Excision of integrated simian virus 40 DNA involving homologous recombination between viral DNA sequences

J. Mol. Biol. (1989) 206, 81-90

Excision of Integrated Simian Virus 40 DNA Involving Homologous Recombination between Viral DNA Sequences Said Dora, Claudia Schwarz and Rolf Knippers Fakultiit j’iir Biologic Universittit Konstanz D-7750 Konstanz. F.R.G. (Received 17 June 1988, and in revised form 11 November 1988) We have investigated the structure of simian virus 40 (SV40) DNA integrated into the genome of transformed mouse mKS-A cells. We have identified at least six independent integration units containing intact or truncated SV40 DNA sequences. One integration unit was isolated from a genomic mKS-A cell library and investigated by restriction enzyme analysis and partial nucleotide sequencing. This integration unit contains one apparently intact SV40 genome flanked on both sides by truncated versions of the SV40 genome. One of the flanking elements contains a large deletion in the SV40 “late” region and an abbreviated SV40 “early” region. This element was efficiently excised and mobilized after fusion of mKS-A to COS cells. The excision products invariably included the entire SV40 early region even though they were derived from an integrated element lacking this part of the SV40 genome. An analysis of this discrepancy led to the conclusion that the early region sequences were acquired by homologous recombination and, furthermore, that homologous excisional recombination was clearly preferred over non-homologous recombination.

Integration of SV40 DNA is reversible. Excision and mobilization of integrated viral DNA occurs when the following conditions are fulfilled: (1) the origin sequence of the integrated virus DNA must be intact (Gluzman, 1981; Conrad et aZ., 1982); (2) a product of an early viral gene, T antigen, the regulator of viral DNA replication, must be functional (Miller et al., 1984); and (3) the permissivity factors of monkey host cells must be supplied. These requirements are achieved experimentally by fusing transformed rodent cells to permissive monkey cells (for a summary, see Tooze, 1982). However, the T antigen in transformed cells is frequently altered mutationally (Rigby & Lane, 1983) and cannot function as a replication initiation protein. To induce excision in these cases, the transformed rodent cells must be fused to the monkey COS cell line (Gluzman, 1981), which produces an active T antigen coded for by chromosomally integrated SV40 DNA that cannot be mobilized itself as it lacks an intact’ origin sequence. The excision pathway appears to proceed in three phases: (1) an interaction of T antigen and the origin induces replication in situ of the integrated SV40 DNA and adjacent cellular DNA sequences; it is possible that the integrated viral origin is activated several times, leading to a complex

1. Introduction The DNA of simian virus 40 (SV40) can integrate into the genome of infected non-permissive rodent tissue culture cells. When the “early” viral coding region remains intact after integration, a viral gene product, the large T antigen, is expressed and may induce the transformation of the infected cell to immortalization and tumorigenicity (for a review, see Tooze, 1982). Analyses of DNA from transformed cells have shown that the integration of SV40 DNA is frequently accompanied by multiple recombination events, leading to complex patterns of several partially deleted, inverted or otherwise rearranged SV40 DNA elements organized as tandem integration units or as integration units interspersed with cellular DNA sequences. The simple insertion of a single SV40 DNA molecule appears to be the exception rather than the rule. Moreover, integration requires no site specificity or long stretches of sequence identity between viral and cellular DNA. However, short regions of incomplete nucleotide sequence similarities are found frequently around the sites where viral DNA and cellular DNA are linked (for reviews of earlier literature, see Tooze, 1982; Stringer, 1982; Savageau et al., 1983: Bullock et al., 1984, 1985; Mounts & Kelly, 1984; Huber et al., 1985; Gish & Botchan, 1987). 0022-2836/89/OXKB-10

