Mutation Research, DNA Repair, 274 (1992) 201-210 © 1992 Elsevier Science Publishers B.V, All rights reserved 0921-8777/92/$05.00

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MUTDNA 06500

Homologous recombination of adenovirus D N A in mammalian cells: enhanced recombination following UV-irradiation of the virus A n d r e w J. R a i n b o w a n d Jos~ E. Castillo Departments of Biology and Radiology, McMaster Unit'ersity, Hamilton, Ont, L8S 4KI. Canada (Received 3 September 1991) (Revision received 28 February 1992) (Accepted 15 March 1992)

Keywords: Adenovirus DNA, homologous recombination; Viral DNA. recombination

Summary We have used adenovirus as a molecular probe to examine the recombination of viral DNA following infection of mammalian cells. The technique gives a quantitative measure of homologous recombination between adenovirus type 2 (Ad2) and Ad51~MTR3. Ad5PyMTR3 is an insertion mutant of Ad5 containing polyoma virus (Py) DNA inserted into a deleted El region of the Ad5 genome. Cells were coinfected with Ad2 and Ad5PyMTR3 and at an appropriate time after infection, viral DNA was extracted from the infected cells, digested with restriction endonuclease and electrophoresed through an agarose gel. Although Ad2 and Ad5 have more than 99% DNA homology, they differ sufficiently in their restriction endonuelease patterns, such that recombinant viral DNA molecules containing the Py insert could be detected and quantified by Southern blotting and hybridization to a radioactive Py DNA probe. Using this method we are able to detect and quantitate recombinant viral DNA molecules containing the Py insert which are present at frequencies down to at least 1 in 100. Recombination was detected in Chinese hamster ovary cells, monkey kidney cells, human HeLa cells, normal human fibroblasts and SV40 transformed human fibroblasts. In experiments using HeLa cells, the frequency of recombination between the Py insert on Ad5PyMTR3 and a number of unique restriction en~me sites on Ad2 increased with the distance from the I~ insert to the restriction site. Also in HeLa cells, recombination increased with increasing amounts of viral DNA synthesis and with increasing UV dose to the virus. UV-irradiation of both coinfecting viruses with 1500 J / m 2 resulted in a more than 100-fold reduction in the amount of viral DNA synthesized and about a 3-fold increase in the frequency of recombination.

Although genetic recombination is a fundamental process in the life cycle of the cell, the molecular mechanisms of the recombination pro-

Correspondence: Dr. Andrew J. Rainbow, Departments of Biology and Radiology, McMaster University, Hamilton, Ont. Lgs 4KI, Canada.

cuss in mammalian ceils are far from understood. Early studies on recombination in mammalian cells were hampered to some degree by a lack of suitable genetic markers and easy methods for scoring the recombinants. However, exploration of homologous DNA recombination pathways in mammalian cells is now being carried out at the molecular level (for a recent review see Kucherla-

2~ pati and Smith, 1988). Recombination in mammalian cells has been examined using plasmid substrates and mammalian nuclear extracts, as well as by the transfection of cells with plasmids carrying selectable marker genes (Kucherlapati and Moore, 1988). Unfortunately, current DNA transfection techniques still have limited quantitative reproducibility often making precise measurement difficult (Thompson, 1988). More recent innovative studies of recombination in mammalian cells have examined recombination between integrated homologous sequences (Tsujimura et al., 1990). The recombinational pathways of mammalian cells have also been studied by genetic analysis of viral progeny following mixed infections with two different viral mutants each containing a distinct genetic marker. Such studies have involved recombination of herpes simplex virus (HSV) (DasOupta and Summers, 1980), adenovirus (Ad) (Young and Fisher, 1980; Williams et al., 1974; van der Lubbe et al., 1989) and Simian Virus 40 (SV40) (Gentil et al., 1983; Dubbs et al., 1974; Sarasin et al., 1983) in both normal and DNA-repair-deficient cells. In a number of these studies, UV-irradiation of the parental virus was reported to increase the recombination frequency as it does in bacteriophage (Baker and Haynes, 1967). Other evidence that UV-damaged DNA is recombinogenic in mammalian cells comes from observations of increased sistor-chromatid exchanges in UV-irradiated cells (Friedberg, 1984) and the stimulation of recombination between integrated homologous sequences by UV-light and other carcinogenic agents (Wang et al., 1988; Tsujimura et al., 1990). Human adenovirus has been used successfully as a probe to study the DNA-repair pathways in various mammalian cell types (Rainbow, 1981, 1989; Bennett and Rainbow, 1988; Jeeves and Rainbow, 1983; Colicos et al., 1991; Defais et al., 1983). Although adenovirus may code for functions which arc involved in recombination, this virus probably relies to some extent on the recombinational machinery of the host cell (Young and Fisher, 1980). in most genetic systems, the frequency of recombination between any two markers is proportional to their genetic distance apart and, in cases where direct correlation with

