of amplified bands superiniposed on the background smear of single copy Since these fragments arise from an amplicon that is only onc-sixteenth the size of the E.coli chromosome (4,000 kb), it seemed possible to identify the restriction fragmcnt(s) containing the origin(s) of rcplication in the DHFR amplicon by labelling cells with [3H]thy~nidineat the beginning of the S period. To achieve the requircd synchronous entry into S, exponentially growing CHOC 400 cells were arrested in the G o period by starvation for the essential amino acid, isoleucine. The block was then reversed by refeeding complete medium containing the drug, aphidicolin, which inhibits at least three DNA polynierases(15).The rationale of this protocol is that cclls can traverse the GI period in the presence of the drug, arrive at the beginning of S, and probably initiate replication at origins. However, chain elongation should be inhibited by the drug. Once released from aphidicolin, only regions iiiiiiiediately flanking origins should be labelled by a short pulse of radioactive thymidine. CHOC 400 cells were subjected to this protocol and labelled in the first 30 min of the S period with high levels of [ “C]thymidine. When the genomic DNA was purified, digestcd with EcoRI, and separated on an agarose gel, labelling was restricted primarily to only four out of more than 50 restriction fragments from the arnplicon(l6).These fragments were 11. -6.2, and 3.5 kb in length (the 6.2 kb band was later shown to bc a 6.2h.1 kb doublet). This result showed for the first time that in a defined region of the chromosome, DNA replication initiates within preferred sequences. To determine whether the four earlylabelled fragments (ELFs) arise from one or more than one origin, the 11 and 6.2 kb amplified bands were excised from a gel, labelled in vitro with [’2P]dCTP, and used to probe a cosmid library prepared from CHOC 400 genomic DNA(I7). All of the clones recovered arose from a single region of the amplicon and defined a 28 kb initiation locus downstream from the 3‘ end of the DHFR gene (Fig. 2A)(I7’. The resolution of the in viva labelling approach was limited in part by background labclling of single copy sequences from the rest of the genome, and also to less-than-ideal synchrony (i.e., cells enter S over a 15-20 min window, whereas replication forks move at -3 k b h i n ) . At the time, there sirnply was 110 better way to achicve a higher degree of synchrony. However, it was possible to eliminate the background by a clever in-gel renaturation technique developed by Roninsoni18)in which the gel is subjected to alternating cycles of the following treatments: denaturation, renaturation for a time short enough to allow only the amplified bands to rehybridize, and S1 nuclease digestion to rid of singlestranded, single copy sequences. Early-labelled DNA from CHOC 400 cells was subjccted to this protocol, the radioactivity of individual fragments was

quantified by densitometry of X-ray films, and relative specific activity was plotted as a function of map position. This much more pristine view again suggested that early labelling in the amplicon is confined to a broad region at least 28 lib in length(I9) (Fig. 2B). However, even with this improvement in the signal-to-noise ratio, the resolution is still limited because many of the amplified fragments in the gel are part of doublets or triplets, the members of which could map different distances from the origin. Thus, these doublet and triplet bands provide no useful information for the graph and the resolution is lowered accordingly. To get around this problem, the experiment was modified to include a 300-fold excess of a non-labelled cosinid containing the entire initiation locus. The in-gel hybridization times were drastically cut so that only those sequences from the ainplicon that are driven into duplexes by excess cosmid are recovered as double-stranded DNA. This more precise cvaluation actually suggested the presence of wo rough peaks of labelling separated by about 22 kb within the prcviously identified 28 kb initiation locus (coinciding roughly with fragment F’ and the right end of C in Fig. 2A)(”). Thus, there appcared to be two closely-spaced origins in this zone (henceforth termed ori-p and ori-y; ori-a lies 200 kb upstream in thc much larger amplicon of another drug-resistant Chinese hamster cell line). Burhans et u L . ( ~ ” ) adopled a diffcrent approach in an attempt to localize more precisely the initiation site(s) in the amplified DHFR domain. Synchronized CHOC 400 cells were labelled either in vivo or in vitrn with radioactive precursors, the DNA replicating in the first few minutes of the S period was adsorbed to BND-cellulose(21),and the partially single-stranded DNA was selectively cluted with caffeine. Alternatively, unlabelled DNA was purified from early S phase cells, purified on BND-cellulose, and labelled in vifro by nick-translation. These ‘early-replicating’ DNA fractions were then used as hybridization probes on subcloned XbaI fragments from the DHFR initiation locus. Each Fraction preferentially illuminated a single 4.3 kb XbaI fragment located in the 6.2 kb EcoRI fragment (F’) near the left end of the 28 kb initiation locus (see map, Fig. 2A). Thus, this study suggested that thcre is a well-defined initiation locus lying about 15 kb downstream from the DHFR gene that coincides with the on-p region detected by in-gel renat~ration(’~). Ori-y was not detected, in part because clones from that region were not analyzed. It is interesting to note that END-cellulose chromatography results in a 30-fold purification of replicating DNA, but only -3% of the DNA eluting from the column with caffeine actually contains forks(”). Thus, it is peiplexing that thc same results were obtained by Burhans et (11. whether the probc was labelled intrinsically in v i w (and which therefore must have been replicating) or in vitro by nick-translation with [32PP]dCTP(in which case only -3% of the DNA is replicating).