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pattern of localized polytenization (the “onion skin model”; Botchan et al., 1979); (2) there is recombination involving sections of the replicated DNA and resulting in the excision of the integrated SV40 DNA; and (3) the excised DNA may continue to replicate as an extrachromosomal element. When an integrated single copy is activated, replication forks invade adjacent cellular sequences, and excisional recombination leads to excision products containing the SV40 origin and neighboring viral and cellular DNA sequences (Bullock et al., 1984, 1985). In contrast, activation of tandemly integrated multi-copy SV40 elements results in a more uniform set) of excision products (Botchan et al., 1979; Hanahan et al., 1980; Huber et al., 1985) that could arise by homologous recombination involving secGons of the integrated viral sequences. This result need not mean necessarily that DNA regions of homology are preferred recombination sites. It is also possible that replication forks invade the origin-proximal SV40 DNA sequences for some distance and come to a stop before they meet the flanking cellular DNA. Recombination between viral DNA sect,ions would be the only possible consequence. We are investigating the structure of a mutant T antigen, produced in the SV40-transformed mouse cell line mKS-.A (Dubbs et al., 1967). In the course of these studies we have isolated a genomic mKS-A DNA segment encompassing a central, apparently complete, SV40 genome flanked on both sides by truncated SV40 elements linked to cellular DNA. One of these flanking truncated SV40 elements contains a functional SV40 origin that is activated after fusion of mKS-A cells to COS cells, resulting in a major class of uniform excision products of about 3.2 kbt. The distance between the origin and the flanking cellular DNA is less than 1.5 kb. We asked whether or not these cellular DNA sequences appear in the excision products. We found that large sections of viral DNA, but no cellular DNA, were acquired during excisional recombination. We argue that homologous recombination is clearly preferred over non-homologous recombination during the excision of SVPO DNA.

2. Material and Methods (a) Cells

together in a 9 cm dish and incubated for 24 h in Dulbecro-modified Eagle’s (DME) medium with 100/(, (w/v) newborn calf serum. The cultures were then washed with DME medium without serum and briefly treated with 507; (v/v) polyethylene glycol 1000. Cultures were then washed with 12.5% (w/v) dimethylsulfoxide in DME medium, washed again with DME medium alonca and incubated under normal growth conditions for 48 to 60 h. The low molecular weight DNA was recovered from the supernatants by the Hirt (1967) procedure. (r) Analysis of mobilized 8 V&I-l)NA Pilot experiments had shown that each of the 2 major SV40 DNA species in the Hirt supernatants (see below)

contained 1 RamHT restriction site. Conseyuently. Hirt supernatant DNA was digested with this endonuclease and ligated to BarnHI-restricted plasmid pATl53 DNA. The resulting bacterial clones were screened according to standard procedures (Maniatis et al.. 1982) for the presence of SV40 sequences using nick-translated (Rigby et al., 1977) 32P-labeled SV40 DNA as a probe. Plasmids with SV40 DNA inserts were prepared and investigated by restriction mapping and partial nucleotide sequencing. For this purpose some SV40 DNA restriction fragments were subcloned in the M13mp18 and mp19 cloning systems (Messing, 1983) and sequenced using the dideoxy chain-termination technique of Sanger et aZ. (1977). (d) Analysis of integrated viral IINA Total cellular DNA from mKS-A cells was isolated as described (Maniatis et al., 1982). Samples of 2Opg DNA were digested with restriction endonucleases (BoehringerMannheim) according to the suppliers’ recommendations, electrophoresed in 0.8% (w/v) agarose gels and transferred to nitrocellulose filters according to Southern (1975). Hybridization to nick-translated “P-labeled SV40 DNA was performed under the conditions of high stringency as described (Hames & Higgins, 1985). For cloning of genomic DNA, mKS-A DNA was partially digested using the restriction endonuclease SauSA. DNA fragments of 15 to 20 kb were ligated to the arms of the lambda-derived phage vector EMBLS (Frischauf et al., 1983). The packaged phages of 3 libraries were combined, giving a total of 7.2 x 10’ phage clones, and amplified. Phages of the combined libraries were screened with 32P-labeled (nick-translated) SV40-DNA. Six strongly positive clones were isolated and expanded for DNA preparation (Maniatis et al., 1982). All isolated phage clones showed identical DNA restriction maps. Parts of the inserted DNA were subcloned in the plasmid vector pAT153 for more detailed restriction mapping. Selected restriction fragment)s were further subcloned in M13mpl8 and mp19 and used for nucleotide sequencing as described above.