physical genomes has been made, is related to the physical distance separating them. Adenovirus is no exception (Ginsberg and Young, 1977). We are studying the nature and extent of homologous recombination of adenovirus in mammalian cells. The method we have developed gives a quantative measure of homologous recombination between Ad2 and Ad5PyMTR3 at the level of the viral DNA. Ad5PyMTR3 is a recombinant virus containing 5.23 kilobases (kb) of polyoma virus (Py) DNA inserted into a deleted E1 region between map positions 1 and 9.4 on the Ad5 genome (Davidson, 1989). Ad2 and Ad5 have greater than 99% DNA homology and recombine with high frequency (Boursnell and Mautner, 1981; Hassell and Weber, 1978). However, they differ sufficiently in their restriction endonuclease patterns, such that following coinfection of cells with Ad2 and Ad5PyMTR39 recombinant viral DNA molecules containing the Py DNA insert can be detected and quantified by digestion with restriction endonuclease, gel electrophoresis, Southern blotting and hybridization to a radioactive Py probe. This technique avoids the potential problem that not all recombinant viral DNA molecules will be assembled into infectious virus and thus bias the recombination frequencies based on genetic analysis of progeny. The use of restriction enzyme differences between different serotypes of adenovirus to study recombination in mammalian cells has been reported earlier (Williams et al., 1975; Young and Silverstein, 1980; Young et al., 1984; Ryan and Rainbow, 1986). In this report we describe the method we have developed and show that UV-irradiation of the parental virus increases the proportion of recombinant viral DNA molecules produced following infection of HeLa cells.

Materials and methods Cells Stock cultures of human HeLa cells and human 293 cells were grown as monolayers in screw-cap bottle (Falcon Plastic) and placed in a 5% CO 2 incubator at 37°C and 90-100% humidity, The growth medium was Eagle's alphaminimal essential medium (alpha-MEM) supplemented with 10% newborn calf serum together

203

with antibiotics. Human 293 cells are ~ transformed human line originally obtained by transfection of human embryonic kidney cells with sheared human adenovirus type 5 DNA (Graham et al., 1977) and were obtained from Dr. F.L. Graham, Departments of Biology and Pathology, McMaster University, Hamilton, Ont. L8S 4K1, Canada.

Viruses and plasmids The viruses used in this study were a wildtype strain of Ad2 and the recombinant virus Ad5PyMTR3 (also designated as Ad5MT7). AdSPyMTR3 is a recombinant virus deleted in the E1 region from map positions 1 to 9.4 on the Ad5 genome (Davidson and Hassell, 1987). Inserted into this deleted region is a 5.23-kb DNA fragment encoding the entire polyoma virus genome with the exception of the middle T antigen intron. Besides the middle T coding region, the functional sequences of the insert also include the Py early promoter, the enhancer elements, the wild-type origin of DNA replication and the early Y-processing signals. The construction of this virus has been described previously (Davidson, 1989). An initial stock of Ad5PyMTR3 was kindly supplied by Dr. J. Hassell, Institute of Biotechnolo~ and Molecular Biology, McMaster University, Hamilton, Ont. L8S 4K1. The original Ad5PyMTR3 stock was plaque purified 3 times on human 293 cells and new stocks of both Ad2 and Ad5~MTR3 were grown and titered on human 293 cells as described previously (Graham et al., 1977). pMK16RCPy was contructed by inserting the Py sequences from pdPxl3BlaST1 into pMK16 (Kahn et ai., 1979). pdPxl3Bla3STl was a gift from Dr. J. Hassell, Institute of Biotechnology and Molecular Biology, McMaster University, Hamilton, Ont. L8S 4KI and was derived from pdPxl3Bla3 in a manner similar to that described previously (Bautch et al,, 1987). The BamHl fragment from pdPxl3Bla3STl containing Py sequences was inserted into the BamH! site of pMK16 to produce pMKI6RCPy.

