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Cell-cycle-dependent repair of damage in alpha and bulk DNA on monkey cells
71
Mutation Research, 166 (1986) 71 77 DNA Repair Reports Elsevier MTR 06160
Cell-cycle-dependent repair of damage in alpha and bulk DNA of monkey cells Steven A. L e a d o n * a n d Philip C. H a n a w a l t Department of Biological Sciences, Stanford University, Stanford, CA 94305 (U.S.A.) (Received 21 October 1985) (Revision received 14 January 1986) (Accepted 14 January 1986)
Summary Excision repair of bulky chemical adducts in alpha DNA of confluent cultures of African green monkey cells has previously been shown to be deficient relative to that in the overall genome. We have compared the removal of adducts produced by treatment with aflatoxin B1 (AFB1) and N-acetoxy-2-acetylaminofluorene (NA-AAF) from alpha DNA sequences in synchronized and exponentially growing cultures of monkey cells. Proficient removal of AFB 1 adducts in alpha DNA was observed in exponentially growing cultures. However, as the cultures approached confluence, adduct removal from alpha DNA became deficient. Cells synchronized by subculturing confluent cultures exhibited proficient removal of adducts from both alpha and bulk DNA when treated in early G 1 or late S / G 2 while those cells treated in early S phase did not remove adducts from either alpha or bulk DNA. We conclude that the accessibility of chemical adducts to repair in alpha chromatin is influenced by the growth state and the cell cycle stage.
The covalent binding of chemical carcinogens to DNA in living cells elicits processes through which the alterations are recognized and the affected nucleotide sequences are replaced by repair replication using the undamaged DNA strand as template to restore the intact genome (Hanawalt et al., 1979). Such processes serve to protect the cells from the lethal, mutagenic and/or carcinogenic consequences of otherwise persisting DNA damage. However, the eukaryotic genome is comprised of a heterogeneous and dynamic population of domains with respect to both function and chromatin structure (Reeves, 1984). The biological consequences of DNA damage and the efficiency of its repair probably depend upon the state of the * Present address: Lawrence Berkeley Laboratory, Building 934, University of California, Berkeley, CA, U.S.A.
chromatin in the respective domains. In actively dividing cells, the chromatin structure undergoes a number of changes to facilitate gene expression and prepare for DNA synthesis which might affect the processing of DNA damage (Pardee et al., 1978; Reeves, 1984). When human fibroblasts are temporarily prevented from entering S phase, the UV-induced mutation frequency is decreased (Maher et al., 1979; Enninga et al., 1985) while cell survival is enhanced (Simmons, 1979; Chan and Little, 1979; Nagasawa et al., 1982). Konze-Thomas et al. (1982) reported that the UV-induced mutation frequency was strongly influenced by the length of the period between irradiation and the beginning of S phase. UV survival, however, has not been found to be dependent on the stage in the cell cycle in which the cells were irradiated (Konze-Thomas et al., 1982; Chan
0167-8817/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
