Genetica 94: 255-266, 1994. @ 1994KluwerAcademicPublishers. Printedin the Netherlands.

255

The quest for a human ori Arturo Falaschi & Mauro Giacca International Centre for Genetic Engineering and Biotechnology, AREA Science Park, Padriciano 99-34012 Trieste, Italy Receivedand accepted30 August 1994 Key words: DNA replication, origin, competitive polymerase chain reaction, transcription, lamin B2

Abstract

Attempts at identifying DNA replication origins in human cells have beenperformed with a variety of molecular genetic and biochemical approaches, with often controversial results. The combination of bromodeoxyuridine labelling, immunopurification of newly synthesized labelled DNA, measurement of the relative abundance of markers in this DNA by quantitative competitive PCR, has allowed the identification within 450 bp of the start-site of DNA replication located at the human lamin B2 gene. The origin is located near the non-transcribed spacer between two highly transcribed genes and shows evidence of a number of specific protein-DNA interactions, the most prominent of which disappears when the cells are differentiated into a non-proliferating state.

Introduction

Just over 30 years ago, Jacob, Brenner and Cuzin (1963) proposed the model of the replicon, a DNA structure capable of replicating autonomously within a cell; according to this model, the rate of DNA replication is regulated by the modulation of the frequency of activation of a defined genetic element within the replicon, named the origin Of DNA replication (ori). The replicon model has victoriously'withstood the test of time and has been satisfactorily proven in prokaryotes, where, in particular, the Escherichia coli ori C system has been dissected by genetic means and convincingly reconstituted in vitro with pure molecules by Arthur Kornberg and collaborators (Kornberg & Baker, 1992). Whereas in prokaryotes the chromosome corresponds to the replicon, the classical work of Huberman and Riggs (1968) showed that each linear eukaryotic chromosome is composed of many tandemly organized replicons, activated (each once and only once) at different times of the cell cycle. According to this proposal, these more loosely defined eukaryotic replicons are also characterized by an origin conceptually similar to the prokaryotic one, from which two oppositely moving semiconservative

forks emanate. The size of the eukaryotic replicon is approximately 100 kb in metazoans and 10 to 20 kb in yeast. Although these authors first showed this functional organization of eukaryotic DNA replication regulation in human cells cultured in vitro, the expected consequent identification of replication origins in "such cells has proven very difficult and irksome, and has eluded the expectations also raised by the success obtained in a simple eukaryote, Saccharomyces cerevisiae. In this organism, in fact, the functional test for autonomously replicating sequences (ARS) has allowed the identification of replication origins that exert this function whether inserted in a plasmid or present in a chromosome (Stinchcomb, Struhl & Davis, 1979; Brewer & Fangman, 1987; Newlon, 1988); furthermore, a description of the activation process of an ARS ofS. cerevisiae has also been initiated at the molecular level, showing that precise and specific protein-DNA interactions probably underlie the origin activation event (Bell & Stillman, 1992; Diffley & Cocker, 1992; Bell, Kobayashi & Stillman, 1993).

256

Fig. I. Schematic representati•n •f the pr•c•ss •f a•tivati•n •f •ri. Several c•pi•s •f a speci•c initiat•r pr•tein bind t• mu•tip•e sites in the •ri region. This binding, together with an active transcriptional event in the neighborhood and with the action oftopoisomerase II, induces distortion of the region and opening of the DNA duplex in correspondence of a nearby AT-rich tract, to which a heliease and a priming complex can bind. The helicase extends the opened area, and priming of at least one leading strand starts; then, the replicative complex initiates continuous replication. When the first lagging strand Okazaki fragment is palmed and initiated, it is extended as the leading strand in the oppositedirection.

