Origins

of replication:

Joyce 1. Hamlin,

timing

James P. Vaughn,

and chromosomal Pieter A. Dijkwel,

position

Tzeng-Horng

Leu

and Chi Ma University

of Virginia

School

of Medicine,

Charlottesville,

Virginia,

USA

Several new methods have been used to localize replication initiation sites in mammalian chromosomes. The results of these studies argue strongly for the presence of defined sequence elements that function much like the origins in the genomes of simple microorganisms. However, relatively disparate results from in vivo and in vitro studies suggest that initiation reactions in mammalian chromosomes may have unique features, possibly related to a more complicated chromosomal architecture. Current

Opinion

in Cell Biology

Introduction

A typical mammalian cell duplicates its chromosomes during an 8 h interval in a cell division cycle lasting 16-36 h. The replication process in mammalian cells is a formidable one: more than 2 m of DNA fiber is packaged into multiple chromosomes, and both the DNA and a sophisticated array of chromosomal proteins must be duplicated with fidelity to ensure that the structural and functional integrity of each chromosome is maintained. Replication occurs within the context of an elaborate nuclear architecture consisting of a proteinaceous scaffolding or matrix to which the DNA is attached at periodic intervals, facilitating supercoiling and higher-order chromatin structure. Autoradiographic studies on mammalian chromosomes show that replication is a multifocal process in which some regions replicate early in the DNA synthetic (S) period and others replicate late [ 1,2]. It is reasonable to assume that the control of replication in mammalian chromosomes will be exerted at the level of individual initiation points (origins>. There are approximately 50000 origins per diploid nucleus [2], each of which could be unique. Only since the advent of recombinant DNA technology has it been possible to analyze replication intermediates in a defined chromosomal domain, and thus to address the question of whether origins in mammalian chromosomes are fixed genetic elements. Experimental approaches designed to localize and characterize origins of replication are patterned after studies in bacterial systems, and fall into three general categories: in viva labelling protocols designed to detect the positions along the chromosome at which nascent chains are initiated; identification of fragments that are capable of autonomous replication and which, by de-

414

1991,

3:414-421

duction, must contain the c&acting elements necessary for initiation; and dismemberment of the cell into constituents in in zJitr0 replication systems, with the aim of identifying both the cis and transacting components responsible for initiation. These approaches have been particularly successful in identifying origins in the simple eukaryotic organism, Saccbaromyces cerevisiue, and have contributed enormously to our understanding of the replication process in animal cells, particularly the enzymology involved in nascent chain elongation. Some of the relevant work published in the last year suggests, however, that models based on simple systems such as bacteria, yeast, and viruses may begin to break down for mammalian systems. This review will focus exclusively on the quest for, and the characterization of, mammalian chromosomal origins of replication, particularly studies on the dihydrofolate reductase locus in Chinese hamster ovary (CHO) cells, which is growing in popularity. The reader is directed to several excellent, recent reviews for discussions of mammalian viruses, yeast, and other eukaryotic organisms [ 3-51. localizing origins intermediates

replicating

DHFR-dihydrofolate

reductase; MAR-matrix-attached PCR-polymerase chain

@ Current

Biology

replication

The diagram in Fig. 1 illustrates the expected intermediates in the vicinity of a genetically tied, bidirectional origin of replication, based on the models of .&%ericJ& coli and simian virus (SV) 40. After ligation of the shortlived Okazaki fragments that characterize lagging-strand DNA synthesis, a collection of nascent fragments of different lengths should be found, each of which is roughly centered around the origin. If methods could be devised to analyze either the distribution of nascent chains, the direction of fork movement, or the structure of the double-

Abbreviations sequence; BrUdR-bromodeoxyuridine;

ARS-autonomously

by characterizing

region; OBR-origin reaction; SV-simian

Ltd ISSN 0955++74

CHO-Chinese hamster ovary; of bidirectional replication; virus.

Origins

stranded replication intermediates in a particular locus, an origin could be localized relatively precisely.

I

1

I

I

,

Fig. 1. A hypothetical bidirectional origin (0) is shown in a region of the genome represented by contiguous restriction fragments A-C. Replication intermediates are shown below as a symmetrically expanding bubble containing a spectrum of centered, nascent fragments. Replication intermediates in restriction fragments A and C would always be single-forked structures, whereas intermediates in fragment B would contain either single or double-forked structures. Except for the top two, it is seen that the Okazaki fragments (represented by dashed lines) switch template strands at this fixed origin of replication.