Coinfection of HeLa cells by AdSMT7 and Ad2 HeLa cells were seeded into 24-well plastic Linbro tissue-culture plates (Flow Laboratories

Inc., Hamden, CT) with 0.5 mi of growth medium per well. 24 h later the monolayers were confluent, at which time the growth medium was aspirated off the cells. Each well was then inoculated with 0.2 ml of an appropriate amount of both Ad2 and Ad5PyMTR3 which had been diluted in alpha-MEM without serum. Coinfection with both viruses was usually carried out at a multiplicity of infection (MOI) of approximately 50 pfu/cell for Ad2 and 10 pfu/cell for AdSPyMTR3. The different inputs for the two parental viruses were chosen in order to skew the production of Pycontaining recombinants towards the minority parent. The virus was allowed to adsorb to the cells for 90 rain and then each well was overlayed with 1 ml of fresh, pre-warmed growth medium. At various times after infection, cells were harvested and the viral DNA isolated.

Analysis of viral DNA Viral DNA from infected cells was isolated by the Hirt extraction procedure (Hirt, 1967) with slight modifications. Growth medium was aspirated off the infected cell monolayer and then subsequently overlayed with 0.5 ml of a lysing solution containing 10 mM Tris, pH 8, 10 mM EDTA, 0.6, SDS and 1 mg/ml of pronase (Boehringer Mannheim, CA). Cells were lysed for 1 h at 37°C and the sample was subsequently transferred to a 1.5-ml Eppendorf centrifuge tube using a wide bore l-ml pipet. 0.25 ml of 3 M Na acetate (pH 7) was added to the sample, gently mixed and allowed to precipitate overnight on ice. After centrifugation in the cold, the supernatant was then extracted sequentially with phenol, phenol :chloroform and chloroform and the resulting aqueous phase was precipitated with absolute ethanol. The DNA pellet was dried and redissolved in 30-100 ~tl of sterile double-distilled water. The viral DNA was then digested with an appropriate restriction endonuclease and electrophoresed through an 0.8% agarose gel at 100 V for 24 h at 4°C. The DNA fragments were transferred to Gene Screen Plus membrane (DuPont Canada) using the Southern blot method (Southern, 1975) and then hybridized to .~2p. labelled pMK16RCPy, pMKI6RCPy was radioactively labelled with 32p by nick translation using a

204

standard nick translation kit from Bethesda Research Laboratories and unincorporated nucleotides were removed by passage through a Sephadex G-50 spun column (Maniatis et al., 1982). The radioactive nitrocellulose membrane was washed, dried and exposed to Dupont Kronex 4L film with intensifying screens at -80°C.

Analysis of autoradiographs Autoradiographs were scanned with a laser microdensitometer and the area corresponding to parental and recombinant bands were determined for each scan. The relative amount of radioactively labelled DNA corresponding to each band was then determined by comparison with standard calibration curves of area versus relative amount of radioactively labelled DNA. A series of 15-20, 2-fold dilutions of Ad5PyMTR3 DNA were slot blotted onto nitrocellulose and subsequently hybridized to "~2p-labelled pMK16RCPy under identical conditions to those employed for analysis of the viral recombinants. The radioactive membrane was exposed to film using intensifying screens under the same conditions and time of exposure as used in the analysis of viral recombinants. A standard calibration curve of peak area versus relative amount of DNA was obtained by scanning the autoradiograph,