The SV40-transformed mouse kidney cell line mKS-A was isolated and described by Dubbs et al. (1967). CO&l cells are CVl monkey cells transformed by an origindeleted SV40 mutant DNA (Gluzman, 1981). (b) Cell fusion The fusion

of mKS-A

cells and CO&l

cells was

performed essentially as described by Botchan et al. (1979). About

lo6 cells of each cell line were seeded

t Abbreviations:

kb, 10’ base-pairs; bp, base-pairs.

3. Results and Discussions (a) SV4U DNA integrates in the mKS-A cell genome

We have screened a genomic library of mKS-A DNA, cloned in the EMBL3 phage vector, for sequences hybridizing with SV40 DNA. We have isolated a phage clone termed L-SV 2.1, which was characterized by restriction mapping, Southern blotting and partial nucleotide sequencing.

Excision of Integrated S V40 DNA

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Figure 1, Restriction map of the insert in the EMBLS clone L-SV 2.1. (a) Open region, cellular DNA; cross-hatched. SV40 DNA. Restriction sites: B, BumHI; H, HindIII. The numbers refer to the SV40 nucleotides (Tooze, 1982) as shown in (b). The triangles indicate the positions of functional SV40 origins. The insert was subdivided by BumHI restriction to give 3 sections. The BamHI fragments were cloned separately in the plasmid vector pAT153 for precise restriction mapping. Fragments of the plasmid clones were then subcloned in Ml3 vectors for sequencing. The region between the Hind111 restriction site at nucleotide 5171 of section A and the right-hand viral-cellular junction was sequenced. No gross structural alterations were found in the section A DNA. A deletion of about 2000 bp (encompassing the region between SV40 nucleotides 144 and 2113) was detected in the SV40 section C DNA. The right-hand SV40 DNA element was found to be linked to cellular DNA at SV40 nucleotide 3765 as indicated. The section B DNA and the left-hand viral-cellular junction were not sequenced. Southern blotting suggested that the BgZI site close to the left end of the integration unit corresponded most likely to SV40 nucleotide 5235. But note the absence of the Hind111 site at SV40 nucleotide 5171. The sections labeled a, b, and c of cellular DNA sequence were used as hybridization probes in some of the experiments to be described below. (b) A restriction map of SV40 DNA. We show the restriction sites relevant t,o the work reported here. The SV40 numbering system of Tooze (1982) was used. The hatched bar within the SV40 map indicates the early coding region for large T antigen. The hybridization probes used in the experiment of Fig. 2 are indicated as bold segments. The results of these experiments show (Fig. 1) the following. (1) The approximately 17 kb long inserted DNA in the EMBL3 clone L-SV 2.1 includes tandem SV40 elements that can be divided into three restriction. The central sections by BamHI section A appears to contain a full-length SV40 genome, as all the restriction fragments indicated in Figure l(a) have exactly the same sizes as the corresponding fragments from wild-type SV40 DNA (Fig. l(b)). The origin and the early coding region up to the BamHI site in section A were sequenced. We detected a number of point mutations but no gross structural alterations. The point mutations in the viral genomic control region were deletions and nucleotide exchanges as described below. We also found eight nucleotide

exchanges (but no deletions) in the “early”, T antigen-coding region. Most of these mutations were neutral or conservative except for an exchange at codons 636 and 637 leading to an Asp-Glu to Asn-Asp replacement in the predicted amino acid sequence. We argue elsewhere that this exchange may be, at least partially, the reason for the replication-negative phenotype of the T antigen, expressed in mKS-A cells (Dora et al., unpublished results). (2) Section B (on the left of the section A:L; Fig. l(a)) lacks the entire “late” region of the SV40 genome (as demonstrated by restriction mapping and Southern

blotting;

not shown).

We expect

some

rearrangements around the SV40 origin of replication as the Hind111 restriction site at SV40 nucleotide 517 1 is lacking in this region.