However, in an asynchronous population of cells that doubles every 24 h and at an elongation rate of 3 kb min- 1 [2], a 3 kb restriction fragment will contain a replication fork less than 0.1% of the time. Thus, attempts to analyze replication intermediates at a suspected origin using specific hybridization probes on Southern blots are relatively difficult without either amplifying the signal emanating from an origin or employing synchronizing regimens that substantially increase the percentage of replication intermediates in the locus at the time of sampling. One way to amplify the signal from a given initiation site (if origins are fixed) is to study replication intermediates in the amplified domains (amplicons) of drug-resistant cells. The 240 kb dihydrofolate reductase (DHFR) amplicon from the methotrexate-resistant cell line, CHOC 400, is amplified approximately 1000 times, and has been cloned in its entirety in overlapping cosmids [6]. The replication pattern of the amplified locus has been studied in several laboratories by a variety of methodologies [7-121, and there is substantial in vim labelling evidence for the presence of two roughly defined zones of initiation (on-j3 and on-y) separated by approximately 20 kb lying downstream from the 3’ end of the DHFR gene [9,10,12]. In only one of these studies was it possible to localize initiation zones in the single-copy DHFR

of replication

Hamlin, Vaughn, Dijkwel, Leu, Ma

locus in drug-sensitive CHO cells [ 121: a switch between leading- and lagging-strand synthesis occurs at origins of replication, and the rough location of strand-switching was determined by taking advantage of the observation that nucleosomes partition to the leading strand of replication under conditions of limited protein synthesis [ 131. In a novel approach, the polymerase chain reaction (PCR) has been used to detect and amplify bromodeoxyuridine (BrUdR)-labelled nascent chains in the vicinity of the putative upstream origin (or@) in the DHFR locus in CHO cells [7,14*]. These data support earlier suggestions that a bidirectional origin resides approximately 15 kb downstream from the 3’ end of the DHFR gene (S-121. The PCR amplification method was also used to study nascent chain distribution in the 5’ flanking region of the c- myc gene in human cells [ 151. The results of the latter study suggest that an origin resides roughly 1.5 kb upstream from exon 1, in a zone encompassing fragments reported to replicate autonomously in human cells [ 16**,17*=]. This PCR-based method is attractive because it is sensitive enough to detect single-copy origin regions, even in nascent strands in exponentially growing cells, obviating the need for cell-synchronizing regimens. Apparent disadvantages are that the resolution is limited to a 2-3 kb region around an origin, and repetitive elements in the genome residing near sequences used as probes can interfere with the analysis in some cases (even if the probes themselves are not repetitive) [ 151. In addition, in both reports, the approximate positions of the initiation sites were already known from work in other laboratories, and only three or four probes were used in each of the PCR studies cited above to relocalize these origins. It will be important to show that the technique can successfully identify initiation sites in a region of DNA whose origins have not been previously mapped. The high copy number of the DHFR amplicons (1000) in CHOC 400 cells has facilitated analysis of replication in this locus by several different methods in the past, but most recently by two complementary twodimensional gel electrophoretic techniques that were developed to study replication intermediates in the yeast genome [l&19]. In the neutral-neutral method [18], restriction fragments containing single replication forks can be distinguished from fragments containing replication bubbles (origins> on the basis of anomalous migration in the gel. In the neutral-alkaline method [ 191, the direction of fork movement in a chromosomal region can be ascertained by examining the distribution of nascent fragment sizes by two-dimensional gel analysis. In both methods, by hybridizing sequentially with probes that recognize a series of restriction fragments in a genomic region of interest, the position of an origin can be determined. When the two methods were used to analyze the replication pattern of the amplified DHPR domain in synchronized CHOC 400 cells, the results did not fit the simple picture of a single, tixed initiation site lying downstream from the DHFR gene [200*]. Rather, replication bubbles were detected in many contiguous and overlapping fragments spanning more than 30 kb of DNA and encompassing both of the initiation zones previously detected in this locus [9,10,12]. Furthermore, replication forks were seen

415

416

Nucleus

and gene expression

to move in both directions in all of the rest&ion ments in this broad initiation zone [20**].

frag-

Thus, two rather tierent two-dimensional gel methods both lead to the heterodoxic suggestion that initiation occurs at many random positions scattered over a 30-35 kb region in the DHFR locus. Experiments on exponentially growing CHOC 400 cells also suggest the presence of many initiation sites scattered over a broad region [20-l. Thus, the synchronizing regimen per se (which uses aphidicolin to prevent’entry into the S period after reversal of a Gl block) apparently does not induce an aberrant mode of initiation. In addition, recent studies in which the neutral-neutral two-dimensional gel-mapping method has been adapted to the single-copy DHFR locus in CHO cells suggest that delocalized initiation is not unique to amplified DNA [ 211. A similar phenomenon seems fo occur in the amplifying chorion locus in the follicle cells of developing Drmopbila embryos. The neutral-neutral twodimensional gel-mapping method shows clearly that initiations occur in more than one fragment in the native chorion locus, and outside of the actual c®ulatory element itselfwhen it is transposed to a new chromosomal location [22-l. The strengths of the two-dimensional gel mapping methods are that they analyze replication intermediates fairly directly in vivo and can be used on either exponentially growing or synchronized cells. The latter attribute allows questions to be asked about origin usage. For example, the neutral-alkaline method has been used to show that replication forks move almost exclusively from 3’ to 5’ through the DHFR gene 30 min after entry into the S-period in synchronized CHOC 400 cells, by which time a fork could not have moved out of the amplicon in which it originated. However, forks move in both directions through the gene in exponentially growing cells UP Vaughn et al, unpublished data). This result argues that only some of the potential ‘origins’ in the DHFR amplicons of CHOC 400 cells actually fire, with the result that many amplicons are replicated passively by forks emanating from origins in adjacent amplicons. The neutral-neutral replicon mapping method was also recently used to show that only some of the potential origins are active in a cloned oligomeric bovine papilloma virus [23]. Both two-dimensional gel mapping methods have disadvantages. For example, it is difficult to accurately measure the relative numbers of forks and/or bubbles in different fragments. Thus, it has not been possible to completely reconcile in vivo labelling data suggesting the presence of two closely spaced zones of labelling in the DHFR domain [9,10,12] with the two-dimensional gel results that argue for a broad zone of initiation encompassing both of these zones [20-*I. In addition, both methods are new, the gel patterns are complex, and their interpretation is necessarily deductive. It is therefore conceivable that some of the observed patterns that are interpreted in the light of the intermediates expected on the basis of bacterial and viral models, may actually represent novel structures arising from a mode of replication unique to mammalian chromosomes. Finally, because of the extreme complexity of mammalian genomes and the small percentage of any given sequence that is replicating

