173
Mutation Research, 62 (1979) 173--181 © Elsevier/North-Holland Biomedical Press
SURVIVAL OF APURINIC SV40 DNA IN THE D-COMPLEMENTATION GROUP OF XERODERMA PIGMENTOSUM
R.D. KUDRNA, J. SMITH, S. LINN and E.E. PENHOET *
Department of Biochemistry, University of California, Berkeley, CA 94720 (U.S.A.) (Received 25 October 1978) (Revision received 19 February 1979) (Accepted 27 February 1979)
Summary The survival of depurinated Form I SV40 DNA was studied in normal human fibroblasts and in D-complementation Xeroderma pigmentosum (XP) fibroblasts. Survival was measured with an infective center assay. Heat-acid and methyl methanesulfonate (MMS) were used as depurinating agents. After 3 hrs of depurination by heat--acid treatment, infectivity in normal cells was less than 15% of the controls compared to more than 50% for the XP D cell strains. Similar results were obtained with MMS-treated DNA. These results are contrary to expectation since apurinic endonuclease activity, which is presumed to be involved in the repair of apurinic sites, is much lower in XP D cell strains than in normal cell strains. Our results indicate that another mechanism for the repair of apurinic sites could exist.
Xeroderma pigmentosum (XP) is a human genetic disease in which afflicted persons are highly susceptible to the induction of skin cancers by sunlight; neurological disorders are also present in some cases [30]. Cell strains derived from skin biopsies from XP patients have low levels of DNA-repair synthesis induced by UV-irradiation [3,5,7,16,30] and have abnormally low levels of thymidine dimer excision from their DNA [9,16,28,32]. Cell hybridization studies define at least 7 complementation groups [2]. These groups vary in their competence to repair UV-induced DNA damage, however this competence is n o t necessarily group-specific [2]. It has been concluded from these and other studies that XP cells are defective at an early stage of the repair process, most likely at the endonuclease incision step [6,8,32]. Attempts to characterize definitely enzyme defects have * To whom reprint requests should be addressed.
174
failed [8], but it has been reported that extracts of XP cells are able normally to excise t h y m i n e dimers from exogenous DNA but not from chromatin [26]. Although differences in UV-endonuclease activity between XP and normal cells have n o t as y e t been demonstrated, we recently showed that apurinic endonuclease activity might be altered in some XP cell strains [18]. Extracts of XP cells from several strains of the D-complementation group have one-sixth of normal activity and a higher Km for substrate was observed in extracts from cell strains of the A and D groups. Further investigation by Kuhnlein et al. [19] revealed that two species of apurinic endonuclease activity could be resolved by phosphocellulose chromatography, a high Km and a low Km species. The D strain cells lack the low K m species. How these data might relate to the inability of XP strains to repair UV-induced damage or to the clinical aspects of the disease is n o t known. To study the possible significance of the differences in apurinic endonuclease in vivo, we have adopted a host-cell reactivation system using an infective center assay for SV40 DNA. SV40 DNA is infectious in m o n k e y cells and human cells; m o n k e y cells are permissive and human cells are semipermissive for viral production. Moreover, SV40 DNA can be treated with various DNAdamaging agents prior to infection thus eliminating the need to treat whole cells or whole viruses with these agents and thus possible uncharacterized types of damage. If apurinic endonuclease activity observed in normal human-cell extracts is necessary for the repair of apurinic sites in vivo, then the survival of apurinic SV40 DNA in XP group D strains may be decreased compared to the normal cell strains. The present paper investigates this idea. Materials and methods
Cell culture Cell line BSC-1 was obtained courtesy of Dr. J o h n Riggs, California State Department o f Health. Cell line CV1-P was obtained from M. Dieckmann, Stanford University. BSC-1 and CV1-P are African Green Monkey Kidney (AGMK) cell lines. Cell strain 424 is a foreskin fibroblast strain obtained from the Navy Biological Laboratories, Oakland, CA. All other cell strains were obtained from the American Type Culture Collection (ATCC). Table 1 lists the human cell strains and their properties. Cells were utilized between passages 10 and 25. Human cell strains were grown in 150-cm 2 Corning T-flasks in Dulbecco's modified Eagle's (DME) medium (Flow Laboratories) containing 10% fetalbovine serum (Pacific Biologicals) and no antibiotics. BSC-1 and CV1-P cell lines were grown in DME containing 5% fetal-bovine serum and no antibiotics. All cell strains were grown at 37°C in 5% CO~. As confluency was reached, human-cell strains were split 1 : 4, and the BSC-1 and CV1-P cell lines split 1 : 10. For survival experiments cells were split into 100 m m Falcon culture dishes, 1 T-flask into 15 dishes for normal human fibroblasts and 1 T-flask into 10 dishes for the XP and ataxia telangiectasia cell strains. Isolation o f S V 4 0 DNA Wild-type SV40 obtained from Dr. Paul Berg, Stanford University, was twice plaque-purified by infecting confluent monolayers of CV1-P cells at a multiplic-
175
TABLE 1 HUMAN CELL STRAINS USED
Cell line 424 CRL CRL CRL CRL
1157 1160 1200 1343
Genotype
(XP6BE) (XP5BE) (XP7BE) (SePan)
Normal XP D G r o u p XP D G r o u p XP D G r o u p AT B Group
T h e n o m e n c l a t u r e for XP p a t i e n t s is d e s c r i b e d b y Cleaver a n d B o o t s m a [8] a n d the a s s i g n m e n t of c o m p l e m e n t a t i o n g r o u p b y K r a e m e r et al. [ 1 7 ] . T h e A T B is d e s c r i b e d b y P a t e r s o n e t al. [ 2 9 ] .
