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Two proteins of 220 kD and 230 kD bind to UV-damaged SV40 minichromosomes in irradiated monkey kidney cells
Mutation Research, 227 (1989) 227-231 Elsevier
227
MUTLET 0275
Two proteins of 220 kD and 230 kD bind to UV-damaged SV40 minichromosomes in irradiated monkey kidney cells Marianne L. Brown-Luedi and Thomas C. Brown* MRC Radiobiology Unit, Chilton, Didcot, 0Xl l ORD (Great Britain) (Accepted 24 July 1989)
Keywords: DNA-repair proteins; SV40; UV damage; Chromatin
Summary We exposed SV40-infected monkey kidney cells to 0, 30 or 150 J / m z of UV-radiation, and isolated viral minichromosomes at various times after irradiation. Analysis of minichromosome-associated proteins by SDS-PAGE revealed the presence of two proteins, of 220 kD and 230 kD associated with minichromosomes from irradiated cells, but not from unirradiated controls. The larger protein was the less abundant, and was most evident in preparations from more heavily irradiated cells. Neither protein was associated with minichromosomes isolated 30 min after irradiation, but were apparent in minichromosome preparations isolated 1-4 h after UV treatment.
Many proteins that participate in DNA-excision repair bind only weakly to native DNA, but bind strongly to damaged DNA (Lindahl, 1982; Sancar and Sancar, 1988; Chu and Chang, 1988; Jiricny et al., 1988). Discrimination based on binding specificity is probably essential for enzymatic activity (Sancar and Sancar, 1988). Mindful of this correlation between binding specificity and possible repair activity, we have developed a way of detecting proteins that bind selectively to UVdamaged SV40 chromatin in situ in virus-infected cells. Correspondence: Dr. Marianne L. Brown-Luedi, MRC Radiobiology Unit, Chilton, Didcot, OXI 1 0RD (Great Britain). * Present address: PKF/LD, F. Hoffmann-La Roche, 124 Grenzacherstrasse, 4002 Basle (Switzerland).
SV40 is well suited to studies of eucaryotic DNA metabolism. In the infected cell, viral DNA is associated with nucleosomes (Jacobovits et al., 1980; Saragosti et al., 1980) and other proteins in a structure remarkably like eucaryotic cell chromatin (DePamphilis and Wassarman, 1980). Many of the proteins known to participate in DNA replication (Tsubota et al., 1979; Otto et al., 1979) and chromatin metabolism (Poirier et al., 1986) are associated with minichromosomes. SV40 DNA is also repaired in situ in carcinogen-treated host cells (Williams and Cleaver, 1981; Brown et al., 1987). Viral and cellular DNA are probably repaired by the same mechanisms (Abrahams et al., 1976). We therefore conceived that proteins mediating repair would be associated, at least transiently, with SV40 minichromosomes.