Identifying initiation sites based on distinct architectural properties Some insight into this problem was afforded by another study on the DHFR locus in which cross-linking was used to

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Fig. 2. Linear map of the 240 kb DHFR amplicon. Panel A shows the DHFR and 2BE2121 genes and two preferred zones of carlylabelling(21)(the centers of which are labelled with an I). The direction of transcription of the two genes is indicated with arrows above the scale. A 40 kb region encompassing the initiation locus is expanded below tn show EcoRT fragments. Note that the 4.3 kb XbaI fragment discussed in the text is located within the 6.2 kb EcoRl fragment. The hatched box represents a prominent matrix-attachment region (MAR)("). Panel B plots the relative specific activity of different fragments ax a function oftime of labelling and tnap position in the amplicon. The solid, open, and hatched circles represent the data for samples that were labelled for 30, 60, and 90 min after release from aphidicolin (see text). Note that the F+F' point i n the 90 tnin sample is lower than it should be because of the linearity of the film was exceeded in this particular experiment. Shown below the graph is a series of recombinant cosmids that was used to construct the 240 kb map ofthe aniplicon.

arrest replication forks near origins(13).Either CHOC 400 or parental drug-sensitive CHO cells were synchronized at the Gj/S boundary by the isoleucine/aphidicolin protocol described above. The cultures were incubated with psoralen and were irradiated with ultraviolet light to induce crosslinks at 2-5 kb intervals. Thc cells were lhen released from the aphidicolin block and allowed to synthesize DNA in the presence of a specific label for one hour. The DNA was purified and denatured lo release nascent fragments from the template. The smallest fraction of labelled nascent DNA was then harvested from a sucrose gradient and used to probe clones from the amplicon. The theory of this experiment is that small labelled DNA can only be derived from the immediate neighborhood of origins, since label incorporated at growing forks that were far away from origins will end up in high molecular weight

DNA (Fig. 1B). When the labelled low molecular weight fraction (termed X-DNA) was used as a hybridization probe on cosmids and plasmids from the DHFR amplicon, it preferentially illuminated the 6.2/6.1 kb doublet and 11 kb EcoRI fragments, which contain ori-p and ori-y. respectively. [Because the 3.5 kb and 1.5 kb fragments in between them are relatively small and thc signals correspondingly weakk,it is not clear to what extent they were represented in lhc earlyreplicating X-DNA probe; see map, Fig. 2A.l Surprisingly, however, the 0ri-P locus (but not the ori-y locus) was also illuminated by the X-DNA fraction from parental CHO cells, which contain only two copies o f the DHFR locus. This result strongly suggested that there is a repetitive element in or ncar ori-P that is present in both the CHO and CHOC 400 genomes. It further suggests that had Burhans ef al.(20)used BND-cellulose-purified CHO DNA as