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Figure 2. Southern blots of restricted mKS-A I)PU’A. Samples of cellular DNA (20 pg) were restricted with the following endonucleases: XbaI (lanes I), Sac1 (lanes 2), BglII (lanes 3), BumHI (lanes 4), EcoRI (lanes 5). or BgZI (lanes 6). The first 3 endonucleases do not cut SV40 DNA. The restriction enzymes BumHI, EcoRI and RgZI cut the SV40 genome at unique sites (see Fig. l(b)). The restricted DPU’A was separated on 0.6% agarose gels. The DNA was transferred to nitrocellulose filbers and hybridized to nick-translated 32P-labeled SV40 DKA (a). to the 321'-labeled Sk'40 BgZI-Z’aqI fragment (b), or to the 32P-labeled SV40 Hind111 D fragment’ (c). The hybridization probes are shown in Fig. l(b). We show the autoradiograms after 2 days of exposure with intensifier screens. (3) The entire nucleotide sequence of the SV40 part of section C (on the right; Fig. l(a)) has been

determined. This part includes an apparently functional origin of replication (see below) and about 1200 bp of the promoter-proximal early coding region. The genetically late SV40 region is truncated by a large deletion of the SV40 DNA sequence between nucleotide 144 and nucleotide 2113. To exclude the possibility of cloning artefacts and to find out whether there was more than one SV40 integration unit in the mKS-A genome, we restricted mKS-A DNA and carried out Southern blots for hybridization with SV40 DNA sequences. Using restriction endonucleases XbaI, BgZII and SacI, which do not cut SV40, we obtained five and six hybridizing bands, respectively, suggesting a minimum of six SV40 integrates (Fig. 2(a)). Only four of these bands hybridized to the SV40 region between nucleotides 5235 (BgZI site) and 4739 (Tuqt site) (Fig. l(b)), which includes parts of the viral origin of replication, suggesting that not all SV40 integrates contained an intact origin of replication. Furthermore, only three of the five BgZIt restriction fragments hybridized to the SV40 element between nucleotides 4002 and 3476 (the HindIII-D fragment, Fig. l(b)) showing that not all SV40 integration units contained an intact early coding region. The mKS-A DNA was also restricted with endonucleases that recognize a unique site in the SV40 genome. We found that the map of the SV40 elements in L-SV 2.1 DNA (Fig. l(a)) is consistent,

with the genomic blot data of Figure 2. For example, hybridization of BumHI restricted genomic mKS-A DNA with the BgZI-TaqI probe (Fig. l(b)) revealed two closely spaced bands, approximately 5.2 kb in size (Fig. 2(b)), which most probably correspond to sections A and C of the insert shown in Figure l(a). Only one of these two bands hybridized to the HindIII-D probe (Fig. 2(c)), and this band may correspond to section A of the EMBLS clone L-SV 2.1. One of the additional two bands in BamHI-restricted mKS-A DNA corresponds most likely to section B of the insert (Fig. l(a)), the other band must originate from another as yet unidentified integration unit. Digestion of mKS-A DNA with the singly cutting enzyme BamHI gave (among others) DNA fragments of 1.4 and 1.2 kb that hybridized to the BgZI-TaqI probe but not to the HindIII-D probe (Fig. 2(b) and (c)). Identical fragments were identified in BgZI-restricted mKS-A DNA. These results suggest that at least three SV40 elements with large internal deletions are integrated as tandem units in one of the six (or more) integration sites (see below). Taken together, the data of Figure 2 indicate that, the mKS-A genome harbors a minimum of six XV40 DNA integration units that are separated from each other by stretches of cellular DNA, and, furthermore, that deletions and rearrangements of SV40 DNA must have occurred during the establishment of the transformed mKS-A cell line. The structure of the integrated SV40 DNA is now quite stable. as results such as those shown in

Excision of Integrated SV40 DNA

85

Figure 2 were obtained in Southern blot experiments performed four years and many cell generations ago (Dora et al., unpublished results). The blotting data are also essentially similar to those reported by Huber et al. (1985), who had previously investigated mKS-A cell DNA by Southern blotting. (b) Mobilization