0165-7992/89/$ 03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
228 Viral minichromosomes can be isolated under gentle, nondenaturing conditions (Turler and Beard, 1985), and contain equal amounts of DNA and protein. In experiments described in this paper, we sought conditions of isolation that would stabilize and preserve protein-DNA associations unique to damaged SV40 minichromosomes. This approach does not rely on the reconstitution of specific protein-DNA interaction in vitro, but requires instead that specific protein-chromatin associations in situ be stable under our conditions of isolation. Materials and methods
We cultured CV-1 African green monkey cells in DMEM (Gibco) supplemented with 5°70 fetal bovine serum, penicillin and streptomycin. Subconfluent ( - 80%) monolayers in 9-cm dishes were infected with SV40 at 5 plaque-forming units (pfu) per cell and incubated for 29-30 h. Media were then removed, saved as pooled conditioned media, and replaced with 5 ml of Tris-buffered saline (Brown and Cerutti, 1986). Cultures in saline were then exposed to 0-150 J / m z of UV radiation (254 nm) at a rate of 0.76 J / m 2 per sec. Saline was then replaced with 7 ml of the pooled conditioned media, and the cultures were incubated under normal culture conditions for 30 min to 4 h before lysis. Lysis and minichromosome preparation followed established procedures (Turler and Beard, 1985). Briefly, cells were scraped from the plate and lysed with 8 strokes of a Dounce homogenizer (loose pestle) in hypotonic buffer containing 10 mM Tris, pH 7.5, 5 mM KC1, 7 mM ~mercaptoethanol and 0.1 mM MgCI2. Nuclei were pelleted and incubated overnight, on ice, with 0.5 ml of lysis buffer. Minichromosomes were purified by centrifugation in continuous 5-20% sucrose gradients, in hypotonic buffer. Minichromosomes sedimented to the middle of 5-ml gradients after 1 h at 35 000 rpm at 4°C in a Beckman 50.1 rotor. Gradients were fractionated from the bottom by peristaltic pumping into 10 samples, and the protein compositions of the frac-
tions were analyzed by SDS-PAGE (Laemmli, 1970) using 5-12.5% acrylamide gradient gels. Protein gels were silver stained (Merril et al., 1981; Dzandu et al., 1984; de Moreno et al., 1985), using a band-enhancement technique (Lukiw and McLachlan, 1988). Results and discussion
Silver-stained gels revealed about 100 protein bands in minichromosome samples from unirradiated and UV-treated cells. Many of the strongest bands were probably from intact ribosomes that cosediment with minichromosomes in the presence of Mg2÷ ions (Poirier et al., 1986). However, individual proteins not associated with large structures do not appreciably sediment under these conditions of centrifugation. Fig. 1 presents protein profiles of minichromosome preparations from ceils exposed to 0, 30 and 150 J / m 2 of UV light, and incubated for 2 h. Comparison of gradient fractions 4, 5 and 6 reveals slight differences in protein composition across each gradient that are unrelated to UV treatment. Two differences are apparent in protein profiles of UV-irradiated and control samples. (1) Samples from cells treated with 30 J / m 2 contain a protein band corresponding to 220 kD which is not visible in unirradiated controls. This 220-kD protein is apparent in all 3 gradient fractions of the UV-treated sample. (2) Samples from cells treated with 150 J / m z also contain this 220-kD protein, but in addition show smaller amounts of a 230-kD protein, also present in all 3 central fractions of the gradient. These proteins were not found in any of the gradient fractions of equivalent preparations from UV-irradiated cells not infected with SV40 (not shown), indicating that the UV-dependent proteins are associated with minichromosomes rather than with other large structures. Neither protein was observed when minichromosomes were isolated in the presence of 1 mM EDTA instead of 0.1 mM MgC12 (not shown). Fig. 2 presents a time course of protein association, comparing the central fraction 5 of unitradiated and UV-treated preparations of cells lysed
229
0 J/m 2
f4
f5
30 J / m 2
f6
M
f4
f5
f6
150 J / m 2
M
f4
f5
f6
kD
200
116. 97. 77. 66 56 43
1
2
3
4
5
6
7
8
9
lane
Fig. 1. Identification of a 220-kD and a 230-kD protein associated with SV40minichromosomesisolated from UV-irradiated monkey ceils. Silver-stained SDS-PAGE profiles of proteins associated with SV40minichromosomes, from sucrose gradient fractions 4, 5 and 6 (f4, fS, f6) of preparations from unirradiated cells (lanes 1, 2, 3) and cells exposed to 30 J / m 2 (lanes 4, 5, 6) or 150 J / m 2 (lanes 7, 8, 9). Molecular weight markers (lanes M) from BDH. Arrows in lane 9 indicate the positions of the 220-kD and 230-kD proteins.