72 and Little, 1982; Enninga et al., 1985). A deficiency in the excision repair of bulky chemical adducts in alpha DNA, a 172-base-pair (bp) highly repeated D N A sequence, was observed in confluent cultures of African green monkey cells (Zolan et al., 1982; Leadon et al., 1983). The repair of furocoumarin photoadducts in alpha D N A was only about 30% of that in the bulk DNA, even though the initial levels of damage, the time course of excision repair and the repair patch size were indistinguishable for the two D N A classes. In addition, D N A interstrand crosslink formation was restricted in alpha D N A (Zolan et al., 1984), In contrast, damage produced by 254-nm UV was repaired equally well in alpha and bulk DNA. These results suggested that some feature of the alpha D N A conformation a n d / o r its chromatin structure inhibited the recognition and repair of furocoumarin damage but not UV damage. We have also reported a much slower loss of aflatoxin B~ (AFB1) adducts from alpha D N A than from bulk DNA, representing a deficiency in the active removal of the primary initial adduct. 2,3-dihyro-2-( N 7-deoxyguanosyl)-3-hydroxyaflatoxin B 1 (Leadon et al., 1983). However, the repair of aguaninic sites produced by the spontaneous loss of A F B ~ - D N A adducts appeared normal. A slightly higher level of N-acetoxy-2-acetylaminofluorene (NA-AAF)-induced D N A adducts was found in alpha D N A than in the bulk of the genome (Leadon and Hanawalk 1984). This was due to an increase in the acetylated C8 adduct of guanine in alpha DNA, Although both the acetylated and deacetylated adducts were removed from the two D N A species, the level of repair was greater in the bulk DNA. Furthermore, the treatment of the cells with UV in addition to either AFB~ or N A - A A F resulted in the active removal of the carcinogen-induced D N A adducts from alpha D N A (Leadon et al., 1983; Leadon and Hanawalt, 1984). Thus, either the UV photoproducts or some step(s) in their repair evidently facilitate access for repair enzymes to bulky chemical adducts in alpha chromatin. It is important to note that all of the previously reported studies on repair in alpha D N A have been carried out on confluent cultures. We have now extended our study to examine the repair of bulky chemical adducts produced by AFB 1 and
NA-AAF in alpha and bulk D N A in exponentially growing and synchronized cultures of monkey cells. We find little difference in the initial rate of removal of D N A adducts from alpha and bulk D N A in exponentially growing and synchronized cultures. In addition, no removal of D N A adducts was detected during S phase in either class of DNA. Materials and methods Chemicals
[3H]AFB1 (20 C i / m m o l e ) was purchased from Moravek Biochemicals. [3H]N-acetoxy-2-acetylaminofluorene (9.8 C i / m m o l e ; Midwest Research Institute) was generously provided by T.D. Tlsty, Stanford University. After evaporation of the methylene chloride solvent, the sample was dissolved in anhydrous dimethyl sulfoxide (Pierce Chemical) to a final concentration of 1.5 raM. The $9 microsomal fraction from rat liver was generously provided by A.D. Burrell (General Products Division, I.B.M. Corp., San Jose, CA). Cell cuhure conditions and treatment
BS-C-1 African green monkey cells (ATCC CCL 26) were cultured as previously described (Zolan et al.. 1982), resulting in a cell-doubling time of approximately 30 h. For labeling of cellular DNA, the cultures were split at a ratio of 1 : 1 0 and grown for 7 days in either 4 /~Ci/ml carrier-free [~2P]orthophosphate (New England Nuclear) or 0.05 /~Ci/ml [14C]thymidine (50.5 m C i / m m o l e : New England Nuclear). For synchronization, cells from confluent plates were removed by trypsinization, pooled and counted with a hemocytometer and approximately 1 × 10 6 cells were seeded into 100-mm petri dishes (Falcon). Exponentially growing cells were prepared from cultures split at a ratio of 1 : 10 and grown for 4 days in 2 ~ C i / m l carrier-free [32p]orthophosphate, trypsinized. pooled and replated at a density of 0.5 × 106 cells/100-mm petri dish. These cultures were then incubated for an additional 2 days before exposure to AFB 1. Cultures were exposed to 0.25 /~M [3H]AFB1 in the presence of an activation buffer containing $9 microsomes for 30 min (Leadon et al., 1983) or to 0.15 /xM [3H]NA-AAF for 20 rain at 37°C (Leadon and Hanawalt, 1984).