of S V40 DNA

The SV40 DNA integrate in clone LSV 2.1 contains two, apparently intact, origin sequences around the BgZI restriction sites (Fig. l(a)), and evidence presented below suggests that the origin sequences in sections A and C serve as starting points for DNA replication in situ. Replication forks established at the origin in section C (Fig. 1(a)) are expected to move leftward into the section A viral DNA and rightward into the adjacent cellular DNA, which begins at a distance of less than 1.5 kb from the origin. This situation offers the possibility of testing whether the adjacent cellular sequences appear in the excision products obtained after mobilization of the integrated SV40 DNA in mKS-A/COS cell fusion experiments. Cellular DNA sequences are expected to be present in these excision products unless they are eliminated during the recombinational processes necessary to mobilize the in situ replicated SV40 DNA. An alternative possibility would be that replication forks are prevented from crossing the SV40-cell DNA boundary. But this possibility appears to be less likely, as shown by the work of Bullock et al. (1984). To investigate these points we have fused mKS-A cells to (:OS-1 cells as a source for permissivity factors and functional T antigen (see Introduction). The SV40 sequences mobilized in the fused cells were recovered as low molecular weight DNA in Hirt supernatants. They were then separated by agarose gel electrophoresis and visualized by hybridization with 32P-labeled SV40 DNA. We show in Figure 3 the results of five independent experiments to demonstrate the reproducibility of the results. In all experiments, we obtained two major excision products, namely a 3.2 kb DNA and a 5.2 kb DNA, in addition to a number of minor SV40 DNA species of different size classes. As discussed below, the mobilized SV40 DNA elements of different size classes most probably originated from different SV40 DNA integration units. To investigate the structure of the mobilized SV40 DNA in more detail, we cloned the BamHIrestricted Hirt supernatant DNA in the BamHI site of the plasmid vector pAT 153. In three independent experiments, we obtained between 50 and 60 plasmid clones each. The plasmid clones were screened for inserts hybridizing to SV40 DNA and to the cellular DNA sequences derived from the flanking regions of the LSV2.1 integration unit (see Fig. l(a)). We isolated a total of 13 plasmid clones with SV40 DNA inserts. Three clones contained SV40 DNA elements without viral origins. The

.5+2 kb

-3.2 kb

Figure 3. Mobilized SV40 DNA sequences as low molecular weight DNA in mKS-A/CO&i heterokaryons. We show t,he results of 5 independent experiments. Hirt supernatants were prepared 48 h after fusion of’ mKS-A and CO&l cells (see Materials and Methods). The low molecular weight) DNA was restricted using endonuclease RamHI. Pilot experiments had shown that the indicated major excision products contained unique BumHI sites. The DKA was separated on a 0+37/, agarose gel, transferred to nitrocellulose filters and hybridized to 32P-labeled SV40 DKA. We show the autoradiogram of the filter after 24 h exposure with an intensifying screen.

inserts in these three clones were identical. They were about 0.7 kb in size and corresponded to a rearranged SV40 DNA section around the viral region between SV40 nucleotides 2000 and 3000 (Fig. 1(b)). The structure and origin of these viral DNA segments were not investigated in detail. Ten clones contained the viral origin replication. The structure of these insertions will be described below. The majority of the plasmid clones carried cellular DNA segments of unknown sequence. They were most likely derived from fragmented nuclear DNA present in the Hirt supernatants or from mitochondrial DNA. The appearance of fragmented cellular DNA in Hirt supernatants is not necessarily connected to the mobilization of integrated viral DNA, as a significant amount of nuclear DNA fragments can be found in Hirt supernat,ants of normal cultured cells. In the present context it may be more important to point out that no plasmid clone carried cellular

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Figure 4. Nucleotide sequences around the origins of replication. The SV40 numbering system of Tooze (1982) is used (see Fig. l(b)). The wild-type SV40 sequence (upper line. SV40) is the published sequence of Fiers et al. (1978). The mobilized elements SD3, SD4, and SD5 are described in the text and shown in Fig. 5. Pu’umbers in brackets indicate the position of common mutations found in all 3 classes of investigated mobilized elements. Asterisks indicate the mutations found in the SD5 element as well as in the section C sequence of the L-SV 2.1 insert (Fig. l(a)), but not in the SD3 and SD4 element,s. DNA sequences derived from the regions flanking the SV40 DNA integration unit (probes a, b and c in Fig. l(a)). It is, of course, possible that these sequences could not be cloned by the particular procedure used. To find out whether flanking cellular DNA sequences were mobilized in mKS-A/ COSl heterokaryons, we hybridized a Southern blot like that shown in Figure 3 to nick-translated flanking cellular DNA sequences (probes a, b and c of Fig. l(a)). We could not detect’ distinct hybridizing DNA bands (data not shown), suggesting that a significant excision of cellular DNA sequences adjacent to the L-SV2.1 integration unit did not occur. (c) Excised viral DNA elements As mentioned above, we obtained from three independent experiments a total of ten plasmid clones carrying SV40 DNA inserts with viral origins. Six of these clones contained a 3.2 kb DNA insert, one clone had a 5.2 kb insert, whereas the remaining inserts were smaller, two clones with a 1.4 kb and one clone with a 1.2 kb insert. The structures of the cloned mobilized SV40 DNA sequences were determined by restriction mapping and nucleotide sequencing. All 3.2 kb inserts, the SD5 group of sequences, were identical, even though they were recovered from the Hirt supernatants of three independent experiments performed at different times and with different cell passages. The cloned SD5 sequences corresponded to the major 3.2 kb excision products shown in Figure 3 as a characteristic BamHI-Hind111 fragment of about 640 bp, present in the SD5 element (see below), was also detected as a major restriction fragment after BamHI and HindITT