ity of infection (M.O.I.) of 10 -4. For DNA preparation, confluent monolayers of BSC-1 cells were infected at an M.O.I. of 0.1 and incubated 8--10 days at which time approximately 90% of the cells showed cytopathic effects. A Hirt supernatant [13] was prepared and extracted once with chloroform--isoamyl alcohol (24 : 1) to remove protein and the DNA was precipitated with cold 95% ethanol. The ethanol precipitate was dissolved in 10 mM Tris--HC1, pH 8.0 and banded by centrifugation in an SW 50.1 rotor at 40 000 rpm for 24 h at 17°C in cesium chloride (Gallard Schlesinger, special biochemical grade, density 1.56 g/cm 3 containing 100 pg/ml ethidium bromide). Form I DNA (closed, circular) was found in the denser band and was removed by tapping the side of the centrifuge tube with a l-cc tuberculin syringe and a 23-gauge needle. The DNA was diluted 1 : 5 with 10 mM Tris--HC1, pH 8.0 and the ethidium bromide removed by extracting twice with isoamyl alcohol. The DNA was then precipitated twice with 2 vol. cold 95% ethanol and dissolved in 10 mM Tris-HC1, pH 8.0 and dialyzed against 10 mM Tris--HCl, pH 8.0. For most preparations, m o r e than 80% of the SV40 DNA was Form I as determined with 1% agarose gels. [3H]Thymidine-labeled SV40 DNA was prepared as above except that 24 h after infection of BSC-1 monolayers, the medium was replaced with fresh medium containing 10 pCi/ml of [3H]thymidine (Schwarz/Mann, specific activity 55 Ci/mMole).
DNA-damaging treatments SV40 DNA (10 pg/ml in 10 mM Tris--HC1, pH 8.0) was irradiated with a G15T8 Westinghouse Sterilamp at dose rate of 24 erg/mm2/sec in a 60-mm culture dish. Dosage was determined using a Latarjet meter and by ferrioxalate actinometry [14]. Acid depurination was as described by Lindahl and Andersson [22]. SV40 DNA, 11 pg/ml in 10 mM Tris--HC1, pH 8.0, was brought to pH 5.0 by the addition o f 0.1 vol. of 1.0 M NaCl, 0.1 M sodium citrate pH 4.0 and incubated at 70°C. The number of alkali-labile (apurinic) sites introduced under these conditions was determined using the nitrocellulose filter binding assay described by Kuhnlein et al. [18]. 3 apurinic sites and less than 0.05 singlestranded nicks per molecule per hour were introduced under these conditions. For treatment with methyl methanesulfonate (MMS), 25 mM MMS and 10 pg/ ml DNA were incubated at 37°C in 10 mM Tris--HC1, pH 8.0. As determined by
176 the filter-binding assay, 4 alkali-labile sites and less than 0.05 nicks per molecule per hour were introduced. For infection with treated DNA, 0.5 pg in 0.05 ml was taken at intervals, immediately diluted to 0.2 ml with Tris-buffered saline [15] pH 7.5 and used to infect a 100-mm plate of cells prepared as described below. The concentration of DNA used lies in the linear range of the dose--response curve for infectivity in human cells (Kudrna, unpublished results and ref. 1). For acid
Mutation Research, 62 (1979) 173--181 © Elsevier/North-Holland Biomedical Press
SURVIVAL OF APURINIC SV40 DNA IN THE D-COMPLEMENTATION GROUP OF XERODERMA PIGMENTOSUM
R.D. KUDRNA, J. SMITH, S. LINN and E.E. PENHOET *
Department of Biochemistry, University of California, Berkeley, CA 94720 (U.S.A.) (Received 25 October 1978) (Revision received 19 February 1979) (Accepted 27 February 1979)
Summary The survival of depurinated Form I SV40 DNA was studied in normal human fibroblasts and in D-complementation Xeroderma pigmentosum (XP) fibroblasts. Survival was measured with an infective center assay. Heat-acid and methyl methanesulfonate (MMS) were used as depurinating agents. After 3 hrs of depurination by heat--acid treatment, infectivity in normal cells was less than 15% of the controls compared to more than 50% for the XP D cell strains. Similar results were obtained with MMS-treated DNA. These results are contrary to expectation since apurinic endonuclease activity, which is presumed to be involved in the repair of apurinic sites, is much lower in XP D cell strains than in normal cell strains. Our results indicate that another mechanism for the repair of apurinic sites could exist.