73 Measurement of A F B l and N A - A A F damage DNA from cells treated with [3H]AFB1 was isolated on 47-mm polycarbonate filters (Nucleopore) (Leadon and Cerutti, 1982). The solutions used for D N A isolation, restriction endonuclease digestion and electrophoresis were adjusted to pH 6.6-6.9 to minimize spontaneous decomposition of the A F B 1 - D N A adducts. DNA isolated on the filters was released by incubation with 8 U Hind III endonuclease (Bethesda Research Laboratories) per t~g DNA for 6 h at 37°C in 20 mM phosphate buffer, 7 mM MgC12, 60 mM NaC1 pH 6.9. The DNA digests were fractionated by electrophoresis on 2% agarose gels in 25 mM histidine, 30 mM MOPS pH 6.6 at 4°C (Leadon et al., 1983). Cells treated with [3H]NA-AAF were lysed, treated with proteinase K (Boehringer Mannhem) and the DNA was isolated by centrifugation in CsC1 gradients (C.A. Smith et al., 1981). The fractions containing the DNA were then dialyzed against 10 mM Tris, 0.1 mM EDTA pH 8, treated with RNAase A at a concentration of 250 btg/ml for 1 h at 37°C and extracted twice with chloroformisoamyl alcohol (24:1) followed by precipitation of the DNA with ethanol. Purified DNA was digested with Hind III for 16 h at 37°C in the buffer accompanying the endonuclease and subjected to electrophoresis on 2% agarose gels in Tris-acetate-NaC1 buffer. The DNA bands were visualized by staining with ethidium bromide and the 172-bp alpha monomer band and the bulk DNA (the brightly fluorescent region near the top of the gel) were excised, removed from the gel fragments by electroelution, and assayed for radioactivity (Zolan et al., 1982). Total adduct frequencies were determined from the 32P-specific activity of the DNA from each sample and the 3H-specific activity of the [3H]AFBl and [3H]NA-AAF preparations. Measurement of rate of DNA synthesis and proportion of DNA repficated For measurement of the rate of total DNA synthesis and the proportion of cells synchronized, [14C]thymidine_labeled cultures were subcultured as described above in the presence of 5 ~M FdUrd and 50/~M BrdUrd. At 3-h intervals, beginning at 6 h after splitting, the cell cultures were pulsed with 10 ttCi/ml [3H]thymidine (80.1 Ci/mmole;
New England Nuclear) for 30 rain. The cells were lysed, treated with proteinase K and the parentaldensity DNA was resolved from hybrid-density DNA (synthesized by semi-conservative replication) by buoyant equilibrium centrifugation with CsC1 as described by C.A. Smith et al. (1981). The rate of DNA synthesis was determined from the amount of [3H]thymidine incorporated into hybrid-density DNA that was acid precipitable. The proportion of DNA that had replicated was determined from the relative amount of 14C-labeled DNA in the hybrid-density peak. For measurements of the rate of thymidine incorporation into alpha DNA, 32p-labeled cells were subcultured and pulsed with 2 /~Ci/ml [3H]thymidine at various times for 30 min at 37°C. The DNA was isolated as described above from CsCI gradients and electrophoresed on 2% agarose gels in Tris-acetate-NaC1 buffer. The DNA bands were visualized by staining with ethidium bromide and the 172-bp alpha monomer and the bulk of the DNA were excised, melted in 0.1 ml 1 N HC1 and assayed for radioactivity. Results
Adduct removal in exponentially growing and confluent cultures Exponentially growing cultures were obtained by subculturing cells after a 4-day growth period for an additional 2 days. At the time of treatment, the cultures were showing an exponential increase with time in the incorporation of [3H]thymidine into their DNA (data not shown). These cultures were exposed to [3H]AFB1 and the removal of adducts from alpha and bulk DNA was measured at various times after treatment. An initial rapid removal of AFB~-DNA adducts from both alpha and bulk DNA was observed in the exponentially growing cultures (Fig. 1). The initial rate of removal of damage from alpha DNA was faster in exponentially growing cultures than in confluent cultures. However, the initial rate of adduct removal from bulk DNA was slower in exponentially growing cultures than in confluent cultures. By 24 h after treatment, a difference in the removal of damage from alpha DNA compared with bulk DNA in exponentially growing cultures was observed while by 48 h post treatment, the rate of
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Fig. 1. Kinetics of the disappearance of total AFBn adducts from alpha D N A and bulk D N A in confluent and in exponentially growing cultures. The cells were prelabeled with 32p and treated with activated [3H]AFB1 (0.25 /xM). Total AFB 1 adducts bound to D N A were determined from the 3 H / 3 2 p ratio: alpha D N A (e) and bulk D N A ( O ) from confluent cultures; alpha D N A (a) and bulk D N A (A) from exponentially growing cultures.
adduct removal from alpha and bulk D N A was similar to .that found in confluent cultures. The similarity in the removal of AFB1 adducts in "exponentially growing" and confluent cultures by 48 h post treatment is most likely due to the reduced rate of D N A synthesis in the exponential cultures which occurs by 4 days after subculturing at the cell densities used in these experiments.