digestion of the Hirt supernantant DNA of Figure 3 (data not shown). The two 1.4 kb inserts, the SD4 sequence, were identical. They were found to be similar to the 1.2 kb insert, the SD3 sequence, which differed from the SD4 group by a 200 bp deletion. Neither the SD5 group nor the SD4 and SD3 groups of cloned sequences contained cellular DNA sequences. To illustrate the similarities and the differences between the SV40 sequences in the SD3, SD4 and SD5 groups of clones we first show the nucleotide sequences around their origins of replication (Fig. 4). We found, first, that all sequences differed from the SV40 wild-type sequence at three sites, a C to T nucleotide exchange at SV40 nucleotide 5209 and deletions of the SV40 nucleotides 3 and 82 (the “common mutations”). Second, the SD5 sequences differed from the SD3 and SD4 sequences at two sites, a deletion of nucleotide 8 and a C to C exchange at nucleotide 44 (the SD5-specific mutations). These data suggest that the three classes of mobilized SV40 DNA elements were derivatives of a common parental sequence, characterized by the three common mutations, and that they have diverged during the establishment of the mKS-A cell line into two groups. During divergence t,htk SD5 sequence acquired the two additional specific mutations. These SD5-specific mutations were also found in the origin region of section C of t’he I,-SV 2.1 integrate (Fig. l(a)) suggesting that the SD5 mobilized element originated from replicated section C DNA. The difference between the groups of mobilized sequences was underlined by the general molecular architecture of the cloned sequences (Fig. 5). The SD5 element was characterized by a full-length

Excision of Integrated SV40 DNA

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Figure 5. Structure of excised and mobilized SV40 DNA elements. The restriction maps were linearized by cutting at the uniyue BumHI sites. Upper line: SV40 wild-type DNA, the origin as well as the early and late genetic regions are indicated (arrows indicate direction of transcription); the numbers indicate SV40 nucleotides that are relevant for comparison with the excised DNA elements. The maps of the excised DNA elements (lower lines) are drawn colinearily

with the wild-type SV40 DNA: bold lines, DNA sequencespresent in the excised elements; bracketed thin lines, deleted DNA. For examnle. deletion A in the SD5 seauence extends from SV40 nucleotide *

1

144 to nucleotide 2113. All DNA

regions represented by bold lines have been sequenced. early region and a deletion of about 2 kb in the late genomic region (Fig. 5). Both the SD3 and the SD4 elements consisted of SV40 sequences that differed from the wild-type SV40 genome by large deletions including the region between nucleotides 2718 and 5033 on the early side, and nucleotides 523 and 2057 on the late side, of the SV40 origin. The SD3 element had an additional deletion of 220 bp in the late SV40 region (Fig. 5). The SD3/SD4 sequences were probably integrated in tandem at one site of the mKS-A genome as the SD3 and SD4 elements have exactly the same size as the BamHI and BgZI fragments seen in the Southern blots of Figure 2. As discussed before, these fragments hybridized to the Bgll-TaqI but not to the HindIII-D probe, exactly as expected for the SD3 and SD4 elements. The SD3/SD4 elements, organized as tandem integration units, could be activated in mKS-S/COS-1 heterokaryons at either the SD3 or the SD4 origin, leading to a set of different, but related, excision products. BamHI restriction of these products resulted in relatively small DNA fragments with relatively high electrophorectic mobility. This may be the reason why they were not discovered in the experiment illustrat#ed in Figure 3. (d) The SD5 excision product The source of the SD5 excision product is more interesting for the present discussion. The SD5 element, one of the most abundant excision products (Fig, 3), was almost certainly derived from the section C of the SV40 DNA integrate shown in Figure 1(a). We conclude this because the section C SV40 element, contained a deletion of the late SV40 region between nucleotides 144 and 2113, the diagnostic feature of the SD5 excision product.