30 min, 1 h, 2 h and 4 h after UV irradiation. Unirradiated controls were always mock irradiated and incubated for the indicated time along with the irradiated cultures. The 220-kD and 230-kD proteins are not observed in minichromosome preparations taken 30 min after UV treatment, but are slightly visible after I h, more abundant after 2 h, and still present after 4 h. It is not feasible to analyse samples at later times because irradiation, especially o f the infected ceils, causes cell detachment and lysis, and loss o f material. We do not know the function of these two proteins, and our supposition that they participate in
D N A repair or in the metabolism of damaged chromatin is based solely on their specific cosedimentation with UV-damaged minichromosomes. It is possible that only small amounts of each protein are associated with minichromosomes, and that larger amounts remain free in the uppermost fraction of the gradient, where the number and diversity of proteins precludes resolution o f individual bands. Both proteins can be visualized using sample isolated from about 20% o f the infected cells on a 9-cm culture dish, and are therefore abundant enough to allow their purification.
230
30 min
1 hr
2 hr
4 hr
J/m 2
kD 200
116 97 77 56 43
lane Fig. 2. Time course of 220-kD and 230-kD protein association with minichromosomes. Protein profiles of minichromosomes isolated 30 min (lanes 1, 2, 3), 1 h (lanes 4, 5, 6), 2 h (lanes 7, 8, 9) and 4 h (lanes 10, 11, 12), from cells irradiated with 0, 30 or 150 J / m 2 as indicated. Molecular weight markers, lanes M. Arrows in lane 9 indicate the positions of the 220-kD and 230-kD proteins.
Acknowledgements We are grateful to Dr. John Thacker Richard
D. Wood
and Dr.
for helpful discussions, and to
Dr. Janet Jones for advice on silver staining. This work was supported in part by CEC Contract No. B16-E-144-UK.
References Abrahams, P.J., and A.J. Van tier Eb (1976) Host-cell reactivation of ultraviolet-irradiated SV40 DNA in five complementation groups of xeroderma pigmentosum, Mutation Res., 35, 13-22. Brown, T.C., and P.A. Cerutti (1986) Ultraviolet radiation inactivates SV40 by disrupting at least four genetic functions, EMBO J. 5, 197-203.
Brown, T.C., P. Beard and P.A. Cerutti (1987) Preferential repair of N-acetoxy acetylaminofluorene lesions in the nuclease-hypersensitive region of Simian Virus 40, J. Biol. Chem., 262, 11947-11951. Chu, G., and E. Chang (1988) Xeroderma pigmentosum group E cells lack a nuclear factor that binds to damaged DNA, Science, 242, 564-567. de Moreno, M.R., J.F. Smith and R.V. Smith (1985) Silver staining of proteins in polyacrylamide gels: increased sensitivity through a combined coomassie blue-silver stain procedure, Anal. Biochem., 151,466-470. De Pamphilis, M.L., and P.M. Wassarman (1980) Replication of eukaryotic chromosomes: a close-up of the replication fork, Annu. Rev. Biochem., 49, 627-666. Dzandu, J.K., M.E. Deh, D.L. Barrat and G.E, Wise (1984) Detection of erythrocyte membrane proteins, sialoglycoproteins, and lipids in the same polyacrylamide gel using a double-staining technique, Proc. Natl. Acad. Sci. (U.S.A.), 81, 1733-1737. Jacobovits, E,B., S. Bratosin and Y. Aloni (1980) A
231 nucleosome-free region in SV40 minichromosomes, Nature (London), 285, 263-265. Jiricny, J., M. Hughes, N. Corman and B.B. Rudkin (1988) A •human 200 kDa protein binds selectively to DNA fragments containing GT mismatches, Proc. Natl. Acad. Sci. (U.S.A.), 85, 8860-8864. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (London), 227, 680-685. Lindahl, T. (1982) DNA repair enzymes, Annu. Rev. Biochem., 51, 61-87. Lukiw, W.J., and D.R. McLachlan (1988) Silver stain image amplification, Trends Genet., 4, 146. Merril, C.R., D, Goldman, S.A. Sedman and M.H. Ebert (1981) Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins, Science, 211, 1437-1438. Otto, B., E. Fanning and A. Richter (1979) D N A polymerases and a single-strand-specific DNA-binding protein associated with Simian Virus 40 nucleoprotein complexes, Cold Spring Harbor Symp. Quant. Biol., 43, 705-708. Poirier, G., T.C. Brown and P.A. Cerutti (1986) Poly ADP ribosylation and DNA strand breakage in SV40 minichromosomes, Carcinogenesis, 6, 283-287.
