Replication Initiates at Multiple Locations on an Autonomously

MOLECULAR AND CELLULAR BIOLOGY, Mar. 1991, p. 1464-1472 0270-7306/91/031464-09$02.00/0 Copyright © 1991, American Society for Microbiology

Vol. 11, No. 3

Replication Initiates at Multiple Locations on an Autonomously Replicating Plasmid in Human Cells PATRICK J. KRYSAN AND MICHELE P. CALOS* Department of Genetics, Stanford University School of Medicine, Stanford, California 94305 Received 6 November 1990/Accepted 26 December 1990

We have used a two-dimensional gel electrophoresis mapping technique to determine where DNA replication initiates on a plasmid which utilizes a fragment of human DNA to replicate autonomously in human cells. Replication was found to initiate at multiple locations on the plasmid carrying the human sequence, in contrast to the pattern seen for an Epstein-Barr virus vector which served as a control with a fixed origin. The family of repeats, a portion of the Epstein-Barr virus origin of replication which is present our plasmid, was shown to function as a replication fork barrier. The nature of the stalled replicative intermediates on the human DNA-based plasmid further indicated that replication did not initiate at a single fixed position each time the plasmid replicated. The results suggest that the replication apparatus used to duplicate DNA in human cells may not have precise sequence requirements which target initiation to specific locations.

During each cell cycle, a mammalian cell initiates replication at thousands of locations throughout its genome (14). It is not clear, however, whether these locations represent fixed genetic elements or more randomly chosen sites with little sequence specificity. Addressing this question requires a technique for physically mapping where replication initiates within a given stretch of DNA. Recently developed two-dimensional gel electrophoresis techniques have been used successfully to determine that some yeast autonomous replication sequences (ARSs) function as fixed origins of replication, both on plasmids and in the yeast chromosome (1, 15, 16). Because of the large size of mammalian genomes, the two-dimension gel techniques do not currently have the sensitivity to detect replication events at single-copy loci. To overcome the sensitivity problem, several groups have developed alternative labeling techniques which have been used to show the presence of putative fixed origins of replication in mammalian cells, notably in the vicinity of the gene for dihydrofolate reductase (DHFR) (3, 9, 18, 25). By contrast, Vaughn et al. (27) have dealt with the problem of sensitivity by using a hamster cell line which has amplified approximately 1,000-fold a portion of its genome which includes the DHFR gene. With this increased amount of signal, they were able to apply the two-dimensional gel mapping techniques to a region of the hamster genome previously implicated as containing one or two specific origins of replication. They discovered, however, that replication appeared to initiate randomly within a 30-kb zone rather than at one or two fixed locations. These conflicting data indicate that additional information is needed before we can understand the nature of replication origins in mammalian cells. We have recently developed a system which allows autonomously replicating human sequences to be maintained as extrachromosomal plasmids in human cells (17). Since plasmid DNA can be selectively extracted from mammalian cells, the problem of low sensitivity due to genomic size is eliminated. In addition, working with a plasmid system allows one to manipulate the sequence of interest with ease. *

A major challenge faced by an autonomously replicating plasmid in a human cell is loss from the nucleus. Generally, a small circular piece of DNA is quickly lost, perhaps during mitosis when the nuclear membrane breaks down. To overcome this problem, we have taken advantage of the ability of portions of the Epstein-Barr virus (EBV) genome to retain linked DNA in the nuclei of human cells (17). This nuclear retention function is provided by the EBV family of repeats and the product of the viral EBNA-J gene. The family of repeats is one of the two functional elements which constitute the viral origin of replication, oriP (23). Approximately 1 kb from the family of repeats lies a 65-bp region of dyad symmetry. Both the family of repeats and the dyad symmetry region contain binding sites for the EBNA-1 protein. When the dyad region is removed from the oriP portion of an EBV vector, the plasmid cannot replicate (23). We have shown, however, that even in the absence of replication, such a plasmid is retained within the nuclei of human cells for a prolonged period of time (17). Such a nonreplicating plasmid, with its nuclear retention function intact, served as the vector into which fragments of the human genome were cloned in a previous study (17). Using this strategy, we were able to isolate a large number of human sequences which allowed the plasmid to replicate. We have now used the two-dimensional gel electrophoresis mapping technique of Brewer and Fangman (1) to determine where replication initiates on one such plasmid carrying a 20-kb human insert. MATERIALS AND METHODS