A different version of a template strand-switching assay a control in their hybridization experiments, they would have has been used by Burhans et L Z ~ . ( ~ to ’ ) attempt to localize also detected preferential hybridization to ori-p. replication start sites more accurately in the DHFR locus. Indeed, in a subsequent study, it was shown that thcre arc CHO or CHOC 400 cells were synchronized with aphidi(among other things) two Alu-like sequences and two very Colin, cells were permcabilized with NP40. and DNA syntheA/T-rich elements in the ori-p region, both of which are sis was allowed to proceed in vitro in the presence of BrUdR highly repcatcd in the CHO gen~me(‘~.~~)). It was also shown and [32P]-labcllednucleoside lriphosphates. Okazaki-sized that these elements (or some precise combination thereof) are fragments were purified from an alkaline agarose gel, the actually enriched in early-replicating DNA, but that their BrUdR-labelled DNA was precipitated with anti-BrUdR synthesis is not confined to early S(2s).Thus, fomially speakantibodies, and the resulting material was used as a hybridizing, since both the X-DNA(l?)and BND-cellulose-purified ation probe on the separated strands of selected M13 subare genomic in origin, it cannot be proven that the clones from the region. repetitive element in the 0ri-P region is active p e r se. Thus. This fraction showed a rather strong differential template experiments in which genornic DNA fractions are used as bias on either side of the ori-p locus. In fact, there appeared to probes are difficult to control and the results are subject to be a six-fold difference in template bias in each of the two differing interpretations. neighboring -250 bp HaeIIl fragments that are centered over Given the uncertainties inherent in the results of the previori-P (although, before corrections were made for backous two approaches. it was important to view the DHFR ground hybridization to elements in the M13 clones theminitiation locus from different perspectives. A novel selves, the bias was only -1.8-fold). Since clones from the approach was taken by Handeli et (modelled after ori-yregion were not utilized in this study, a complete picture studies on SV40(*’)), which relies on the fact that the small of the strand bias throughout the locus is not yet available. Oka7;iki fragments involved in lagging strand synthesis(”) Taken at face value, it therefore appears that at least one switch templates a1 origins of replication (also, interestingly, at termini). CHO cells were incubated fur -24 hr in emetine replication initiation start site in a mammalian chromosome has been localized to within a few hundred base pairs. How(a protein synthesis inhibitor that has been shown lo sclectively inhibit synthesis on the lagging strand ever, two totally independent approaches indicate that the The reduced amount of leading strand DNA that is synthestory may not be so simple. Brewer and F a ~ ~ g r n a ndeveloped (~~) a two-dimensional (2sized under thesc circumstances is labelled with BrUdR and. after isolation, it can be probed with the separated + and D) gel electrophoretic technique that separates replicating restriction fragments from non-replicating fragments on the strands of M13 subclones from a region of interest to dctcrmine the position at which template strand switching occurs basis of diffcring hydrodynamic properties (Fig. 3). Mole(see Fig. 1A). cules are separated in the first dimension largely according to When applied to the DHFR locus in exponentially growmolecular mass, which will necessarily go from In for the ing CHO cells, this method confirmed the presence of two non-replicating version to just less than 2rz. In the second strand-switching zones (origins?) roughly positioned in the dimension, the fragments are separated under conditions that previously defined ori-j3 and ori-y regions. However. the accentuate differences in shape. Depending on whether a hybridization signals became so insignificant as the probes fragment is replicated actively from its own origin (i.e., conapproached the center of each of these loci that it was not tains a bubble as in Figs. 3B and 3C) or passively (contains possible to localize the origins more precisely than f 8 kb. either a single fork or a double-forked terminus, as in Figs. However. within limits, two very different m e t h o d ~ ( ’ ~ 3 ~3A ~ ) and 3D), it will migrate in a characteristic pattern on the suggested the presence of two closely-spaced preferred start gel. In the actual experiment, the DNA from actively dividsilcs or zones in the DHFR locus. ing cells is carefully isolated, digested with a restriction Handeli et a/. also obtained evidence for a terminus enzyme, and separated on the gel. The digest is transferred to located just upstream from the DHFR gene (sce map, Fig. a membrane and hybridized with several probes from a 2A), but this result was not confirmed in an independent region suspected to contain an origin. study in which DNA labellcd with BrUdR in the early S When the DHFR initiation locus in synchronized CHOC period was used to probe cosmids from the IIHFR locus; in 400 cells was analyzed by this a surprising result this study, as the BrUdR pulse time increased, forks appeared was obtained. Not only was a centered bubble pattern to move rnonitonically away rrom 0.i-p through the gene and observed in a fragment containing ori-j3, but in every other well beyond the proposed terminus at the 5‘ end(30). fragment in the 28 kb initiation locus defined by earlier in However, the results of this strand-switching approach vivo labelling studies(’5v19).Furthermore, every fragment have another peculiar feature that is not easily explained, i.e., that contained a bubble pattern also contained a complete adjacent fragments from the same side of the ‘origins’ in this single fork arc. Subsequent studies have shown, in fact, that locus display very differcnt degrees of strand bias (based on bubbles can be detected in every restriction fragment the strength of hybridization signals), when they would be between the DHFR and 2BE2 I2 I genes, regardless of the expected to be identical on the same side of the origin in a reslriction enzyme used (albeit at lower levels in the 10 kb at given replicon. We suspect that this result relates to repetitive each end of this zone; see map, Fig. 2A). Fragments outside sequence elements in the genome that replicate in early S and of the initiation locus displayed only single fork arcs, whose replication pattern (or hybridization patt.ern) may The most likely explanation for this phenomenon is that itself demonstrate a template strand bias. initiation of nascent chains can occur at any of a number of