Xeroderma pigmentosum (XP) is a human genetic disease in which afflicted persons are highly susceptible to the induction of skin cancers by sunlight; neurological disorders are also present in some cases [30]. Cell strains derived from skin biopsies from XP patients have low levels of DNA-repair synthesis induced by UV-irradiation [3,5,7,16,30] and have abnormally low levels of thymidine dimer excision from their DNA [9,16,28,32]. Cell hybridization studies define at least 7 complementation groups [2]. These groups vary in their competence to repair UV-induced DNA damage, however this competence is n o t necessarily group-specific [2]. It has been concluded from these and other studies that XP cells are defective at an early stage of the repair process, most likely at the endonuclease incision step [6,8,32]. Attempts to characterize definitely enzyme defects have * To whom reprint requests should be addressed.
174
failed [8], but it has been reported that extracts of XP cells are able normally to excise t h y m i n e dimers from exogenous DNA but not from chromatin [26]. Although differences in UV-endonuclease activity between XP and normal cells have n o t as y e t been demonstrated, we recently showed that apurinic endonuclease activity might be altered in some XP cell strains [18]. Extracts of XP cells from several strains of the D-complementation group have one-sixth of normal activity and a higher Km for substrate was observed in extracts from cell strains of the A and D groups. Further investigation by Kuhnlein et al. [19] revealed that two species of apurinic endonuclease activity could be resolved by phosphocellulose chromatography, a high Km and a low Km species. The D strain cells lack the low K m species. How these data might relate to the inability of XP strains to repair UV-induced damage or to the clinical aspects of the disease is n o t known. To study the possible significance of the differences in apurinic endonuclease in vivo, we have adopted a host-cell reactivation system using an infective center assay for SV40 DNA. SV40 DNA is infectious in m o n k e y cells and human cells; m o n k e y cells are permissive and human cells are semipermissive for viral production. Moreover, SV40 DNA can be treated with various DNAdamaging agents prior to infection thus eliminating the need to treat whole cells or whole viruses with these agents and thus possible uncharacterized types of damage. If apurinic endonuclease activity observed in normal human-cell extracts is necessary for the repair of apurinic sites in vivo, then the survival of apurinic SV40 DNA in XP group D strains may be decreased compared to the normal cell strains. The present paper investigates this idea. Materials and methods
Cell culture Cell line BSC-1 was obtained courtesy of Dr. J o h n Riggs, California State Department o f Health. Cell line CV1-P was obtained from M. Dieckmann, Stanford University. BSC-1 and CV1-P are African Green Monkey Kidney (AGMK) cell lines. Cell strain 424 is a foreskin fibroblast strain obtained from the Navy Biological Laboratories, Oakland, CA. All other cell strains were obtained from the American Type Culture Collection (ATCC). Table 1 lists the human cell strains and their properties. Cells were utilized between passages 10 and 25. Human cell strains were grown in 150-cm 2 Corning T-flasks in Dulbecco's modified Eagle's (DME) medium (Flow Laboratories) containing 10% fetalbovine serum (Pacific Biologicals) and no antibiotics. BSC-1 and CV1-P cell lines were grown in DME containing 5% fetal-bovine serum and no antibiotics. All cell strains were grown at 37°C in 5% CO~. As confluency was reached, human-cell strains were split 1 : 4, and the BSC-1 and CV1-P cell lines split 1 : 10. For survival experiments cells were split into 100 m m Falcon culture dishes, 1 T-flask into 15 dishes for normal human fibroblasts and 1 T-flask into 10 dishes for the XP and ataxia telangiectasia cell strains. Isolation o f S V 4 0 DNA Wild-type SV40 obtained from Dr. Paul Berg, Stanford University, was twice plaque-purified by infecting confluent monolayers of CV1-P cells at a multiplic-
175
TABLE 1 HUMAN CELL STRAINS USED
Cell line 424 CRL CRL CRL CRL
1157 1160 1200 1343
Genotype
(XP6BE) (XP5BE) (XP7BE) (SePan)
Normal XP D G r o u p XP D G r o u p XP D G r o u p AT B Group
T h e n o m e n c l a t u r e for XP p a t i e n t s is d e s c r i b e d b y Cleaver a n d B o o t s m a [8] a n d the a s s i g n m e n t of c o m p l e m e n t a t i o n g r o u p b y K r a e m e r et al. [ 1 7 ] . T h e A T B is d e s c r i b e d b y P a t e r s o n e t al. [ 2 9 ] .