Plasmids and cell culture. The plasmids pLIB41 and p220.2 have been described previously (17). These plasmids were transfected by calcium phosphate coprecipitation (28) into human 293S cells (24), a suspension-adapted derivative of the human embryonic kidney cell line 293 (8). Four days posttransfection, the cells were split 1:10 into selective medium containing hygromycin B (200 ,ug/ml) and grown for approximately 10 days under this selection. Next they were split 1:10 to nonselective medium. (Dulbecco's modified Eagle's medium with 10% fetal calf serum, 100 U of penicillin per ml, and 100 ,ug of streptomycin per ml). Once confluent, these cells were split 1:12 to nonselective medium and harvested 48 to 55 h later, when the cells were approx-

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REPLICATION INITIATION AT MULTIPLE LOCATIONS

imately 50% confluent, in order to maximize the number of log-phase cells. There were approximately 8 x 106 cells per dish at the time of harvest. DNA isolation. Plasmid DNA was harvested from the human cells by a Hirt (13) extraction protocol. For each 100-mm dish harvested, the growth medium was removed from the dish and the cells were lysed by adding 3.5 ml of Hirt buffer (0.6% sodium dodecyl sulfate, 10 mM Tris (pH 8), and 10 mM EDTA). Ten to 30 min later, the lysed cells were scraped off the dish with a rubber policeman. Proteinase K was then added to the lysate to a final concentration of 100 pug/ml, and the tubes were incubated at 42°C for 1.5 to 2 h. Then 1 ml of 5 M NaCl was added per 100-mm dish of extraCt, and the tubes were gently inverted several times and placed at 4°C. After 12 to 48 h, the tubes were spun at 14,500 rpm at 4°C for 2 to 6 h in a Sorvall SS-34 rotor. The supernatant was then collected, and proteinase K was added to a final concentration of 100 ,ug/ml and incubated at room temperature for 1 to 3 h. The samples were then extracted with phenol, phenol-chloroform, and ether. The DNA was then precipitated by adding 0.6 volume of 2-propanol. The DNA pellet was resuspended in TE (10 mM Tris [pH 8], 1 mM EDTA) and precipitated by adding 0.5 volume of 7.5 M ammonium acetate and 1 volume of 2-propanol. The DNA extracted from 12 100-mm dishes of cells was resuspended in 500 ,ul of TE. This DNA was then digested with restriction enzymes and RNase A in a reaction volume of 500 to 700 ,ul. The digest was then precipitated by adding 1/10 volume of 3 M sodium acetate and 1 volume of 2-propanol. The pellet was resuspended in 15 p1l of TE and loaded onto the first dimension of a two-dimensional gel. Two-dimensional electrophoresis. Two different sets of electrophoresis conditions were used, depending on the size of the fragment being tested. The gels for p220.2 and the pLIB41 fragments shown in Fig. 5 were run under the following conditions for fragments smaller than -7 kb. The first dimension was a 0.35% agarose gel run at 33 V for 22 to 24 h at room temperature with no ethidium bromide. The gel was then stained with 0.3 ,ug of ethidium bromide per ml. The size range of interest from the appropriate lane was excised, placed against an already solidified second-dimension gel, and then sealed in place by pouring molten agarose around it. The second dimension was a 0.875% agarose gel with ethidium bromide (0.3 ,ug/ml) run at 75 V for 20 to 24 h at 4°C. The second-dimension gel was equilibrated for 2 to 3 h in running buffer containing ethidium bromide (0.3 ,ug/ml) at 4°C before running. For the fragments of pLIB41 larger than -10 kb (fragments I to V), the electrophoresis conditions were as follows. The first dimension was a 0.28% agarose gel run for 40 to 48 h at 25 V at room temperature. The second dimension was a 0.58% agarose gel with ethidium bromide (0.3 p.g/ml) run for 42 to 48 h at 35 V at room temperature. The second-dimension gel was equilibrated for 1 to 3 h in running buffer containing ethidium bromide (0.3 ,ug/ml) at room temperature before running. The gels were all run in TBE running buffer (108 g of Trizma base, 54 g of boric acid, and 8.4 g of EDTA per liter). The gels measured 12.5 cm wide by 27 cm long. The electrodes in the gel tank were separated by 37.5 cm. Blotting and hybridization. Gels were transferred via vacuum blotting to Zeta Probe (Bio-Rad) nylon membranes by an alkaline transfer protocol described by the manufacturer. The filters were then hybridized by the Blotto protocol described by the manufacturer, with the appropriate restriction fragment which had been labeled with 32P by random