257 Table 1. Expectedfeaturesof a mammalian DNA replicationorigin.

Scaffoldattachment Recognitionby initiation-specificproteins Correlationwithtranscription Susceptibilityto topoisomeraseII action ATrichness Secondaryand unusualstructures

Expected features of a m a m m a l i a n ori

From the results obtained in the in vitro reconstituted system of E. coli (Bramhill & Kornberg, 1988), from the similar ones obtained in some bacteriophage and viral systems (in particular, ~ phage (Marians, 1992) and SV40 (Stillman, 1989; Murakami, Eki & Hurwitz, 1992; Waga & Stillman, 1994)) and from the initial results obtained in S. cerevisiae (Bell, Kobayashi & Stillman, 1993), some general features of the origin activation process begin to emerge (Figure 1). In the first place, an ori must contain recognition sites for an origin-specific DNA protein, which is the key element for origin activation and which is typically represented by the dnaA protein of E. coli or by the T antigen of SV40. The presence of transcriptional elements within or close to the ori region appears to be essential, and an active transcriptional event is likely to be required for the subsequent steps, together with topoisomerase II action. The binding of several copies of the initiator protein, coupled with the effect of nearby transcription and topoisomerase II action, introduces a particular torsional stress in the origin area that leads to the opening of an AT-rich region which is always present in an ori area. This opening allows the entrance of specific helicases which extend the unwound DNA tract and enable the assembly of the replication machinery for (at least) one of the leading strands. Once leading strand synthesis has started and progressed, aided by the function of specific helicases, discontinuous synthesis on the opposite, lagging strand initiates, and the first fragment synthesized retrogradely on the lagging side becomes the leading strand for the fork moving in the opposite direction. Some features that might reasonably be expected to be present in any replication origin may simply be deduced from this generalized model (Table 1). 1) First of all, the origin must be attached to some 'solid' cell structure, which will eventually allow the separa-

tion of the daughter chromosomes into separate ceils: this is assured in bacteria by the binding of the ori to a specific site of the membrane, and is most likely assured in eurkaryotes by the attachment of ori to the chromosome scaffold (Cox & Laskey, 1991). 2) The ori area must bear recognition sites for initiation specific proteins. 3) Transcriptional signals, assuring active transcription at the moment of origin activation, must be present nearby (De Pamphilis, 1988); 4) The ori area must be exposed to topoisomerase II activity (in eukaryotes this enzyme is indeed known to be an important element of the chromosome scaffold (Cook, 1991)); 5) An AT-rich area must be present nearby, where duplex opening may initiate (Umek & Kowalski, 1988; Natale, Umek & Kowalski, 1993); 6) The recognition sites for the initiator protein(s) very often correspond to DNA sequences containing unusual structures, such as palindromes, inverted repeats, direct repeats, and so forth.

Autonomous replication studies

Several years ago, in our laboratory (first at the Istituto di Genetica Biochimica ed Evoluzionistica del Consiglio Nazionale delle Ricerche in Pavia, and then jointly in Trieste and Pavia), we initiated a program aiming at the identification of replication origins in human DNA and of the specific protein-DNA,.interaction underlying the ori activation" process. Initially, we performed extensive attempts at obtaining ARSs in human and other mammalian cells, in analogy with what happens in yeast (Biamonti et al., 1985). We explored almost 3 million bp of human DNA in 4 to 5kb stretches, inserted into plasmids which were transfected into human cells cultured in vitro; the replication challenge was performed in conditions where even a single molecule replicating once per cell cycle would have been detected. No evidence for autonomous replication was obtained, a failure corroborated by similar experiences shared by many laboratories. A number of reports claiming success in obtaining episomes capable of indefinite autonomous replication in cultured mammalian cells, at a rate comparable to that of the host chromosomes (Table 2), have either not withstood closer scrutiny (Gilbert & Cohen, 1989), have failed to be reproduced in other laboratories (Burhans et al., 1990; Caddle & Calos, 1992), or possiblyrefer to properties peculiar to particular viral systems rather than to the organism (Krysan, Haase & Calos, 1989; Krysan & Calos, 1991). For a review of the possible reasons