ity of infection (M.O.I.) of 10 -4. For DNA preparation, confluent monolayers of BSC-1 cells were infected at an M.O.I. of 0.1 and incubated 8--10 days at which time approximately 90% of the cells showed cytopathic effects. A Hirt supernatant [13] was prepared and extracted once with chloroform--isoamyl alcohol (24 : 1) to remove protein and the DNA was precipitated with cold 95% ethanol. The ethanol precipitate was dissolved in 10 mM Tris--HC1, pH 8.0 and banded by centrifugation in an SW 50.1 rotor at 40 000 rpm for 24 h at 17°C in cesium chloride (Gallard Schlesinger, special biochemical grade, density 1.56 g/cm 3 containing 100 pg/ml ethidium bromide). Form I DNA (closed, circular) was found in the denser band and was removed by tapping the side of the centrifuge tube with a l-cc tuberculin syringe and a 23-gauge needle. The DNA was diluted 1 : 5 with 10 mM Tris--HC1, pH 8.0 and the ethidium bromide removed by extracting twice with isoamyl alcohol. The DNA was then precipitated twice with 2 vol. cold 95% ethanol and dissolved in 10 mM Tris-HC1, pH 8.0 and dialyzed against 10 mM Tris--HCl, pH 8.0. For most preparations, m o r e than 80% of the SV40 DNA was Form I as determined with 1% agarose gels. [3H]Thymidine-labeled SV40 DNA was prepared as above except that 24 h after infection of BSC-1 monolayers, the medium was replaced with fresh medium containing 10 pCi/ml of [3H]thymidine (Schwarz/Mann, specific activity 55 Ci/mMole).
DNA-damaging treatments SV40 DNA (10 pg/ml in 10 mM Tris--HC1, pH 8.0) was irradiated with a G15T8 Westinghouse Sterilamp at dose rate of 24 erg/mm2/sec in a 60-mm culture dish. Dosage was determined using a Latarjet meter and by ferrioxalate actinometry [14]. Acid depurination was as described by Lindahl and Andersson [22]. SV40 DNA, 11 pg/ml in 10 mM Tris--HC1, pH 8.0, was brought to pH 5.0 by the addition o f 0.1 vol. of 1.0 M NaCl, 0.1 M sodium citrate pH 4.0 and incubated at 70°C. The number of alkali-labile (apurinic) sites introduced under these conditions was determined using the nitrocellulose filter binding assay described by Kuhnlein et al. [18]. 3 apurinic sites and less than 0.05 singlestranded nicks per molecule per hour were introduced under these conditions. For treatment with methyl methanesulfonate (MMS), 25 mM MMS and 10 pg/ ml DNA were incubated at 37°C in 10 mM Tris--HC1, pH 8.0. As determined by
176 the filter-binding assay, 4 alkali-labile sites and less than 0.05 nicks per molecule per hour were introduced. For infection with treated DNA, 0.5 pg in 0.05 ml was taken at intervals, immediately diluted to 0.2 ml with Tris-buffered saline [15] pH 7.5 and used to infect a 100-mm plate of cells prepared as described below. The concentration of DNA used lies in the linear range of the dose--response curve for infectivity in human cells (Kudrna, unpublished results and ref. 1). For acid