Irradiation of virus UV-irradiation of virus was performed using a germicidal lamp (General Electric Germicidal Lamp GgT5) emitting a wavelength of predominantly 254 nm. The method employed was essentially the same as that described previously

Cla 1 Py DHA

AdSPyMT3

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Detection of homologous recombination of adenovirus DNA Fig. 1 shows the Cla 1, BamH 1 and EcoR 1 restriction endonuclease sites for both Ad 2 and Ad5PyMTR3. Digestion of Ad5PyMTR3 DNA with Cla 1, BamH 1 or EcoR 1 results in DNA fragments which contain all or part of the Py DNA insert of 38.1, 23.8 and 29.2 kb respectively. Following coinfection of cells with both Ad5PyMTR3 and Ad2, homologous recombination between the Py insert of Ad5PyMTR3 and the second Cla 1 restriction site at 51.92 m.u. on Ad2 should result in a recombinant viral DNA molecule which when digested with Cla I yields a recombinant DNA fragment of 20.9 kb containing the Py insert. By a similar analysis, digestion of recombinant viral DNA molecules with BamH 1 should result in recombinant DNA fragments of 12.9 and 17.6 kb which contain the Py insert; and digestion with EcoR 1 should result in recombi-

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(Rainbow, 1989). Stock viruses were diluted with cold alpha-MEM without serum and an aliquot of virus suspension no greater than 1.6 ml was irradiated in a 35-mm diameter Petri dish (Falcon plastics) with the dish cover removed, kept on ice, with constant swirling during the irradiation. Appropriate amounts of Ad2 and AdSPyMTR3 were UV-irradiated simultaneously in the same Petri dish. Under these condition the incident dose rate was about 4 j / m 2 / s e c as determined using a J-225 shortwave U.V. meter (Ultraviolet Products, San Gabriel, CA).

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Fig, I. Restriction endonucleuse cleavage maps of Ad2 and AdSPyMTR3, Cleavage sites are shown for the restriction endonucleases Cla I, BamH 1 and EcoR !. Fragment sizes are shown in kb and the Py DNA insert of AdSPyMTR3 is shown as a thicker

black porlion of the viral DNA. Map unit locations of the restriction enzyme sites for Ad2 are: Cla 1, 2.55 and 51,92; BamH l, 29.72, 42.86 and flU.12, EcoR !, 59,38, 71,33, 76A7, 83,61 and 89.78 re.u, By convention, the total Ad2 8enome has 100 m.u. and enzyme sites are expressed as distances from the left end.

205

nant DNA fragments of 23.2 and 27.5 kb which contain the Py insert. In order to determine if such homologous recombination events could be detected, HeLa cells were coinfected with Ad2 and Ad5PyMTR3. At an appropriate time after infection, viral DNA was extracted from the infected cells, digested with restriction endonuclease and electrophoresed through an agarose gel. A typical ethidium bromide-stained gel from such an experiment is shown in Fig. 2B. It can be seen that the visible DNA fragments obtained are consistent with the restriction enzyme patterns of Fig. 1 for cells infected with Ad5PyMTR3 alone (lanes 4, 5 and 6), cells infected with Ad2 alone (lanes 7, 8 and 9) and cells coinfected with both Ad5PyMTR3 and Ad2 (lanes 1, 2 and 3). The gel of Fig. 2B was Southern blotted and probed with radioactively labelled Py DNA from pMKI6RCPy to obtain the autoradiograph shown in Fig. 2A. Viral DNA fragment sizes corresponding to the Py containing fragments from the parental Ad5PyMTR3 are visible in the autoradiograph for DNA extracts from cells infected with Ad5PyMTR3 alone (lanes 4, 5 and 6) as well as for DNA from cells co-infected with Ad5PyMTR3 and Ad2 (lanes 1, 2 and 3). As expected, no hybridization of pMK16RCPy was detected for DNA extracted from cells infected with Ad2 alone. Recombinant bands are clearly visible in the autoradiograph for DNA extracts from coinfected cells (lanes 1, 2 and 3), whereas such recombinant bands are absent for DNA extracted from cells infected with Ad51~MTR3 alone (lanes 4, 5 and 6). The Cla 1 digest (lane 1) shows both the 38.1 kb parental and the 20.9 kb recombinant band as expected. The BamH I digest (lane 2) shows the 23.8 kb parental band as well as the recombinant bands at 12.9 and 17.6 kb. Recombination between the second (42.86 m.u.) and third (60.12 m.u.) BamH 1 sites on Ad2 results in a recombinant DNA fragment containing the 1~ insert of 23.8 kb which comigrates with the parental band. The EcoR 1 digest (lane 3), shows the 29.2 kb parental band and one of the recombinant bands at 23.2 kb. The other expected 27.5 kb and 29.2 kb recombinant bands are considerably less abundant than the 23.2 kb recombinant and either comigrate with the 29.2