at the time of sampling (even in synchronized cells), an enormous quantity of cells must be harvested for each analysis. An in vitro method for identifying initiation sites has recently been adapted from the original studies of Okazaki [24] and has been applied to an analysis of the DHFR locus in CHO cells [25-l. The method depends upon the observation that lagging strand Okazaki fragments switch template strands at an origin of replication. Small nascent DNA fragments were isolated from synchronized CHO cells labelled in vitro with BrUdR and 3*P-dCTP, and were then hybridized to the separated plus and minus strands of paired Ml3 clones containing fragments from the previously defined upstream ori- locus in the DHFR domain [25-l. Surprisingly, the majority of initiations in vitro seemed to occur in a narrow zone of approximately 500 bp centered within the previously defined or@ region. The same result was apparently obtained with both synchronized and logarithmically growing CHOC 400 cells. Taken at face value, this important study suggests that initiation at chromosomal origins is, in all respects, very much like that observed in simpler model systems such as SV40. Several features of the Okazaki strand-switching assay are quite remarkable. The most surprising thing is that sign& cant amounts of initiation appear to occur at this origin in lWo. This follows from the fact that the assay measures labelled fragments that hybridize to the 500 bp region containing the putative origin only if they are labelled during the in vitro replication reaction. Thus, initiation is occurring in the absence of any protein synthesis or added initiation proteins. It is also surprising that there are enough Okazaki fragments of sufficiently high specific radioactivity to actually record a hybridization signal at the ‘origin’ on the slot blots used to detect hybridization. The maximum number of Okazaki fragments there could possibly be in a hybridization probe prepared from 2 x 108 cells (the number used in this study) is 4 x 108 (two per cell), if every origin in this locus contained an Okazaki fragment at the time of sampling. This amounts to a respectable 2OOpg of specific probe if the fragments are 500 nucleotides long. However, the amount of probe recovered would be considerably reduced if all origins did not fire relatively synchronously during the 1.5 min of the in vitro replication assay, and this kind of synchrony is simply not attainable with cultured cells. In fact, the data show clearly that many replication forks had travelled more than 60 kb from the proposed origin of bidirectional replication (OBR) by the time of sampling, arguing that initiation had already occurred at many origins during sample preparation and probably during the aphidicolin block itself. However, there appears still to be enough probe to give a specific and strong signal on the dot blots used to detect hybridization. It is difficult to reconcile the results of this strand-switching assay with some of the in vi00 studies on the ori-j!l locus. The linding that Okazaki fragments appear to switch template strands in a narrow zone even in exponentially

Origins

growing CHOC 400 cells [25**] is particularly thoughtprovoking, because two-dimensional gel analysis on log cells indicates that many amplicons are replicated passively by forks from adjacent amplicons (JP Vaughn et al, unpublished data). This phenomenon would necessarily scramble the leading and lagging strands among amplicons so that there would not be any strand bias for Okazaki fragment hybridization. What could be the reason for the discrepancy between the in vitro strand-bias experiments [25”], which suggest that initiation in the DHFR domain occurs almost exclusively within a 500 bp zone, and the in vivo twodimensional gel methods [20**1, which suggest that initiation occurs at many random sites scattered over a 30-35 kb zone? An obvious possibility is that one or the other method (or its interpretation) is fallacious. For example, it has been suggested that the number of initiations occurring in the 500 bp zone encompassing the OBR (defined by the strand-switching assay) has been severely underestimated in the two-dimensional gel analysis because the assay is not very quantitative [ 25**]. However, in recent experiments in our own laboratory, it has been possible to quantitatively retain the fragile bubble structures that characterize initiation reactions, and in these DNA preparations (in which the bubble arcs are very prominent), the amounts of bubble arcs are virtually indistinguishable between two adjacent fragments of exactly the same size, one of which contains the putative OBR (PA Dijkwel et al., unpublished data). This result cannot be explained by the presence of a single OBR in this region. While the strand-switching assay has been used convincingly to map initiation sites in the E. coli and SV40 chromosomes replicating in vivo, to our knowledge it has not been shown to accurately predict the position of a known origin in vitro. By the same token, both twodimensional gel replicon-mapping methods accurately predict the locations of several yeast autonomously replieating sequence (ARS) elements [18,19,26,27] and the polyomavirus origin [20**]. As yet, neither method has been used to successfuliy map a ‘standard’ mammalian chromosomal origin (because there currently is none). Thus, some caution should be exercised in interpreting the results obtained with either of these rather complicated and still rather novel mapping procedures. Indeed, part of the disparity in results may arise from a failure to fully appreciate some of the fine points of the two different assay systems. However, we consider it more likely that the disparities between the two systems reflect unanticipated differences between in vivo and in vitro modes of replication. For example, as currently practiced, the DNA prepared for the twodimensional gel-mapping studies on mammalian cells is supercoiled until it is digested with a restriction enzyme prior to analysis on the gel [21], whereas the DNA template for the in vitro replication assay is undoubtedly nicked and relaxed because the cells are permeabilized in the presence of magnesium [25**]. Perhaps interaction of a transacting factor with a fixed origin in the context of the constrained, supercoiled chromosome causes an entire looped domain contain-