Furthermore, the SD5-specific point mutations (Fig. 4) were also found in the origin region of section C DNA. However, the SD5 sequences and the section C sequence differed by the presence of the entire early SV40 DNA region in the SD5 excision product and the lack of this region in the inserted section C SV40 DNA. The most likely explanation for this discrepancy will now be discussed. We consider first the situation after an association of T ant,igen and permissivity factors with the origin in the section C sequence of the I>-SV 2.1 integration unit. Two replication forks will be established moving bidirectionally into the adjacent DNA sequences. The left-hand fork must proceed at least for about 2000 bp up to around SV40 nucleotide 4000 of the central SV40 DNA section A (Fig. 6). The branches of the left-hand replication fork are essential partner for excisional strands recombination. The right-hand fork is expected to travel a similar distance. We assume this because the SV40 origin is known to be activated in a symmetrical manner. During ring-to-ring replication in permissive host cells the replication forks meet each other half way on the circular viral DNA at a point exactly opposite to the origin (for a review, see DePamphilis & Bradley, 1986). A bidirectional and symmetrical activation of the origin of an integrated SV40 DNA element is also likely, as shown by the results of previous studies on the mobilization of single-copy SV40 DNA integrates (Bullock et al., 1984). It’ would also be consistent with the general organization and functioning of eukaryotic replicons (Huberman & Riggs, 1968; Hand, 1978). Our assumption that the right-hand replication fork can invade adjacent cellular sequences (Fig. 6) was supported by experimental evidence. We have

a 2,,3'LL

5000

Figure 6. A possible excision pathway. The parallel lines represent the right half of the L-SV 2.1 insert DNA (Fig. 1). Open area, SV40 DNA sequences; hatched area, cellular sequences, SV40 nucleotide numbers, the diagnostic deletion (between nucleotides 144 and 2113) and the origin (triangle) are indicated. After replication in situ, SV40 sequences between SV40 nucleotides 4000 and 5000 are engaged in homologous recombination (arrows). Replicated cellular sequences do not participate in recombination. The result of excisional recombination is the circular SD5 element containing

the entire early region.

prepared high molecular weight DNA from the Hirt pellets of heterokaryons at various times after fusion of mKS-A and COS cells. The DNA was denatured, dot-blotted on nitrocellulose filters and to 32P-labeled SV40 DNA or to h bridized 3PP-labeled DNA probes from flanking cellular sequences (probes a and b, Fig. l(a)). We found, as expected (Botchan et al., 1979), that the amount of hybridizable SV40 DNA increased several-fold with time after fusion (Fig. 7). We also found an increase of DNA that hybridized to the cellular probe a fragment (Fig. l(a)), an approximately 800 bp piece of DNA, located at a distance of 1040 bp from the right SV40-cellular DNA junction in the l-SV2.1 integration unit (Fig. 7). More distantly located cellular DNA participated less well in replication, as the amount of DNA hybridizing to probe b barely increased after mKS-A/COS cell fusion (Fig. 7). Probe b DNA corresponds to a section of flanking cellular DNA located almost 2 kb from the SV40cellular DNA junction (Fig. l(a)). Replication appears to be required as a condition for excisional recombination, which may be stimulated by strand discontinuities in the replicated DNA branches. However, cellular DNA sequences could not be detected in the six independently

obtained and cloned SD5 DNA elements, indicating that only viral DNA sequences were engaged in excisional recombination (Fig. 6). Thus, recombination between homologous sequences was clearly preferred over recombination between non-homologous sequences. The SD5-element was a major product found in of mKS-A/COS heterothe Hirt supernatants karyons, indicating that it was not only mobilized by replication in situ and homologous excision but also multiplied by extrachromosomal replication. This is somewhat surprising as the origin of the SD5 element deviates from the wild-type SV40 origin by deletions of SV40 nucleotides 3 and 28 (Fig. 4). Previous studies had shown clearly that both sites are important for the function of the SV40 origin during replication in vivo and in vitro (Deb et al., 1986; Dean et al., 1987). However, we could show that SD5 DNA replicated efficiently in vivo after transfection into COS cells as well as in the in vitro replication system of Li & Kelly (1985) (Dora et al., unpublished data). It is possible therefore that deletions of these nucleotides have less drastic effects on the origin function than do the base substitutions investigated by Tegtmeyer and his colleagues (Deb et al., 1986; Dean et al., 1987).