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1.7 Fig. 2. Restriction endonuclease analysis of DNA extracted from cells 48 h after infection with Ad2 and/or AdSPyMTR3. Lanes 1, 2 and 3 show DNA extracts from HeLa cells infected with both viruses at 5{} pfu/cell for Ad2 and I0 pfu/cell for AdSPyMTR3. Lanes 4, 5 and 6 show DNA extracts from human 293 cells infected with Ad5PyMTR3 alone ;tt 10 pfu/cell. Lanes 7. 8 and 9 show DNA extracts from HeLa cells infected with Ad2 alone at 10 pfu/cell, Lanes 1.4 and 7 show Cla I digestions: lanes 2. 5 and 8 show BamH I digestions: and lanes 3.6 and 9 show EcoR I digestions. (A) Shows an autoradiograph of the Southern blot probed with radioactive I~ DNA and includes the size of the fragments which hybridize (B) Shows the ethidium bromide-stained gel including the Ad2 restriction endonuclease fragment sizes for the BamH I and EcoR 1 digests in lanes 8 and 9. DNA fragments less than 1.5 kb had run off the gel and cannot be seen in this photograph.

kb parental band or are not in sufficient abundance to be detected on this autoradiograph. Failure to detect the 27.5 kb recombinant band may result also, in part at least, from a reduced

206

ability of the genomes to recombine in this region of considerable heterology encoding the structural hexon protein (Boursnell and Mautner, 1981). Autoradiographs were scanned with a laser microdensitometer. Fig. 3 shows a typical scan obtained for the BamH 1 digested DNA extract of lane 2, in Fig. 2. The area under the optical density profile for each band was measured and the relative amount of radioactively labelled DNA corresponding to each band was determined by comparison with standard calibration curves. Each recombinant band results from recombination events between the Py insert on Ad5PyMTR3 and the unique restriction enzyme site on Ad2. In Fig. 4 the percentage of Py containing DNA molecules present as a recombinant is plotted for each unique restriction enzyme site as a function of map units along the Ad 2 genome. It can be seen that recombination increased with distance from the l'y insert to the restriction site. The amount of recombination detected was greater at 48 h compared to 24 h after infection. The data of Fig. 4 as well as the results of other experiments (not shown) indicated that the amount of recombination detected increased with increasing amounts of viral DNA synthesis as has been reported previously (Young and Silverstein, 1980).

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Distance from Py Insert in kb Fig. 4. Recombination as a function of distance of available homology in kb from the Py insert. The percentage of Py containing recombinant molecules is plotted as a function of distance in kb from the Py insert on Ad5PyMTR3 to a number of unique restriction sites on Ad2 for DNA extracted from infected cells at 24 h and 48 h after infection, The data points at 7.3, 12, 15.3 and 18 kb correspond to the amount of recombination between the Py insert on AdSPyMTR3 and the first BamHI site (29,72 re.u,), the second BamH ! site (42.0fi m,u,), the second Cla 1 site (51.92 m.u,) and the first EcoR I site (59,38 m,u,)on Ad2 respectively, The data points at 12 kb incorporate the amount of recombination between the Py insert on AdSPyMTR3 and the first BamH I site (29,72 m,u,) as well as that between the first (29.72 m,u.) and second (42,80 re,u,) BamH I site on Ad2,

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Using this technique we are also able to detect recombination of Ad following the infection of Chinese hamster ovary cells, monkey kidney cells, normal human fibroblasts as well as human fibroblasts transformed with Simian virus 40 (data not shown),

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Homologous recombinationfollowing infection with UV.irradiated virus

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Distance In mm Fig. 3. Microdensitomcter tracing of the autoradiograph, Shows a typical laser microdensitometer scan for a single lane of the autoradiogruph obtained from the Southern blot, DNA was extracted from HeLa cells infected with both Ad2 and Ad5PyMTR3 and digested with BamH I as shown in lane 2 of Fig, 2.