of replication

Hamlin, Vaughn, Dijkwel, Leu, Ma

ing 15-20 kb of DNA around an OBR to melt, whereas, in vitro, only 40-50 bp of DNA would be destabilized by interaction of a c®ulatory origin with an initiation protein. Clearly, new approaches wiU have to be devised to look at mammalian origins in different ways, with a broadminded attitude that recognizes that marnmaUan origins may be more complex than those of viruses, yeast, or bacteria. In addition, more origins will have to be studied from many different angles for purposes of comparison. In this regard, an additional initiation locus has recently been identified that lies approximately 250 kb upstream from the DHFR gene in the 450 kb amplicon of a methotrexate-resistant Chinese hamster lung fibroblast [28]. This origin was detected by using BrUdR-labelled early replicating DNA as a hybridization probe on a series of overlapping clones spanning this large amplicon. Attempts rescue

to isolate

origins

by phenotypic

Following the examples set in simpler systems, many attempts have been made to rescue the c&acting elements responsible for origin function by identifying sequences capable of autonomous replication in mammalian cells. Genomic fragments are cloned into a vector (usually contaming a selectable marker) and are transfected into a suitable host ceU line. The genomic inserts can either be an entire genomic library, or sequences suspected to contain an origin of replication based on independent in vivo studies. By analogy to the assay devised for ARS elements in yeast, fragments supplying origin function are expected to transform cells with high frequency. A variation on this theme is a short-term assay in which low molecular weight DNA is sampled over the period of several days after transfection, and the ability of the transfected DNA to replicate autonomously is determined by its resistance to the enzyme DpnI. This enzyme cannot cleave its recognition sequence (GATC) when the A residue is not methylated, as in DNA that has replicated in mammalian cells, but readily digests the transfected bacterial DNA These approaches have been attempted ad nauseam in several laboratories and have largely been unsuccessful, accounting for the paucity of such reports in the Uterature. However, there are a few exceptions. Using a protocol designed to induce branch migration of small nascent strands centered around origins of replication [29], several monkey sequences have been isolated, some of which have been reported to replicate autonomously when reintroduced into human or monkey cells [30]. A sequence near the 5’ promoter of the human c-myc gene that was previously implicated as a chromosomal origin of replication by in vivo labelling studies [ 311 has also been reported to persist as an autonomously replicating element for more than 300 ceU generations and to increase in mass by 500-lOOO-fold over this time period [ 17.1. A different laboratory also reports the episomal maintenance of a sequence from the

417

418

Nucleus

and gene expression

human c-myc promoter region (unfortunate@, not the same sequence as above), both in tissue culture cells and by transmission through several generations of transgenic mice [ 181. This result implies that the cloned c- myc fragment supplies both replication and partition functions. The latter sequence is also reported to bind to the c-m. protein, which is suggested to regulate replication in this system 1321.

These data therefore seem to suggest that the way is now open for critical genetic studies that can mutate cloned origins in vitro and determine the key c&acting (and eventually tiumacting) elements required for origin function. But, there continues to be an element of doubt about the identity of these fragments and the authenticity of the assay systems. For example, it is dilficult to reconcile the fact that two different but adjacent c-myc fragments are reported to replicate autonomously in two different laboratories. In addition, none of the clones discussed above has been shown to replicate reproducibly in a laboratory other than the one in which it was cloned. Most disconcerting is the recent observation that virtually any human sequence can replicate in either HeIa cells or in 293 cells (a human line transformed with the adenovirus Ela protein), provided that the fragment is long enough [33-l. Furthermore, most laboratories that have attempted to get their favorite origin fragment to replicate in the DpnI-resistance assay or in long-term selection schemes have been frustrated by the inability to reproduce either assay from experiment to experiment. The vector sequences all by themselves can be shown to replicate and to become Dpnl-resistant at a disarmingly high level (but usually non-reproducibly so). However, we appreciate that many breakthroughs in science have had dill?& births. For a variety of reasons, it is possible that certain bona fide chromosomal origins (but, unfortunately, not necessadly our favorites) may replicate better than others. The particular recipient cell line may make a difference, as might the particular vector construction, the assay system, and even the experimenter. A recent report on the ability of cloned DNA sequences to replicate after injection into frog eggs suggests that compartmentalization may actually shield sequences from interaction with the replication machinery [ 341. Therefore, even the mode of transfection may make a difference. The importance of a reliable phenotypic assay to a true understanding of mammalian origins argues that these and related studies should be given our full attention. It will be extremely important for someone (preferably in the first year of a 5-year grant) to systematically attempt to design a phenotypic assay that allows the direct isolation of the c®ulatory elements necessary for origin function. Properties regulators

of origins

and potential

tram-acting

A comparison of sequence elements in known microbial, viral, and yeast origins suggests two common features: binding sites for initiator proteins in discreet regions of