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dimension of this gel system is idcntical to that of the BrewedFangman technique, separating fragments according to mass. However, the second dimension of the gel is run in alkali, which releases nascent strands that m i p t c according to size. The digest is transferred to a membrane and hybridized with appropriate probes. The diagram in Fig. 4 shows that the nascent strands centered over an origin range in length from a few nucleotides to replicon-sized. A small probe from one end of a restriction fragment in a replicon will detect only the largest nascent strands if the probe is from the origin-distal end, but fragments of all sizes when it is from the origin-proximal end. The direction of fork movement through any given fragment can thus be determined, and by 'walking' along an extended locus of interest with several different probe pairs, the rough location of an origin can be determined. An analysis of the DHFR locus by this method showed clearly that forks move in both directions in fragments within the initiation This would result from initiations occurring at different sites within the initiation zone in different amplicons or cells, Conversely, forks move only away

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two-dimensional gel electrophoretic method of Brewer and Fangman(32).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 intermedkdtes. Panel A. A complete simple Y or fork arc (0) resulting from a fragment that is replicated passively from an outside origin. Curve u represents the diagonal of non-replicating fragments from the genome as a whole. B. The pattern obtained when a fragnient with a centered origin of replication is probed (curve c). Bubbles migrate more slowly at all extents of replication than do forks in a fragment of equal mass (bj. C . The presence of an off-centered ongin in a fragmcnt gives rise to an incomplete bubble arc ( c ) ,which then reverts to the Pork arc when the bubble expands beyond the righthand restriction site. resulting in a fork arc 'break'. D. When two forks approach each other in a fragment either symmetrically or asymmetrically, curves r and d are obtained, respectively. If there i s a fixed terminus in a fragment, the collected X-shaped structures would result in a concentrated spot somewhere on curvef. Recombination structures would also fall along curvcf:

sites scattered over the entire initiation locus. Thus, in onc amplicon, a fragment will be replicated passively by a single fork emanating from an initiation site in a neighboring fragment, but in another amplicon, it will contain a bubble resulting from an internal initiation. The same picture was obtained whether log or early S phase CHOC 400 cells were examined(33),arguing that artifacts of the synchronization protocol are an unlikely cause of t h s unexpected result. Delocalized initiation was also observed in synchronized CHO cells, arguing that it is not an artifact of the amplification process per se(22J,.Finally, replication bubbles are observed in the initiation locus only during the early S as would be expected of an early firing origin. To examine this phenomenon by an independent method, a second 2-D gel was used to determine the direction of fork movement through this locus. The first