Hela cells were coinfected with both Ad2 and Ad5PyMTR3 using unirradiated virus as well as virus which had been UV-irradiated. At various times after infection, viral DNA was extracted from the infected cells, digested with BamH 1 and analysed by electrophoresis through agarose, Southern blotting and subsequent hybridization to a radioactive Py containing probe. Two autoradiographs from such an experiment are shown in Fig. 5. Reduced amounts of viral DNA synthesis

207

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'~ Fig. 5. Autoradiographs of a Southern blot for DNA extracted from cells infected with UV-irradiated Ad. HeLa cells were coinfected with Ad2 at 50 pfu/cell and Ad5PyMTR3 at 10 pfu/cell with either UV-irradiated or unirradiated virus. DNA was extracted at different times after infection, digested with BamH 1 and eletrophoresed on an agarose gel. The gel was Southern blotted and hybridized to a 32p-labelled pMKI6RCPy probe. Autoradiographs were obtained after exposure of the radioactive membrane to film for either (A) !1 h or (B) 168 h. Lanes 1, 2 and 3 show DNA extracts from cells infected with unirradiated virus and collected at 24, 36 and 48 h after infection respectively; Lanes 4, 5 and 6 are for infections with virus exposed to 500 J/m 2 and collected at 48, 72 and 96 h after infection respectively; Lanes 7, a and 9 are for infections with virus exposed to 1000 J / m 2 at 48, 72 and 96 h after infection respectively; Lanes I0, I1 and 12 are for virus exposed to 1500 J / m 2 at 72, 96 and 120 h after infection respectively.

and increased amounts of recombination were seen clearly in the autoradiographs for cells infected with virus exposed to 1500 J / m 2. The autoradiographs were scanned with a laser microdensitometer and the relative amount of DNA in each band was determined. The relative amount of DNA detected in the 23.8 parental band is plotted as a function of time after infection in Fig. 6 and shows a delay in viral DNA synthesis for UV-irradiated Ad as has been reported previously (Mak and Mak, 1974). It can be seen also that redhced amounts of viral DNA were synthesised in cells infected with UV-irradiated virus for all the UV exposures tested. The maximum amount of viral DNA detected in the infected cells is plotted as a function of UV exposure to the infecting virus in Fig. 7. Results were consistent with an exponential decrease in

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Hours After infection Fig. 6. Viral DNA synthesis in HeLa cells infected with UV-irradiated Ad. DNA was extracted from infected cells at different times after infection, digested with BamH I and electrophoresed on an agarose gel. The relative amount of Ad5PyMTR3 viral DNA synthesized was determined by Southern blotting and hybridization to a "12P-labelled pMKI6RCPy probe. Shows pooled resells for two experiments. UV fluence to the virus: * , no UV: z , 500 j/m2; e, 1000 j/m2; o, 1500 J/m 2.

viral DNA with increasing UV exposure to the virus with a D~) value of about 300 J/m 2. The maximum detectable percentage of Py containing molecules present as the recombinant 12.9 kb band is plotted as a function of U~t

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UV Fluence to Virus (J/m2) Fig. 8. Recombination of Ad after infection with UV-irradiated Ad. From autoradiographs of BamH 1 digested DNA as shown in Fig. 5. the percent recombination between the Py insert on AdSPyMTR3 and the first BamH I restriction site at 29.72 mu on Ad2 (corresponding to the 12.890-kb recombinant DNA fragment) was determined. The maximum percent recombination obtained is shown as a function of UV exposure to the infecting viruses. Pooled results for 2 Expts.

exposure in Fig. 8. It can be seen that the percentage of Py containing molecules present as the 12.9-kb recombinant increased with UV exposure to the virus. UV-irradiation of the infecting virus with 1500 J/re" resulted in about a 3-fold increase in recombination.