DNA bending; and direct repeat sequences that lie within well defined boundaries of an unusual anti-bent domain [35*], The question, then, is whether mammalian origins will have the same properties. Even though several candidate origins have been identifid recently, the one that has been studied in detail this year is the ori- locus lying just downstream from the DHFR gene in CHO cells. Figure 2(a) shows the relationship of this initiation locus to the broad zone of initiation detected in viva by twodimensional gel mapping methods [20**]. Several subfragments from this region that have been implicated as predominant initiation zones by various in vivo labelling protocols [7-12,14*,20**,25**] are shown in Fig. 2(b). A 6 kb region encompassing the putative origin of bidirectional replication has been sequenced by two groups [36,37] and, even though there is no genetic evidence at present to suggest which, if any, sequence elements might be critical for origin function, several features are noteworthy. The strand-switching site (OBR) is flanked immediately upstream by an inverted repeat sequence that would be moderately stable if folded into a hairpin-like structure [36,37]. Upstream from the inverted repeat lies an Alulike element flanked on its 5’ side by a long string of thymidines. About 1 kb downstream from the proposed OBR is a 280 bp HuetIl fragment that contains, beginning from the 5’ end: an OTF-1INFIJ.I binding site [38**]; a binding site for a newly identi!ied protein that associates with an ATP-dependent helicase [38**], and which enhances bending of the DNA in this region [3!P]; a short region of bent DNA [36,37]; and a T-rich segment that signals the beginning of another Alu-like element. One kb futther downstream lies an H3/XmnI fragment that has been shown to replicate autonomously in yeast [40], undoubtedly because of the presence of several yeast ARS consensus sequences mapping in a region that is more than 75% A-T-rich. Finally, 500 bp further downstream lies a small stretch of potential Z DNA followed by a sequence that can form a triple helix under appropriate conditions [37]. Thus, there are several elements in the 6 kb region encompassing the putative OBR that could be involved in origin function. The cautionary note is that any 6 kb stretch of DNA would be expected to display a panoply of elements that might appear (and even behave as if) biologically relevant, but until genetic studies implicate certain of these sequence elements in origin function, they remain in the categoxy of computer-generated oddities. The most interesting elements (and the nearest to the OBR) are those that bind proteins with properties expected of transacting factors involved in initiation of replication [38**,39]. It is not known whether these proteins have anything to do with origin function, nor whether they relate to any other cellular proteins that have been identified as being important in initiating replication in SV40 and polyoma virus. However, replication factor A, which is a multisubunit cellular protein that apparently interacts with T antigen during initiation, has re-

Origins

(a)

I

of replication

Hamlin,

Vaughn,

Dijkwel,

Leu,

Ma

I

0

160

L

180

200

220

240 kb I

Eco

R

xba I

(b) ‘4.3’ B I 0

(c)

1

x

‘1.86’kb

kb (actually

2

B

4.5 kb)

(actually H

P

1.9 kb)

l-l3

I

HH3

HP

4

H H

H3

AL” T

5

R AL”

H3 RX

H3 Yeast

WT

IR API

Bent

6kb

DNA

RIP60

ARS

1

I

I

Z /Triplehehcal region

Fig. 2. (a) The 240 kb dihydrofolate reductase (DHFR) amplicon showing the positions of the DHFR gene, a second transcription unit (2BE2121), and the centers of the two rough peaks of early labelling detected in I101 and I121 (each indicated by I). In two-dimensional gel replicon-mapping studies [20**1, initiations are detected throughout the broad zone indicated by the bracket and expanded in the map below. (b) The position of a 4.3 kb Xbal fragment (4.5 kb based on DNA sequence) which was previously reThe 1.85 kb 8amHI/Hindlll fragment beported to be the earliest replicating fragment in the upstream ori-p locus 181 is shown. low it maps in the center of the upstream peak of labelling detected in [lo]. The two junctions of the two small Haelll fragments define the proposed origin of bidirectional replication (OBR) in the oriregion. The interesting sequence elements discussed in the text are diaerammed in tc). and restriction sites are indicated above the line (X, Xbal; B, BamHI; P, Pvull; H3, Hindlll; H, Haelll). ”

cently been shown to be phosphotylated in a cell-cycledependent manner [41*]. In addition, a human protein factor (W-S) that activates Gl phase extracts to replicate 5V40 in vitro has been shown to contain a homologue of the p34 cdc2 kinase from Scbirosaccbaromyces pombe [42*]. A cdc2like protein has also been implicated in the initiation of replication in Xenopus luevis in vitro replication extracts [ 431. However, in the latter case, there is no convincing evidence for site-specific initiation [ 441. If any of these proteins are, indeed, involved in initiation at cellular origins (or in a cascade leading to initiation), it may soon be possible to link together the burgeoning field of cell cycle control with those molecular events that occur at specific origins to effect strand-separation and initiation of nascent chains. Effects of nuclear