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primer extension. The blots were washed according to the manufacturer's instructions and then exposed to XAR-5 film for 2 h to 7 days. RESULTS Two-dimensional gel technique. In an exponentially growing population of cells, a small percentage of DNA restriction fragments will have replication forks within them. Brewer and Fangman (1) have developed a two-dimensional agarose gel electrophoresis technique for revealing the identity of these replicative intermediates. These gels are called neutral-neutral gels because both dimensions are run under nondenaturing conditions. There are three basic types of replicative intermediates that can exist in a restriction fragment: a siihple Y, a bubble, and a double Y. A simple Y is formed by the presence of a single replication fork passing through the fragment. A bubble occurs when two replication forks are present in the fragment and are moving away from each other. The presence of a bubble indicates that replication initiated within the given restriction fragment. A double Y is.also formed by two replication forks in the fragment, but in this case the forks are moving towards each other until they ultimately meet at a replication terminus. To map the replication forks on our autonomously replicating plasmids, DNA was extracted from exponentially growing human cells and digested with a restriction enzyme. The digested DNA was run on the first-dimension agarose gel, which separates DNA mainly according to size. Under these conditions, the replicative intermediates trail behind linear fragments according to their extent of replication. At this point, no distinction can be made between the different replicative intermediates. The entire lane from the first dimension gel is then excised, turned 900, and incorporated into a second agarose gel. This second-dimension gel is run so that the three different types of replicative intermediates migrate with distinctive patterns. The gel is then Southern blotted and hybridized with a probe which will detect the fragment of interest. Figure 1 shows the patterns observed for the different types of intermediates. By determining which types of replicative intermediates different restriction fragments contain, one can map the replication origins used on a plasmid. If a replication fork barrier (2) is present in a fragment, then replicative intermediates will accumulate at a particular size and reveal themselves on the blot in the form of a spot

of increased hybridization intensity along an otherwise fairly uniform arc. By comparison with size standards run in the first dimension, one can estimate the location of this replication fork barrier on the restriction fragment being analyzed. Plasmids and cell lines. Two different autonomously replicating plasmids were used in this study. The first is p220.2, a 9.0-kb plasmid that uses the EBV origin of replication, oriP, and the product of the EBNA-1 gene to maintain itself extrachromosomally in human cells (Fig. 2A) (4). This plasmid also has the hygromycin resistance gene, a selectable marker in human cells, and pBR322 sequences for cultivation of the plasmid in Escherichia coli. p220.2 replicates once per cell cycle and is maintained at an average copy number of 50 per cell in human 293S cells (unpublished data). This plasmid serves as a control to demonstrate the ability of the two-dimensional gel technique to identify a fixed origin of replication under the experimental conditions used in this study. The other plasmid used in this study is pLIB41 (17). To

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