Sancar, A., and G.B. Sancar (1988) DNA repair enzymes, Annu. Rev. Biochem, 57, 29-67. Saragosti, S., G. Moyne and M. Yaniv (1980) Absence of nucleosomes in a fraction of SV40 chromatin between the origin of replication and the region coding for the late leader RNA, Cell, 20, 65-73. Tooze (Ed.) (1980) DNA Tumor Viruses, Cold Spring Harbor Laboratory Press, New York. Tsubota, Y., M.A. Waqar, J.F. Burke, B.I. Milavetz, M.J. Evans, D. Kowalsky and J.A. Huberman (1979) Association of enzymes with replicating and nonreplicating Simian Virus 40 chromosomes, Cold Spring Harbor Symp. Quant. Biol., 43,693-704. Turler, H., and P. Beard (1985) Simian Virus 40 and Polyoma virus: growth, titration, transformation and purification of viral components, in: B.W.J. Mahy (Ed.), Virology: A Practical Approach, IRL Press, Oxford, pp. 169-192. Williams, J., and J. Cleaver (1978) Excision repair of ultraviolet damage in mammalian cells, Evidence for two steps in the excision of pyrimidine dimers, Biophys. J., 22, 265-279. Williams, J., and J. Cleaver (I 979) Removal of T4 endonuclease V sensitive sites from SV40 DNA after exposure to ultraviolet light, Biochim. Biophys. Acta, 562,429-437. Communicated by J.M. Gentile
227
MUTLET 0275
Two proteins of 220 kD and 230 kD bind to UV-damaged SV40 minichromosomes in irradiated monkey kidney cells Marianne L. Brown-Luedi and Thomas C. Brown* MRC Radiobiology Unit, Chilton, Didcot, 0Xl l ORD (Great Britain) (Accepted 24 July 1989)
Keywords: DNA-repair proteins; SV40; UV damage; Chromatin
Summary We exposed SV40-infected monkey kidney cells to 0, 30 or 150 J / m z of UV-radiation, and isolated viral minichromosomes at various times after irradiation. Analysis of minichromosome-associated proteins by SDS-PAGE revealed the presence of two proteins, of 220 kD and 230 kD associated with minichromosomes from irradiated cells, but not from unirradiated controls. The larger protein was the less abundant, and was most evident in preparations from more heavily irradiated cells. Neither protein was associated with minichromosomes isolated 30 min after irradiation, but were apparent in minichromosome preparations isolated 1-4 h after UV treatment.
Many proteins that participate in DNA-excision repair bind only weakly to native DNA, but bind strongly to damaged DNA (Lindahl, 1982; Sancar and Sancar, 1988; Chu and Chang, 1988; Jiricny et al., 1988). Discrimination based on binding specificity is probably essential for enzymatic activity (Sancar and Sancar, 1988). Mindful of this correlation between binding specificity and possible repair activity, we have developed a way of detecting proteins that bind selectively to UVdamaged SV40 chromatin in situ in virus-infected cells. Correspondence: Dr. Marianne L. Brown-Luedi, MRC Radiobiology Unit, Chilton, Didcot, OXI 1 0RD (Great Britain). * Present address: PKF/LD, F. Hoffmann-La Roche, 124 Grenzacherstrasse, 4002 Basle (Switzerland).