258 Table 2. Approaches for the identification of DNA replication origins - Replication studies.

Method

Region studied

Main conclusion

References

c-myc upstream region

autonomous replication ofplasmids containing the region

(McWhinney & Leffak, 1990)

nascent DNA strands extruded from replication bubbles or random DNA sequences

selection of ars sequences

(Frappier & Zannis-Hadjopoulos, 1987) (Landry & Zannis-Hadjopoulos, 1991) (Wu, Zannis & Price, 1993)

random DNA sequences cloned in EBV-derived vectors

autonomous replication for inserts > 12,000 bp

(Krysan et al., 1989) (Krysan & Calos, 1991)

density substitution Dpn I resistance in vitro replication

underlying the failure of this functional approach for the identification of a mammalian ori, see Falaschi et al., 1993.

Physical cloning of ori-containingDNA fragments In view of the overall failure of the autonomous replication studies, we decided to attempt the physical isolation of the ori. The problem is in itself formidable, since it entails the identification and cloning of a sequence that may be as short as a few tens of bp out of a 3 billion bp tangle, without the assistance of any biological selection procedure. In order to attempt the isolation of human DNA fragments which were likely to contain the ori, we first applied a strict synchronization procedure obtained by two subsequent cycles of aphidicolin treatment (Tribioli et al., 1987) to cultured myeloid HL-60 cells; the cells were thus piled up at the G1-S border and were then allowed to enter S for a limited period of time in the presence of bromodeoxyuridine (BrdUrd). Through this procedure, all the DNA synthesized immediately after the aphidicolin step-down, corresponding to the first originsactivated at the beginning of the S-phase, was density-labelled. The BrdUrdsubstituted DNA was totally purified from the bulk of the DNA through repeated CsC1 gradients, and then cloned. In this way, a DNA library of fragments corresponding to early replicating regions (conceivably containing the ori element) was obtained, The overall features of this library were the absence of enrich-

ment for repetitive DNA sequences with respect to total human DNA, the absence of mitochondrial DNA and the enrichment for snap-back Cot=0 DNA, i.e. DNA with a very fast renaturation kinetics, suggesting the presence of several short complementary stretches (Tribioli et al., 1987). The longest sequence of this library, named B48 (a 1500 bp long fragment), was further characterized and became the object of the subsequent studies. In the first place, by repeating the synchronization and BrdUrd labeling procedure for different times, we isolated the DNA which was synthesized at different moments of the S-phase, and could confirm, by slot blot hybridization, that the B48 region was indeed replicated exclusively in the first hour of the S-phase (Tribioli et al., 1987). A further refinement of this method (see below; Biamonti et al., 1992a) indeed led to the conclusion that this region is replicated within the first minute after entry in Sqbhase. This conclusion suggested that it is very likely that the ori itself corresponds to, or is located close to, the cloned B48 fragment. With the purpose of determining its precise localization, a 14 kb region, containing B48 roughly in its middle, was cloned from a human genomic library, and a procedure was developed to map the movement of the replication forks within this area. The cloned fragment belongs to a single-copy region localized in the subtelomeric portion of the short arm of human chromosome 19 (band 13.3). This area contains the gene encoding for lamin B2 and a second highly transcribed gene (provisionally named ppvl), encoding for a still unidentified product of 75 amino acids containing a possible Zn finger; the two genes are arranged in tandem and spaced by a

259 600 bp area (see Figure 3, panel B for a close view of the tail of the lamin B2 gene and of the non-transcribed spacer) (Biamonti et al., 1992a).