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from the initiation locus in flanking regions in which initiation was not detected by the 2-D gel method of Brewer and Fangman (e.g., in the DHFR and 2BE2121 genes). Yet another method for measuring the quantities and sizes of nascent strands in a chromosomal locus has been developed that use^ the polymerase chain reaction to amplify the signal from nascent BrUdR-substituted DNA from single copy loci in exponentially growing cells(36). While the method has promise, initial results were disappointing since, in one report, only three probes were used to analyze thc Cmyc locus, one of which was useless because of its proximity to a repetitive sequence element(36).Nevertheless, when the same method was applied to the ori-0 locus, the results supported the earlier suggestion that initiation occurs in a relatively circumscribed region centered over ori-p(”7). Again, however, only three probes were used, and it is known that there are many repetitive sequence elements in this region. Thus, when studied by different techniques, initiation of replication in the DHFR locus appears to occur either at relatively fixed sites or at many random sites scattered over a broad zone. Since it is virtually impossible to quantitate accurately the number of initiations occurring per unit length of DNA by 2-D gel methods, it is not clear whether slightly more initiations occur over the ori-p and ori-y loci than in flanking regions. If so, this would reconcilc the finding of two low peaks of early labelling in the in-gel renaturation experiments(I9) with the 2-D gel results(33). It is almost inconceivable, however, that the six-fold template bias observed by Burhans et within the two halves of a single 500 bp fragment in the ori-p region, which suggests a fixed origin in this region, could be missed in the 2-D experiments. Thus, there is a discrepancy between the two sets o f data that cannot be reconciled on quantitative grounds alone. The difference couldrelate to the fact that, in the study of Burhans et ~ l . ( ~ lthe ) , Okazaki-sized fragments uscd to determine template strand bias are synthesized in vitro, whereas the 2-D gel studies of Vaughn et a1.(33)are performed on DNA replicating in vivo. It is conceivable, for example, that the ‘origin of bidirectional replication’ (OBR; defined by Burhans et u Z . ( ~ ~ as ) the strand-switching site) really corresponds to (or is close to) the true cis-regulatory element controlling the function of this origin. I n vivo,in the context of the chromatin, the region might initially melt at the OBR, which then serves as a loading site for replication complexes that can course up and down the helix for several kilobases and initiate RNA primers at random sites (for stochastic reasons, more often over the OBR itself). However, in vitro, it may be that chromatin structure is altered (relaxed supcrcoiling, perhaps?) and initiation is confined to regions in the immediate neighborhood of the proposed OBR. Another difference that might play a role is the static versus kinetic nature of the 2-D gel methods and the in v i m strand-switching assay, respectively. In addition, it is interesting that all of the methods that suggest very localized initiation in the DHFR locus (including the strand-switching assay) examine replication intermediates labelled with BrUdR(2’,26,31.371.

Thus far, no other mammalian chromosomal locus has been examined by such a large variety of techniques as the DHFR domain in CHO cells. However, other properties of replication bubbles have been taken advantage of to attempt to identify and isolate other origins of replication. ZannisHadjopoulos et a1.(38’made the initial observation that small nascenl DNA fragments centered over the SV40 origin of replication can be recovered in quantity if replication intermediates are isolated and the nascent strands are caused to branch migrate and extrude by incubation at elevated temperature. This method was extended to monkey cells released from an aphidicolin block in hopes of isolating origins of replication that tirc in the first two minutes of the S Several of these sequences have been cloned and sequenced, and a few clones even appear to be capable of limited autonomous replication after transfection into animal