SV40 is well suited to studies of eucaryotic DNA metabolism. In the infected cell, viral DNA is associated with nucleosomes (Jacobovits et al., 1980; Saragosti et al., 1980) and other proteins in a structure remarkably like eucaryotic cell chromatin (DePamphilis and Wassarman, 1980). Many of the proteins known to participate in DNA replication (Tsubota et al., 1979; Otto et al., 1979) and chromatin metabolism (Poirier et al., 1986) are associated with minichromosomes. SV40 DNA is also repaired in situ in carcinogen-treated host cells (Williams and Cleaver, 1981; Brown et al., 1987). Viral and cellular DNA are probably repaired by the same mechanisms (Abrahams et al., 1976). We therefore conceived that proteins mediating repair would be associated, at least transiently, with SV40 minichromosomes.
0165-7992/89/$ 03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
228 Viral minichromosomes can be isolated under gentle, nondenaturing conditions (Turler and Beard, 1985), and contain equal amounts of DNA and protein. In experiments described in this paper, we sought conditions of isolation that would stabilize and preserve protein-DNA associations unique to damaged SV40 minichromosomes. This approach does not rely on the reconstitution of specific protein-DNA interaction in vitro, but requires instead that specific protein-chromatin associations in situ be stable under our conditions of isolation. Materials and methods
We cultured CV-1 African green monkey cells in DMEM (Gibco) supplemented with 5°70 fetal bovine serum, penicillin and streptomycin. Subconfluent ( - 80%) monolayers in 9-cm dishes were infected with SV40 at 5 plaque-forming units (pfu) per cell and incubated for 29-30 h. Media were then removed, saved as pooled conditioned media, and replaced with 5 ml of Tris-buffered saline (Brown and Cerutti, 1986). Cultures in saline were then exposed to 0-150 J / m z of UV radiation (254 nm) at a rate of 0.76 J / m 2 per sec. Saline was then replaced with 7 ml of the pooled conditioned media, and the cultures were incubated under normal culture conditions for 30 min to 4 h before lysis. Lysis and minichromosome preparation followed established procedures (Turler and Beard, 1985). Briefly, cells were scraped from the plate and lysed with 8 strokes of a Dounce homogenizer (loose pestle) in hypotonic buffer containing 10 mM Tris, pH 7.5, 5 mM KC1, 7 mM ~mercaptoethanol and 0.1 mM MgCI2. Nuclei were pelleted and incubated overnight, on ice, with 0.5 ml of lysis buffer. Minichromosomes were purified by centrifugation in continuous 5-20% sucrose gradients, in hypotonic buffer. Minichromosomes sedimented to the middle of 5-ml gradients after 1 h at 35 000 rpm at 4°C in a Beckman 50.1 rotor. Gradients were fractionated from the bottom by peristaltic pumping into 10 samples, and the protein compositions of the frac-
tions were analyzed by SDS-PAGE (Laemmli, 1970) using 5-12.5% acrylamide gradient gels. Protein gels were silver stained (Merril et al., 1981; Dzandu et al., 1984; de Moreno et al., 1985), using a band-enhancement technique (Lukiw and McLachlan, 1988). Results and discussion
Silver-stained gels revealed about 100 protein bands in minichromosome samples from unirradiated and UV-treated cells. Many of the strongest bands were probably from intact ribosomes that cosediment with minichromosomes in the presence of Mg2÷ ions (Poirier et al., 1986). However, individual proteins not associated with large structures do not appreciably sediment under these conditions of centrifugation. Fig. 1 presents protein profiles of minichromosome preparations from ceils exposed to 0, 30 and 150 J / m 2 of UV light, and incubated for 2 h. Comparison of gradient fractions 4, 5 and 6 reveals slight differences in protein composition across each gradient that are unrelated to UV treatment. Two differences are apparent in protein