Localization of ori within chromosomes

A variety of approaches have been developed in several laboratories, with some limited success, for mapping the localization of ori in mammalian cells (Table 3). The two dimensional (2-D) gel approach (Brewer & Fangman, 1987; Huberman et al., 1987; Nawotka & Huberman, 1988), which has been extremely useful for the study of replication origins in viral and yeast systems, when applied to mammalian cells has given indications that replication initiates in a delocalized fashion within a 50 kb origin area in the dihydrofolate reductase (DHFR) gene replicon (Vaughn, Dijkwel & Hamlin, 1990). This has brought some authors to question the very existence of precise replication origins in higher organisms and to propose that in eukaryotes more complex than S. cerevisiae (including the fission yeast Schizosaccharomyces pombe), diffused broad areas of the chromosome (as long as 3 kb in S. pombe (Zhu et al., 1992) and as long as 50 kb in Chinese hamster) are activated simultaneously as delocalized origins, in sharp contrast with what happens in prokaryotes (Linskens & Huberman, 1990). Other authors have utilized the imbalanced DNA synthesis caused by the addition of emetine (an inhibitor of protein synthesis which is claimed to inhibit the synthesis of Okazaki fragments with an unknown mechanism) to specifically enrich samples of newly synthesized DNA for leading strand fragments. Through hybridization with strand-specific RNA probes, a leading strand polarity switch was identified within a 14 kb region in the Chinese hamster DHFR replicon (Burhans et al., 1991), and within 5 kb in the human fl-globin region (Handeli etal., 1989; Kitsberg etal., 1993). The amplified sequences of the Chinese hamster DHFR gene have also been the object of other physical attempts at ori identification: these studies have brought different authors to initially map the ori within approximately 5 kb (Burhans, Selegue & Heintz, 1986), and then narrow it down to 2 kb (Anachkova & Hamlin, 1989; Leu & Hamlin, 1989); finally, the identification of the switch point of Okazaki fragments polarity (Burhans ~ et al., 1990) allowed the resolution of 450 bp for a proposed ori site. Overall, the most puzzling conclusion that can be drawn from these studies derives from the striking con-

trast between the results obtained by the 2-D gel mapping technique (origin dispersed within 50 kb) and those obtained by the template switch analysis (origin mapped within 450 bp). It should be considered, however, that most of the studies on the DHFR gene suffer from the fact that, by utilizing a DNA region amplified over a thousand-fold, one may be dealing with genome areas which have peculiar properties also in terms of replication organization. Furthermore, the results of the studies which rely on the analysis of 2-D gels are often ambiguous and difficult to interpret, and may suffer from artefacts due to the breakage of the replication intermediates, to multiple reinitiation events in the same cell cycle induced by the synchronization procedure, or to branch-migration. On the contrary, the procedures based on imbalanced DNA synthesis have to rely on the use of inhibitors with an absolutely obscure mechanism of action, which could possibly grossly alter the cellular metabolism and thus also affect the physiological pattern of replication regulation. Moreover, the identification of the switch point of Okazaki fragment synthesis relies on the use of cells which have been treated first with a synchronization procedure and then permeabilized by detergents, two treatments yielding a very reduced rate of DNA synthesis that could also reflect possible perturbation of the initiation process. The first approach which we utilized, in order to localize the ori within the cloned fragment of the lamin B2 gene domain, relied again on the isolation of DNA synthesized at the beginning of S-phase in HL-60 cells. DNA extracted from aphidicolin-synchronized cells synthesized within the first minute of S-phase was denatured and fractionated by size. Nascent DNA fragments of an approximate length of 1000 nt were then selected and hybridized to different DNA probes scattered along the lamin B2 region. The results of these experiements showed that this region was highly enriched in this sample of newly synthesized DNA with respect to other portions of genomic DNA, and therefore confirmed that this area was indeed replicated in the first minute of the S-phase; furthermore, the marker located in the middle of the area under study, roughly corresponding to the original B48 isolate, was more enriched (about 50 fold) than the flanking ones (20 to 30 fold), suggesting the localization of ori near this area (Biamonti et al., 1992a). It should be considered that, for all the mapping experiments utilizing nascent DNA strands, the selection of the size of the DNA to analyze further is extremely important: short stretches can be contain-

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261 inated with Okazaki fragments and hence scattered all around the genome; larger fragments may be contaminated with the bulk of the DNA. Therefore, in this, and in all the subsequent experiments of this type, we selected DNA sizes ranging between 600 and 2500 nucleotides. Furthermore, due to the poor representation of nascent DNA as compared to the amount of the total bulk of the DNA, the size-fractionated samples are also likely to be contaminated by randomly fragmented parental DNA. Consequently, we devised a procedure for the purification of newly synthesized DNA (which is labeled by a short pulse with BrdUrd) through a chromatographic step based on a column containing a monoclonal antibody against BrdUrd DNA (Contreas, Giacca & Falaschi, 1992).