cell^'^^^. However, the sequences had little in common with each other and the method has not been widely accepted, at least in part because it is a ‘shot-gun’ approach. If the results of the two-dimensional gel analyses outlined above are valid, then the extruded material that was cloned could be located anywhere within a 15-20 kb zone surrounding a bonafide cisregulatory origin o f replication, and only a small subset might derive from a cis-regulatory origin sequence itself. The lcsson learned from the Chinese hamster DHFR locus suggests that an extensive analysis of one or two of these chromosomal fragments along with their surrounding sequences will be required to determine whether they behave in all respects as origins in their appropriate chromosomal context. A last approach to identifying replication origins is based on observations that when DNA is labelled with radioactive thymidine at the beginning of the S period, the DNA that is tightly associated with the nuclear scaffolding or matrix is the first to be labelled(41).Furthermore, this label remains with the matrix, whereas a brief label delivered to log cells is ultimately chased away into the DNA loops. Based on these and related data, complicated models have bccn developed in which DNA is spooled through a matrix-affixed replication complex as it is synthesized, with the origin remaining permanently attached(”). Thus. matrix-associated DNA has been isolated from cells at the beginning of S, and these sequences have been cloned and sequenced(43).To date, this shot-gun approach has yielded little information of either a general or specific nature about origins. In this regard, however, it is interesting to note that a sequence located approximately midway between ori-0 and ori-y in the DHFR locus appears to be permanently and tightly attached to the matrix(44J.Could it be that the real origin of replication in the DHFR locus is not at ori-p and/or ori-y at all, but rather in between at the matrix-associated region?

Improved synchronizationprotocols A problem alluded to earlier is the inability to obtain a truly synchronized population of mammalian cells. Even if suitable temperature-sensitive initiation mutants were available, such mutants have generally not been as useful in studies on

mammalian cells as they have in bacteria, primarily because the viable permissive and non-permissive temperatures are not different enough to allow clean arrest andor release. By the same token, none of the inhibitors available until now inhibits initiation per se. The most useful ones all inhibit chain elongation in one way or another. Thus, by definition, if one traps a population of mammalian cells traversing G1 with drugs such as hydroxyurea, methotrexate, 5'-fluorodeoxyuridine, or aphidicolin (one of the more efficacious inhibitors), it should be understood that initiation - the event one is trying to study ~. probably occurs at many early firing origins in the presence of these drugs. Only recently in our laboratory have we begun to appreciate how leaky these agents actually are. The 2-D gel technique of Brewer and Fangman shows clearly that CHOC 400 cells arrested in high concentrations of aphidicolin nevertheless have significant numbers of replication forks in regions several kb away from the ori- p h i - y region(44). However, a most exciting recent finding is a drug that appears to inhibit mammalian cells just prior to initiation. Lalat~de(~') has investigated the action of the plan1 amino acid, mimosine, by fluorescence-activated cell sorting, and has concluded that it affects cells in the late G I phase a few hours before the S period. We have utilized mimosine to collect CHOC 400 cells after release from isoleucine-free medium and, using 2-D gels, have shown that virtually no initiation occurs in the DHFR locus in the presence of the drug. Upon release, a very synchronous burst of initiation e n s ~ e s ( ~ Recent ~ . ~ ~ )work . in our laboratory suggests that milnosine may- actudlly inhibit initiation pel-se, as opposed to the Gj/S Thus, in our minds, this simple compound has opened a new era in the study of control of replication in mammalian cells. -4lthough it is not yet clear what mimosine does at the molecular level, it should be the drug of choice in studies on the cis- and trans-acting factors that control initiation in mammalian chromosomes (happily, it is also 100 times less expensive than aphidicolin).

Perspectives for the future Many novel approaches will be stimulated by the availability of new inhibitors such as mimosine. powerful 2-D gel techniques, and very sensitive and defined in vitro replication assays. In addilion, a host of factors that are being identified in somewhat tangential studies, e.g., oncogenes, anti-oncogencs, cyclins, etc., may prove to be directly or indirectly involved in initiation reactions. It seems likely lhat studies in the next few years will unmask the true nature of the DHFR initiation locus. It is also likely that many new origins will be localized and characterized. Comparisons among them will undoubtedly shed light on those elements required for origin function, just as the study ofmany genes eventually led to the identity of conserved regulatory sequence elements required for initiation of transcription. Acknowledgements Work in the author's laboratory in the last ten years was sup-

ported by grants from the National Institutes of Health and the American Cancer Society. I would like to thank all of the members of my laboratory, past and present, for their devotion to the replication project and their always stimulating ideas and discussions.

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