architecture

The possible consequences of the sophisticated pa&ging of mammalian chromosomal DNA on the replication and transcription processes must finally be considered. Anyone who has lysed a cell and witnessed the explosive uncoiling of the DNA must appreciate the constraints imposed and the consequent potential energy that can be contained by protein-DNA interactions. The DNA is thought to be attached periodically to a subnuclear proteinaceous scaffolding or matrix at speciiic scaffold- or matrix-attached regions (SAR/MA&), many of which have

been circumstantially implicated in control of either trarscription or replication on the basis of their locations near promoters or putative origins [45]. Because the size of the chromosomal loops formed between two h4AR.sis approximately equal to size estimates for replicons in mammalian cells, it has been suggested that the loops may correspond to replicons, that the replication machinery is affixed to the matrix, and that DNA is spooled through the replication complex [ 461. Suggestive evidence for this view has been obtained by showing that 95% of replication forks partition with the matrix when the DNA loops are removed with a restriction enzyme [47]. While arguments can be made that single-stranded regions in forked structures could aggregate non-specifically with matrices during isolation procedures, this finding has allowed sufficient enrichment of replication intermediates to analyze a single-copy mam malian initiation locus by two-dimensional gel mapping methods for the first time [21]. Our instincts tell us that the somewhat controversial matrix field will not be sorted out until new methods of analysis are brought to bear on the whole question of the geometty of the replication and transcription processes. Biases and perspectives In summary, tremendous progress has been made in recent years in localizing mammalian initiation sites, par-

419

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Nucleus

and gene expression

titularly the ori-fi locus in the DHFR doma& of CHO cells. Most laboratories are continuing to proceed along pathways prescribed by prokaryotic and eukaryotic viral models, and the rate constant of data accumulation has markedly increased. The results obtained for the DHFR locus in particular appear to be somewhat contradictory and unsettling. However, we are confident that when the dust settles, we will have a more realistic view of what really happens at the molecular level when the cell decides to double its DNA Acknowledgements Work in the authors’ laboratory was supported by NIH grants GM26108 and CA52559 to JL Hamlin. We thank all the members of the Hamlin laboratory, past and present, for intellectual and experimental input over the years.

and recommended

References

reading

Papers of special interest, published within the annual period of review, have been highlighted as: . of interest .. of outstanding interest 1.

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2.

HUBERMANJ4 RIGGS AD: On the Mechanism Replication in Mammalian Chromosomes. J Mel 32~327-337.

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Dm JFX, Snw B: The Initiation of Chromosomal DNA Replication in Eukaryotes. Trenak Genet 1990, 6~4271337.

4.

Uhmt RM, LLNSKENS MHK, KOWALSKID, HUBERMANJA: New Beginnings in Studies of Eukaryotic DNA Replication Origins. Bicxbim Biophys Acta 1989, 1007:1-14.

5.

CAWBEIL

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LOONEYJE, HAMUNJH: Isolation of the Amplified Dibydrofolate Reductase Domain From Methotrexate-Resistant Cbinese Hamster Ovary Cells. Mol Cell Biol 1987, 7~56~577.

7.

HEINTZ NH, m JH: An Amplilied Chromosomal Sequence that Includes the Gene for Dihydrofolate Reductase Initiates Replication within Specific Res’tiction Fragments.

JL Eukaryotic DNA Replication. 1986, 55:733771.

Proc

Nat1 Acud

Sci LISA 1982,

Annu

Ret* Biochem

79:4083-4087.

8.

BURHANS WC, SEIIGUE J, HERXZ NH: Replication Intermediates Formed During Initiation of DNA Synthesis in Methotrexate-Resistant CHOC 400 Cells are Enriched for Sequences Derived thorn a Specific, Amplified Restriction Fmgment. Biochemisty 1986, 25&l-449.

9.

ANACHKOVAB, hhUlN JL: 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 Bid 1988, 9:532-540.

10.

LEU TH, w JL: High-Resolution Mapping of Replication Fork Movement Through the Amplilied Dihydrofolate Reductase Domain in CHO Cells by In-Gel Renaturation halysis. MoI Cd Biol 1989, 9523531.

11.

BURHANSWC, SUECUE JE, HEINIZ NH: Isolation of the Origin of Replication Associated with the Amplified Chinese Hamster Dihydrofolate Reductase Domain. Proc Nat1 Acud Sci USA 1986, 837790-7794.

HAN~Eu S, KLul 4 MEUIH M, CEDARH: Mapping Replication Units in Animal Cells. Cell 1989, 57:909-920. LMNE AJ, KANG HS, BIUHELMERFE: DNA Replication in SV4013. infected Cells. Analysis of Replicating sV40 DNA / Mel Bid 1970, 50:54F568. VASSI~, LT, BURHANSWC, DEPA~~PHIUSML Mapping an Ori14. gin of DNA Replication at a Single-Copy Locus in Expo . nentially Proliferating Mammalian Cells. Mel Cell Biol1990, 10:468%689. A new method based on PCR technology that allows replication initiation sites to be roughly mapped by determining the distribution of nascent strands. V~SSILEVLT, JOHNSONEM: An Initiation Zone of Chromoso15. mal DNA Replication Located Upstream of the c-myc Gene in Proliferating HeLa Cells. Mel Cell Bioll990, 10:489+4904. 12.