Competitive PCR of nascent DNA for precise ori mapping Close mapping of the replication origin in the lamin B2 gene domain was attempted by the procedure outlined in Figure 2. According to the functional definition of the ori, newly replicated DNA of increasing length emanates bidirectionally from the ori and progressively covers adjacent sequences on the DNA. As a, consequence, the representation of defined markers within a given genomic area in samples of short nascent DNA fragments is inversely correlated with their distance from the origin: the more abundant the marker, the closer it is to the ori. Therefore, by measuring the abundance of selected DNA fragments along a genetic region, it is possible to map precisely the movement of the replicative fork along that region and hence the localization of the ori. Although the results of the hybridization experiments in samples of nascent DNA fragments described at the end of the previous section were extremely encouraging, as they confirmed the likely presence of an ori in the DNA region under study, they could not allow a fine resolution mapping because of the limits of the sensitivity of the hybridization assay itself. It was essential, therefore, to develop a PCR-based measure of the abundance of the different markers, with all the difficulties intrinsic to the use of this technique for precise quantitative studies. It should be considered, in fact, that conventional PCR, although extremely sensitive, does not produce a quantitative result, since the yield of each amplification is affected by a number of uncontrollable variables affecting the reaction (Ferre, 1992).

For the purpose of precise quantification, we developed a quantitative PCR procedure based on the principles of competitive PCR (Gilliland et al., 1990; Siebert & Larrick, 1992). According to this method, PCR amplification is carried out on the DNA template to be quantified after adding a DNA competitor sharing almost exactly the same sequence with the template (including primer recognition sites). Since the two molecular species (template and competitor) compete for amplification, any controllable or uncontrollable variable affecting amplification has the same effect on both; therefore, the ratio between the two final amplification products reflects precisely the input ratio of the two species. There are several advantages in the utilization of this technique; in particular, as the quantification procedure is based on the calculation of the ratio between the amounts of competitor and target products, the technique is unaffected by the overall yield of amplification, allows the experimenter to reach the plateau phase of amplification, and is insensitive to the formation of aspecific amplification products (Bagnarelli et al., 1992; Menzo et al., 1992; Sestini et al., 1994). A simple method for the construction of competitors was developed through an application of the recombinant PCR technology: these competitors are constituted by 150-250 bp fragments corresponding to the genomic amplification products, with the addition of 20 bp in the middle, in order to allow simple resolution by polyacrylamide gel electrophoresis (Diviacco et al., 1992). The competitive PCR technique used on nascent DNA samples for origin mapping was first validated on the SV40 ori. Monkey COS-1 cells were transfected with a plasmid containing the whole of the SV40 genome, and newly replicated DNA was labeled for one minute with BrdUrd, extracted, and size fractionated. Newly synthesized DNA was further purified by immunoaffinity chromatography and challenged for the abundance of several markers located at different distances on either sides of the ori. The results clearly indicated a peak of abundance at the well known site of the ori, and regularly decreasing levels for the markers far away from it (Giacca et al., 1994). Having in this way validated the procedure, different competitors scattered along the human lamin B2 genomic area were constructed and then used to measure the abundance of the corresponding genomic fragments in samples of nascent DNA from synchronized HL-60 cells. In all the experiments conducted with samples recovered at different,periods after entry in

262

Fig. 2. Mapping the localization of a DNA replication origin by quantitation of the abundance of neighboring markers within samples of nascent DNA. Short fragments of nascent DNA, synthesized in the presence of bromodeoxyuridine (BrdUrd) from a bidirectional origin of replication (ori), are isolated from the bulk of parental DNA by size-fractionation on sucrose gradients and further selectively purified by immunoaffinity chromatography with anti-BrdUrd antibodies. Within this population of newly synthesized DNA molecules, the number of molecules containing selected fragments (A through E) scattered within a genomic region are precisely quantified by competitive polymerase chain reaction. The pair of primers (small arrows on top)amplifying from the highest number of molecules in the sample is the closest to the origin (marker C).