Discussion We have developed the use of adenovirus as a molecular probe to examine the fivquency of recombination between Ad2 and AdSPyMTR3 DNA following the infection of mammalian cells. Using this approach we arc able to detect and quantitate recombinant viral DNA molecules containing the Py insert which are present at frequencies down to at least 1 in 100. We are able to measure recombination frequencies following the infection of Chinese hamster ovary (CHO) cells, monkey kidney cells, human tumor cells, normal human fibroblasts as well as SV40 transformed human fibroblasts, in principle the method should be able to detect recombination events in all cell types permissive for Ad infection. The method can also be used in semi-permissive infections if sufficient viral DNA synthe.

sis occurs, as is the case following infection of certain rodent cell types including CHO cells (Eggerding and Pierce, 1986). Furthermore, the technique could be modified to detect recombinant molecules under conditions where the viral DNA has entered the cell but in which no viral DNA synthesis takes place as occurs in some non-permissive infections by adenovirus as well as after infections with DNA negative viral mutants. In adenoviruses, the frequency of recombination increases throughout the course of viral replication (Williams et al., 1974; Young and Silverstein, 1980; Young et al., 1984). The results of the present study show an increase in recombination with time after infection which parallels an increase in the amount of replicated viral DNA. 'This appears similar to the situation with T-even phages, where recombination and replication of DNA are intimately related and there is a steady increase in recombination frequency during the period of active DNA synthesis (Doermann, 1953; Levinthal and Visconti, 1953; Visconti and Delbruck, 1953). This suggests that the viral DNA molecules undergo multiple rounds of recombination as DNA synthesis proceeds. The use of molecular hybridization techniques to study the production of recombinant viral DNA molecules following infection by Ad has been reported previously (Young and Silverstein, 1980; Williams et al., 1975; Young et al., 1984). Using this approach, recombinant viral DNA molecules could be detected before the rise in infectious viral progeny. Such recombinant molecules increased in frequency relative to the parental molecules throughout the exponential period as found in the present study. These results suggest that recombination is dependent, at least to some extent, upon viral DNA replication in adenovirus infected ceils. Also in the present work it was found that the frequency of recombination increased with increasing distance from the Py DNA insert on AOSPyMTR3 to a number of unique restriction sites on Ad2. Results were consistent with a frequency of recombination which was proportional to the length of available homology. UV-irradiation of the infecting adenovirus resuited in a delay in viral DNA synthesis and a reduction in the amount of viral DNA synthe-

2O9

sized (see Figs. 6 and 7). The D n value for viral DNA synthesis of adenovirus in HeLa cells was about 300 J/m-', which is intermediate between the D . value for plaque formation of 210 J / m : and the D o value for the inhibition of cell cloning of 700 J/m-" reported for adenovirus infection of human KB cells (Rainbow and Mak, 19721. This supports previous results which suggest that the inactivation of adenovirus by UV is not at the step of viral DNA synthesis (Mak and Mak, 19741. Experiments using temperature-sensitive mutants of SV40 have shown a strong enhancement of viral recombination following infection with UV-irradiated virus (Dubbs et al., 1974; Sarasin et al., 19831. Other studies have also shown that UV-irradiation of herpes simplex virus results in a marked increase of viral recombinants (Dasgupta and Summers, 1980; Hall et al., 19801. Using deletion mutants of AdS, van der Lubbe et al. 11989) showed that UV-irradiation of adenovirus also results in enhanced recombination. The present work shows that UV-irradiation of the infecting adenovirus results in an increased proportion of recombinant viral DNA molecules in infected HeLa cells. This suggests that the previous studies based on the genetic analysis of progeny which show an enhanced recombination frequency for UV-irradiated virus are not the result of bias in the assemby of viral DNA molecules into infectious virus. Acknowledgements We thank Mary Speagle for excellent technical assistance in some of the experiments. We also thank Dr. John Hassell for supplying the initial stock of AdSPyMTR3 as well as the pdPxl3 Ba3ST1 plasmid. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada. References Baker, R,M., and R.H. Haynes (1967) UV induced enhancement of recombination among lambda bacteriophage in UV-sensitive host bacteria. Mol. Gen. Genet., 1011, 166177. Bautch, V.L., S. Toda, J.H. Hassell and D. Hanahan 119871 Endothelial cell tumors develop in transgenie mice carrying polyoma virus middle T oncogene, Cell, 51. 529-538.