profiles of UV-irradiated and control samples. (1) Samples from cells treated with 30 J / m 2 contain a protein band corresponding to 220 kD which is not visible in unirradiated controls. This 220-kD protein is apparent in all 3 gradient fractions of the UV-treated sample. (2) Samples from cells treated with 150 J / m z also contain this 220-kD protein, but in addition show smaller amounts of a 230-kD protein, also present in all 3 central fractions of the gradient. These proteins were not found in any of the gradient fractions of equivalent preparations from UV-irradiated cells not infected with SV40 (not shown), indicating that the UV-dependent proteins are associated with minichromosomes rather than with other large structures. Neither protein was observed when minichromosomes were isolated in the presence of 1 mM EDTA instead of 0.1 mM MgC12 (not shown). Fig. 2 presents a time course of protein association, comparing the central fraction 5 of unitradiated and UV-treated preparations of cells lysed
229
0 J/m 2
f4
f5
30 J / m 2
f6
M
f4
f5
f6
150 J / m 2
M
f4
f5
f6
kD
200
116. 97. 77. 66 56 43
1
2
3
4
5
6
7
8
9
lane
Fig. 1. Identification of a 220-kD and a 230-kD protein associated with SV40minichromosomesisolated from UV-irradiated monkey ceils. Silver-stained SDS-PAGE profiles of proteins associated with SV40minichromosomes, from sucrose gradient fractions 4, 5 and 6 (f4, fS, f6) of preparations from unirradiated cells (lanes 1, 2, 3) and cells exposed to 30 J / m 2 (lanes 4, 5, 6) or 150 J / m 2 (lanes 7, 8, 9). Molecular weight markers (lanes M) from BDH. Arrows in lane 9 indicate the positions of the 220-kD and 230-kD proteins.
30 min, 1 h, 2 h and 4 h after UV irradiation. Unirradiated controls were always mock irradiated and incubated for the indicated time along with the irradiated cultures. The 220-kD and 230-kD proteins are not observed in minichromosome preparations taken 30 min after UV treatment, but are slightly visible after I h, more abundant after 2 h, and still present after 4 h. It is not feasible to analyse samples at later times because irradiation, especially o f the infected ceils, causes cell detachment and lysis, and loss o f material. We do not know the function of these two proteins, and our supposition that they participate in
D N A repair or in the metabolism of damaged chromatin is based solely on their specific cosedimentation with UV-damaged minichromosomes. It is possible that only small amounts of each protein are associated with minichromosomes, and that larger amounts remain free in the uppermost fraction of the gradient, where the number and diversity of proteins precludes resolution o f individual bands. Both proteins can be visualized using sample isolated from about 20% o f the infected cells on a 9-cm culture dish, and are therefore abundant enough to allow their purification.
230
30 min
1 hr
2 hr
4 hr
J/m 2
kD 200
116 97 77 56 43
lane Fig. 2. Time course of 220-kD and 230-kD protein association with minichromosomes. Protein profiles of minichromosomes isolated 30 min (lanes 1, 2, 3), 1 h (lanes 4, 5, 6), 2 h (lanes 7, 8, 9) and 4 h (lanes 10, 11, 12), from cells irradiated with 0, 30 or 150 J / m 2 as indicated. Molecular weight markers, lanes M. Arrows in lane 9 indicate the positions of the 220-kD and 230-kD proteins.
Acknowledgements We are grateful to Dr. John Thacker Richard
D. Wood
and Dr.
for helpful discussions, and to
Dr. Janet Jones for advice on silver staining. This work was supported in part by CEC Contract No. B16-E-144-UK.
References Abrahams, P.J., and A.J. Van tier Eb (1976) Host-cell reactivation of ultraviolet-irradiated SV40 DNA in five complementation groups of xeroderma pigmentosum, Mutation Res., 35, 13-22. Brown, T.C., and P.A. Cerutti (1986) Ultraviolet radiation inactivates SV40 by disrupting at least four genetic functions, EMBO J. 5, 197-203.