S-phase, a clear peak of abundance was detected for a region c o r r e s p o n d i n g to the original B48 isolate, with decreasing levels o f abundance as one m o v e s away f r o m the origin in either direction, as expected f r o m a bidirectional ori. The peak sequence was enriched

f r o m 1,000 to 10,000-fold o v e r a r a n d o m sequence in the human g e n o m e (Biamonti e t al., 1992b; Giacca e t al., 1994). The success of this procedure led us to consider that, in principle, it could also be applied to non

263

Fig. 3. Origin mapping in asynchronously growing HL-60 cells in the lamin B2 gene domain and features of the ori area. PanelA. Quantitation by competitive PCR of the number of molecules containing different regions scattered within the lamin B2 gene domain within a newly synthesized DNA sample of ~ 1000 nt. As a control, the number of molecules, within the same sample, containing a segment of the insulin gene is shown on the right side. The results are expressed as number of molecules per 106 total Brd-Urd-substituted molecules of the same size on the basis of specific radioactivity. The results are the same described by Giacca et al., 1994. Panel B. Protein-DNA interactions revealed by in vivo footprinting in the lamin B2 ori region. The non-translated tail of the lamin B2 gene, and the intron-exon organization of the 5 t of the ppv 1 transcript are shown (Biamonti et al., 1992a). The arrows indicate DNase I hypersensitive sites. For the other symbols, see text.

264 synchronized cultures, based on the consideration that short stretches of nascent DNA issuing from an origin should be easily separable from the bulk of parental DNA by virtue of their small size. Accordingly, DNA of an asynchronous HL-60 culture was pulse-labeled with BrdUrd, extracted through conventional procedures, denatured and size fractionated, the BrdUrd-substituted DNA was further purified through immunoaffinity chromatography. The abundance of the different markers inthe lamin B2 region was evaluated in a sample of ~ 1,000 nt of average length. As reported in Figure 3, panel A, a clear peak was again observed at the same localization detected by the experiments performed in synchronized cells. This result indicates that the procedure is also applicable to asynchronously growing cell populations, thus avoiding the use of replication (or other) inhibitors, which could always introduce a disturbing factor in these sort of studies, as mentioned above (Giacca et al., 1994). From the data obtained from the synchronized cultures, as well as from those from the asynchronous one, the same conclusion could be drawn: in the lamin B2 gene area the ori is contained within a defined 450 bp DNA stretch. In the asynchronous culture it was estimated that the enrichment for the peak segment corresponds to one copy per 30,000 DNA molecules; in other terms, this means that this specific ori fragment represents 1/30,000 of the total fragments of the same lengths. This value corresponds to that expected from a single origin out of a comprehensive collection of all human ori, considering that an average length of replicon of 100 kb would give a total of approximately 30,000 replicons in the human genome.

Features of the ori region Figure 3 panel B reports a simplified representation of the main features of the lamin B2 ori region, which is comprised between the B48BIS and B48TER markers and which contains the B48 marker. This area corresponds to the end of the lamin B2 gene (which encodes for a ,,~ 2,000 nt untranslated exon) and to the non-transcribed spacer which separates this gene from the downstream positioned p p v l gene and contains an active promoter for the latter; both these genes appear to be highly transcribed in HL-60 cells (Biamonti et at., 1992a; 1992b). The ori region contains at AT-rich tract and a topoisomerase II binding site on its left side, possibly corresponding to a scaffold attachment site (to