16.

SUDO K, OGATA M, SATO Y, IGUCHI-ARIGA SMM. AJUGA H: Cloned Origin of DNA Replication in c-myc Gene Can Function and Be Transmitted in Transgenic Mice in an Episomal State. Nucleic Acirir Res 18:542?5432. One of the few examples in which a cloned sequence from a mam malian genome has been reported to replicate autonomously in mammalian cells after transfection. The same sequence can apparently be maintained for many generations in transgenic mice. MCWHINNEYC, LEFFAKM: Autonomous Replication of a DNA 17. Fragment Containing the Cluomosomal Replication Origin .. of the Human c-myc Gene. Nucleic Acids Res 18:12331242. Another recent example in which a cloned mammalian genomic se quence has been reported to replicate autonomously in animal cells. 18. BRNVER BJ, FANGMANWL The Localization of Replication Origins on ARS Plasmids in S. cereofsiae. Cell 1987, 51:463-471. NAWOTKA KA. HUBERMANJA Two-dimensional Gel Elec19. trophoretic Method for Mapping DNA Replicons. Mol Cell Biol 1988, 8:140%1413. VAUGHNJP, DIJKWELPA HAMU& JL Replication Initiates in a 20. .. Broad Zone in the Amplified CHO Dihydrofolate Reductasc Domain. Cell 1990, 61:107~1087. Application of the two-dimensional gel repUcon mapping methods to a study of the DHFR locus in CHOC 400 cells. The results indicate that, in titq replication may initiate in a broad zone surrounding each origin of replication.

. .

21.

22. .

DIJKX%L PA VAUGHN JP. OLIN JL Mapping ReplicationInitiation Sites in Mammalian Genomes by Two-dimensional Gel Analysis: Stabilization and Enrichment of Replication Intermediates by Isolation on the Nuclear Matrix. MoI Cell Biol 1991, in press.

HECK MM, SPRADUNG AC: Multiple Replication Origins are Used During Drosophila Chorion Gene Amplification. / Cell Biol 1991, 110:903914. Another example of multifocal initiation surrounding an origin of replication. ScHvARTzMANJB, AcolP~ S, MARni+P.+aa.4sL. SCHIWKRAL~~CL: 23. Evidence that Replication Initiates at Only Some of the Potential Origins in Each Oligomeric Form of Bovine Papillomavims Type 1 DNA. Mel Cell Biol 1990, l&3078-3086. 24. KOHARAY, TOHD~H N, J~ANGXW. OKAZ!AKIT: The Distribution and Properties of RNA Primed Initiation Sites of DNA Syn thesis at the Replication Origin of Escherichia Coli Chromosomes. Nucleic Acid Res 1985, 136847~. BURHANSWC, V~SSILEVLT, CADDLEMS, H!ZINIZNH, DEPAMPHIUS 25. .. ML: Identification of and Origin of Bidirectional DNA Replication in Mammalian Chromosomes. Cell 1990. 62:955%5. An approach modelled on studies in bacteria in which Okazaki fragments from the lagging strand of replication are isolated and used as hybridization probes on the separated template strands in order to determine he position at which the Okazaki fragments switch from one template to the other. With a lixed origin of replication, this switch cccurs at or close to the genetic origin.

Origins 26.

27.

LINSKENS MHK, HUREWAN JA: Organization of Replication of Ribosomal DNA in Saccbaromyces cerevisiae. Mol Gel1 Biol 1988, 814927-4935. HUBEW JA, ZHU JG, DAVIS LR, NLWIDN CS: Close Association of a DNA Replication Origin and an ARS Element on Chromosome III of the Yeast, Saccharomyces Cerevisiae. Nucleic Acids Res 1988. 16:637%6384.

28

MA C, DELI TH, HMUN JL cation in the Dihydrofolate Methotrexate-resistant Chinese Biol 10:133&&1346.

29.

23~~1s.~JOPO~I~S

M, PERXO

able Instability of Replication Method for the Isolation of Cell 1981, 27:155-163. 30.

31.

Multiple Reductase Hamster

Origins of RepiiAmpUcons of a Cell Line. Mol Cell RG: The RemarkProvides a General of DNA Replication.

M, MAltTIN

Loops Origins

LEFFAK M: Opposite Replication Polarity of the Germ Line c-myc Gene in HeLa Cells Compared with that of Two Burkitt Lymphoma Cell Lines. Mol Cell Biol 1986, 9586593. SMM, ~TANI T. KIJI Y, AIUCA H: Possible Function of the c-myc Product: Promotion of Cellular DNA Replicdon. EMBO J 1987, 6:236%2371.

32.

IGUCHI-AIUGA

33. ..

HEW&I. SS. KRYSAN PJ, TRAN CT, 01.0s MP: Autonomous Replication in Human Cells is Alfected by Size and Source of DNA blol Cell Biol 1991, 11~2263-2273.