Excision

of Integrated

sv40

XV40 DNA

89

blotting, not shown) that were identical in size with those found in the section A of the L-SV 2.1 integration unit. The appearance of a unit length SV40 DNA excision product after activation of the origin in section A is not surprising, as this section is flanked on both sides by long stretches of SV40 DNA.

4. Conclusion probe a

probe b

We can account for the pathway by which one of the major extrachromosomal SV40 DNA elements, the SD5 sequence, was produced after the fusion of mKS-A to COS cells. An essential part of this pathway appears to be homologous excisional recombination involving the viral sequences in the branches of an in situ replicated SV40 integration unit. Non-homologous cellular sequences that most likely constituted parts of the same replicon were excluded from the recombination process. Our findings that homologous recombination appeared to be clearly preferred over non-homologous recombination may warrant a biochemical search in mammalian cells for an enzyme that preferentially cuts homologous sequences in recombination intermediates. A yeast endonuclease with these properties has recent,ly been described (Parsons & West,, 1988). This work was supported by Forschungsgemeinschaft through SFR 156.

Figure 7. Replication in situ. The fusion of mKS-A and COS-1 cells was performed as described in Materials and Methods. Low and high molecular weight DNA was separated by the Hirt procedure. The high molecular weight DNA in the Hirt pellet was resuspended and sequentially treated with RPu’ase and proteinase K before extraction with phenol/chloroform. Samples of 1 pg and of 2 pg of dialyzed DMA were denatured in 0.20 M-KaOH, renatured in Tris.HCl (pH y), and dot-blotted on nitrocellulose strips. The filter-bound DNA was hybridized to nick-t’ranslated SV40 DNA as well as to cellular probe a and probe b DNA (see Fig. l(a)). We used DNA prepared at 2 h (1), 6 h (2), 12 h (3). 24 h (4) and 48 h (5) after cell fusion.

Finally, we have described in this study the st,ructure of only one of the two major exision products, the 3.2 kb SD5 element, and the structure of the two minor products, the SD3/SD4 sequences. We have not investigated in detail the second major excision product, the 5.2 kb element (Fig. 3). We have only one plasmid clone carrying a 5.2 kb insert, but it is quite likely that the major 5.2 kb excision products shown in Figure 3 resulted from an activation of section A of the L-SV 2.1 integration unit. This conclusion is based on two observations: (1) the origin in section A (Fig. l(a)) has the same structure as the origin in section C and should consequently be of comparable efficiency; (2) restriction of the Hirt supernatants, shown in Figure 3, by BamHI and Hind111 resulted in a number of fragments (identified by Southern

Deutsche

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Hand, R. (1978). (Irll, 15. 317-325. Hirt, B. (1967). J. A!IoZ.Hiol. 26, 365-369. Huber, B., Vskalopoulou. E.. Burger. (“. &, Fanning. E. (I%%). ~i?YdOgy. 146. 188-~202. Huberman, J. A. & Riggs. A. 1). (1968). .I. Mol. Hiol. 32. 327-342. Li. .J. ,J. & Kelly. T. ,J. (1985). No/. (‘PII. Niol. 5, 1238 1246. Maniatis, T.. Fritsch. E. F. bz Sambrook. !J. (1982). Editors of Molecular Cloning: A Lalmatory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Martin. R. (:. & Oppenheim, A. (1977). Cell. 11, 85!f 869. Messing, J. (1983). Methods Enzymol. 101. 20--78. Miller, ,J.. Bullock, P. & Botchan, M. (1984). Proc. A&. Acad. A%%..C!.S.A. 81. 7534-7538.

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Edited by J. Karn