be published). Several inverted and direct repeats are observed in this region (Tribioli et al., 1987). Protein-DNA interactions in the ori region were investigated both by in vitro and in vivo studies. In particular, the in vivo studies were performed utilizing the in vivo footprinting technique (Mueller & Wold, 1989; Pfeifer et aI., 1989) through optimization of the ligation-mediated PCR technology (Demarchi et al., 1992; 1993; Dimitrova, Giacca & Falaschi, 1994). This technique entails treatment of living cells with dimethylsulfate, DNA extraction and specific breakage in correspondence with methylated guanines with piperidine (or, alternatively, DNase I treatment of isolated nuclei followed by DNA extraction and denaturation), primer extension with an oligonucleotide specific for the region of interest, ligation of a linker to the blunt ended fragments generated and, finally, PCR amplification of the fragment ladders. The analysis of the resulting patterns allows the identification of the nucleotides specifically involved in protein-DNA interactions. This technique allowed for the recognition of several protein-DNA contacts in the origin area, which are schematically indicated in Figure 3 panel B. They correspond, within the non-transcribed spacer, to the binding sites for: 1) the b-HLH family of transcription factors; 2) a still unrecognized protein; 3) the NRF-1 factor. A b-HLH protein, spefically binding to the cognate site with the ori region, was purified to homogeneity from human cells and turned out to correspond to transcription factor USF (Csordas Toth et al., .1993); another potential binding protein belonging to the same family is the product of the c-myc oncogene (which, incidentally, is amplified in HL-60 cells). Strikingly, a very large in vivo footprint appears to cover a ~ 70 nt region corresponding to the B48 marker (named HOR for human origin protein in Figure 3 panel B) with the generation of large DNase I hypersensitive area in an immediately upstream ATrich sequence; this footprint completely disappears in terminally differentiated (i.e. non proliferating) HL-60 cells (Dimitrova et al., submitted).

Conclusions In conclusion, it appears that the development of the competitive PCR mapping assay for ori localization, coupled to the protein-DNA interaction studies, finally promises to shed light on the problem of the identification and characterization of a human origin of DNA replication. Nevertheless, several fundamental ques-

265 t i o n s r e m a i n to b e a n s w e r e d , b o t h at t h e D N A level (is an ori a c t i v e w h e n t r a n s p o s e d to a n o t h e r c h r o m o s o m a l l o c a t i o n ? Is t h e s a m e ori utilized in d i f f e r e n t cell types a n d t i s s u e s ? W h i c h are the g e n e t i c d o m a i n s essential for ori f u n c t i o n ? ) a n d at the p r o t e i n level ( w h i c h are the p r o t e i n s i n v o l v e d in ori a c t i v a t i o n ? H o w is this process c o n t r o l l e d ? H o w is r e - i n i t i a t i o n f r o m t h e s a m e ori p r e v e n t e d u n t i l c o m p l e t i o n o f t h e cell cycle?). A n s w e r ing t h e s e q u e s t i o n s is o b v i o u s l y o f p r i m a r y i m p o r t a n c e in o r d e r to fill t h e gap w h i c h actually links the m e c h a n i s m s o f c e l l - c y c l e to t h e p r o c e s s o f a c t i v a t i o n o f D N A replication.

Acknowledgements T h e w o r k r e p o r t e d in this r e v i e w was initiated in P a v i a in c o l l a b o r a t i o n w i t h Prof. S i l v a n o R i v a a n d Dr. G i u s e p p e B i a m o n t i o f t h e Istituto di G e n e t i c a B i o c h i m ica ed E v o l u z i o n i s t i c a del C o n s i g l i o N a z i o n a l e delle R i c e r c h e . T h i s e s s e n t i a l c o l l a b o r a t i o n is c o n t i n u i n g , a n d t h e p r o j e c t is in fact a j o i n t effort of the Trieste a n d P a v i a l a b o r a t o r i e s . T h e p r o j e c t is partially s u p p o r t e d b y g r a n t s f r o m t h e S c i e n c e ( g r a n t No. E R B - S C l * C T 9 2 0 8 2 2 ) a n d H u m a n C a p i t a l a n d M o b i l i t y (grant No. ERBCHRXCT920022) programmes of the European Community.

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