Brown, T.C., P. Beard and P.A. Cerutti (1987) Preferential repair of N-acetoxy acetylaminofluorene lesions in the nuclease-hypersensitive region of Simian Virus 40, J. Biol. Chem., 262, 11947-11951. Chu, G., and E. Chang (1988) Xeroderma pigmentosum group E cells lack a nuclear factor that binds to damaged DNA, Science, 242, 564-567. de Moreno, M.R., J.F. Smith and R.V. Smith (1985) Silver staining of proteins in polyacrylamide gels: increased sensitivity through a combined coomassie blue-silver stain procedure, Anal. Biochem., 151,466-470. De Pamphilis, M.L., and P.M. Wassarman (1980) Replication of eukaryotic chromosomes: a close-up of the replication fork, Annu. Rev. Biochem., 49, 627-666. Dzandu, J.K., M.E. Deh, D.L. Barrat and G.E, Wise (1984) Detection of erythrocyte membrane proteins, sialoglycoproteins, and lipids in the same polyacrylamide gel using a double-staining technique, Proc. Natl. Acad. Sci. (U.S.A.), 81, 1733-1737. Jacobovits, E,B., S. Bratosin and Y. Aloni (1980) A
231 nucleosome-free region in SV40 minichromosomes, Nature (London), 285, 263-265. Jiricny, J., M. Hughes, N. Corman and B.B. Rudkin (1988) A •human 200 kDa protein binds selectively to DNA fragments containing GT mismatches, Proc. Natl. Acad. Sci. (U.S.A.), 85, 8860-8864. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (London), 227, 680-685. Lindahl, T. (1982) DNA repair enzymes, Annu. Rev. Biochem., 51, 61-87. Lukiw, W.J., and D.R. McLachlan (1988) Silver stain image amplification, Trends Genet., 4, 146. Merril, C.R., D, Goldman, S.A. Sedman and M.H. Ebert (1981) Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins, Science, 211, 1437-1438. Otto, B., E. Fanning and A. Richter (1979) D N A polymerases and a single-strand-specific DNA-binding protein associated with Simian Virus 40 nucleoprotein complexes, Cold Spring Harbor Symp. Quant. Biol., 43, 705-708. Poirier, G., T.C. Brown and P.A. Cerutti (1986) Poly ADP ribosylation and DNA strand breakage in SV40 minichromosomes, Carcinogenesis, 6, 283-287.
Sancar, A., and G.B. Sancar (1988) DNA repair enzymes, Annu. Rev. Biochem, 57, 29-67. Saragosti, S., G. Moyne and M. Yaniv (1980) Absence of nucleosomes in a fraction of SV40 chromatin between the origin of replication and the region coding for the late leader RNA, Cell, 20, 65-73. Tooze (Ed.) (1980) DNA Tumor Viruses, Cold Spring Harbor Laboratory Press, New York. Tsubota, Y., M.A. Waqar, J.F. Burke, B.I. Milavetz, M.J. Evans, D. Kowalsky and J.A. Huberman (1979) Association of enzymes with replicating and nonreplicating Simian Virus 40 chromosomes, Cold Spring Harbor Symp. Quant. Biol., 43,693-704. Turler, H., and P. Beard (1985) Simian Virus 40 and Polyoma virus: growth, titration, transformation and purification of viral components, in: B.W.J. Mahy (Ed.), Virology: A Practical Approach, IRL Press, Oxford, pp. 169-192. Williams, J., and J. Cleaver (1978) Excision repair of ultraviolet damage in mammalian cells, Evidence for two steps in the excision of pyrimidine dimers, Biophys. J., 22, 265-279. Williams, J., and J. Cleaver (I 979) Removal of T4 endonuclease V sensitive sites from SV40 DNA after exposure to ultraviolet light, Biochim. Biophys. Acta, 562,429-437. Communicated by J.M. Gentile