MARIN

NJ,

BELOW

RM:

Differential

of Plasmid DNA Microinjected bryos Relates to Replication 11:299-308. 35. .

ECKDAHL

Origins

39. .

have

interesting

CREEPER I

41.

.

Cells 1988,

paper describes a cellular factor required for SV40 initiation in that is phosphotylated in a cell-cycle.dependent manner. If it can be related to any proteins that bind fo the DHFR orilocus (or any other initiation locus), the whole field of ceU cycle regulation could be tied together with initiation of replication.

vitro

D’UR~

G, MARRACCWO

Cycle Control Human Cells

..

RIP60

and RIPlOO,

Mammalian

N, HEINIZ

Proteins

NH:

with

Purification Origin-Specific

of

&

of DNA

MARSHAK DR.

Replication

CeU from Science,

ROBERTS JM:

by a Homologue

of the p34cdc2 Protein Kinasc. 250:78&791. Another interesting cell-cycle-regulated prorein that, if related to DHFR o&a-binding proteins, will provide a link with initiation of replication.

See 138*‘,39’1. 43.

BLO\Y, JJ. NUKE

P: A cdc2-like

Initiation of DNA 62:855-862.

NEIS~N

MS, HEINE

in CHO

B: Cell-Cycle-Regulated Phosphorylation of DNA Replication Factor A From Human and Yeast Cells. Genes Dev 4968-977.

45.

DAILEY 1 CADDE

Domain

This

WG,

Replication

LVKEY RA into Eggs PIENTA

Protein

is Invoked

in Xenopus

Egg Extracts.

Regulated Replication of Xenopus Laevis.

KJ, BARRACK ER, Comv

in the CeU

of Cell

DNA 1980,

DS: The

Role of

of the Nuclear Matrix in the Organization and Function DNA Annu Rev Biophvs Gem 1986, 15:457-475.

important reading to gain insight into the sequence features that appear to be required by all origins that have been srudied in derail so far.

38

Reductase

63329-333.

DIN S, BRIU SJ. FAIRMAN MP, STIUMAN

TI’. ANDERX)N JN: Conserved DNA Structures in of Replication. Nucleic Acids Res 1990, 18:1609-1612.

CADDLE MS, LUWER RH, HEIN-IZ NH: intramolecular DNA Triplexes, Bent DNA, and DNA Unwinding Elements in the Initiation Region of an Amplitied Dihydrofolate Reductasc RepUcon. J Mol Biol 1990, 211:19-X3.

be involved DHFR oria cisregulathe proteins

PK. MA CA, VAUGHN JP, DIJKWEL H: Analysis of the Initiation Locus

Dihydrofolate

Cancer

HARLWD RM, Microinjected 21:761-771.

37.

Activities.

FOREMW’

WIUARD

of the Amplilied

44.

IIU TH, ANACHKOVA B. HAMuN JL Repetitive Sequence Elements in an initiation Locus of the Amplilied Dihydrofolate Reductase Domain in CHO Cells. Genomics 1990, 7:42WW.

Leu, Ma

properties.

&4MUN JI, LEU TH,

Xenopus laevis EmMO/ Cell Rio1 1991,

36.

Dijkwel,

CADDLE

Cells

Compartmentalization

into Efficiency.

Vaughn,

MS, D.um L, HEINIZ NH: RIP60, a Mammalian Origin-Binding Protein, Enhances DNA Bending near the Dihydrofolate Reductase Origin Of Replication. Mel Cell Bid KGQ, 10:6236-6243. This report extends the data in [38**] and shows that one protein can aCNdt)’ enhance bending of an akeady bent DNA sequence near the or&p locus in the DHFR domain.

42. .

An imporrant paper suggesting that any human DNA fragment can replicafe to a limited extent after transfcction into human cells, provided that it is long enough. This result implies that origins are not specific sequence elementi, or that cisregulatory elements occur at very frequent intervals in human DNA

34.

described

PA

CD,

Hamlin,

DNA-Biding and ATP-Dependent DNA Helicase Mol Cell Biol 1990, lOt6225-6235. The first report of specilic DNA-biding proteins that might in the function of a mammalian chromosomal origin (the locus). Although there is no genetic evidence that there is tory element in this particular region of the DHFR domain,

40.

FRAPPIER L, ZANNIS.HADJOPOIILOS M: Autonomous RepUcation of Plasmids Bearing Monkey DNA Origin-Enriched Sequences. Proc Nat1 Acad Sci USA 1987, 846668-6672. J-S

of replication

46.

V~GEISTEIN

and 47.

Eukaryotic

B, Pmu

DNA

DM, COFFEY DS: Supercoiled Loops Replication. Cell 1980, 22~79-85.

VAUGHN JP, DIJKWTL PA

MUUENDER~

tion Forks are Associated with Acids Res 1990, 18:1%51%9.

the

HAML[N JL: RepUcaNuclear Matrix. Nucleic

LHF,

JL HamUn, JP Vaughn, PA Dijkwel. T-H ku, C Ma, Department of Biochemistry, University of Virginia School of Medicine, Box 440, Jordon Hall, Charlottesville, Virginia 22908, USA

421