Bennett, C.B.. and A.J. Rainbow (1988) Delayed expression of enhanced reactivation and decreased mutagenesis of UVirradiated adenovirus in UV-irradiated ataxia telangieclasia fibroblasts, Mutagenesis, 3, 389-395. Boursnell, M.E.G., and V. Mautner (19811 Recombination in adenovirus: Crossover sites in intertypic recombinants are located in regions of homology, Virology, 112, 198-209. Colicos, M.A., Y. Hal-Abroad, K. Valerie, E.E. Henderson and A.J. Rainbow (1991)Construction of a recombinant adenovirus containing the denV gene from bacteriophage T4 which can partially restore the DNA repair deficiency. in xeroderma pigmentosum fibroblasts, Carcinogenesis, 12, 249-255. Dasgupta, U.M., and W,C. Summers (1980) Genetic recombination of Herpes simplex virus, the role of the host cell and UV-irradiation of the virus, Mol. Gen. Genet., 178, 617-623. Davidson, D. (1989) Construction of adenovirus vectors for efficient transfer and overexpression of polyoma virus middle tumor antigen in mammalian cells, Ph.D. Thesis, McGill University. Davidson, D., and J,H. Hasscll 119871 Overproduction of polyomavirus middle T antigen in mammalian cells through the use of an adenovirus vector, J. Virol., 61, 1226-1239. Defais, MJ., P,C. Hanawalt and A. Sarasin (1983) Viral probes for DNA repair, Adv. Radiat. Biol., 10, 1-37. Doermann, A.H. (1953) The vegetative state in the life cycle of bacteriophage: Evidence for its occurence and its genetic characterization, Cold Spring Harbor Syrup. Qu:mt. Biol., 18. 3-11. Dubbs, D.R., M. Rachmeler and S. Kit (1974) Recombination heteen temperature sensitive mutants of simian virus 40. Virology, 57, 161-174, Eggerding, F.A., and W.C. Pierce (1986) Molecular biology of adenovirus type 2 semipermissive infections, I, Vind growth and expression of viral rcplicativ¢ functions during restricted ~dcl~o'~irusinfection, Virology. 148. 97-I I I. Friedberg, E.C. (19851 DNA Repair. Freeman. New York. pp. 55l)-552, Gentil, A.. A. Margol and A. Sarasin (1983) Effect of UVirradiation on genetic recombination of simian virus. 40 mutants, in: E.C. Friedberg and P.C. Hanawall (Eds.). Cellular Responses to DNA Damage, Liss, New York. pp. 385-396. Ginsberg, H.S.. and C.S.H. Young (1977) The genetics of adenoviruses, in: H. FraenkeI-Conrat and R.R. Wagner (Eds.), Comprehensive Virology, Vol. 9. Plenum. New York., pp. 27-88. Graham. F.L.. J. Smiley, W.C, Russell and R. Nairn (1977) Characteristics of a human cell line Iransfi)rmed by DNA from human Adenovirus type 5. J. Gen. Virol., 36. 59-72. Hall, J.D., J.D. Featherston and R.E. Almy (Ig80) Evidence fi)r Ih¢ repair of ultraviolet light-damaged Herpes simplex virus in human fibroblasts by a recombination mechanism. Virology. 105. 4911-5(111. Hassell, LA., and J. Weber 119781 Genetic analysis of adenovirus type 2. VIII. Physical location of temperature sensilive mutations, J. Virol.. 28, 671-678.

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