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Fig. 2. Kinetics of D N A replication in BS-C-1 cells. The cells were prelabeled with either [14C]thymidine or 32p, grown to confluence and then subcultured. At various times, the cell cultures were pulsed with [3H]thymidine for 30 min. (A) -~H incoporation into total D N A (e) or into D N A that was resolved on agarose gels as alpha D N A (a) or bulk D N A (A). (B) lac-labeled cells were subcuhured in the presence of 50 /~M BrdUrd and 5 /zM FrUrd. Parental-density D N A was resolved from hybrid-density (newly replicated) D N A by centrifugation in CsC1 gradients. The percentage of D N A that had replicated was determined from the relative amount of 14C-labeled D N A in the hybrid-density peak.
The more rapid removal of AFB 1 adducts from alpha D N A in exponentially growing cultures compared with that in confluent cultures indicated that regions of the genome which are inaccessible to repair enzymes when the cells are in a quiescent phase might be altered during division to then allow repair to occur. Since the exponentially growing cultures include cells at all stages of the cell cycle, we examined the removal of D N A damage in synchronized cultures in order to test this hypothesis. The rate of [3H]thymidine incorporation was used to determine the onset and duration of S phase in cells in which the D N A had been prelabeled with [14C]thymidine. An increase
in [3H]thymidine incorporation was observed in cells 12 h after subculturing (Fig. 2A). The rate of [3H]thymidine incorporation into the total D N A increased steadily until a peak value was reached 21 h after subculturing. The rate of incorporaton decreased from this point as the cells progressed into G 2 and mitosis. The length of time during which the cells were synthesizing D N A was approximately 15 h and was termed S phase. Little or no synchrony in [3H]thymidine incorporation was observed in the subsequent 40 72 h after subculturing. Using this technique, we found that approximately 90% of the D N A in the cell popula-
75
tion had been replicated by the end of S phase (Fig. 2B). In order to determine when alpha DNA was replicated in relation to the bulk of the genomic DNA, the incorporation of [3 H]thymidine into the 172-bp alpha D N A monomer was monitored in cells in which the DNA had been prelabeled with 32p (Fig. 2A). The bulk of the DNA, which was isolated as a high-molecular-weight band at the top of the gel and is depleted of alpha sequences, showed a pattern of D N A replication similar to that of total DNA. In contrast, alpha DNA was found to replicate late in S phase in agreement with a previous report (Tobia et al., 1972). Adduct removal in synchronized cell cultures The pattern of synchronization of cells observed in Fig. 2 following subculturing was arbitrarily divided into 3 successive 12-h periods: 3 h through 15 h (designated G1/early S phase), 15 h through 27 h (S phase) and 27 h through 39 h (late S phase/G2). At the beginning of each time interval, the cultures were exposed to [3H]AFB1 (0.25 /~M) in the presence of a microsomal activation system. The initial frequency of AFB~-DNA adducts and the amount remaining after 12 h were then measured in alpha DNA and in the bulk of the DNA and compared to the values obtained for confluent cultures (Go). The initial frequencies of D N A adducts for all 3 time periods in synchronized cultures were 1.3-1.8 AFB1 adducts/108 dalton DNA in both alpha and bulk DNA. Similar levels were also observed in confluent cultures. As previously observed, little or no removal of AFB 1 adducts from alpha D N A was observed in G O
TABLE 1 R E M O V A L OF A F B 1 - D N A CHRONIZED CULTURES Phase in cell cycle when cells were treated
G1 S phase Late S / G 2 G o (confluent)
ADDUCTS
FROM
SYN-
Percentage of initial adducts remaining after 12 h Alpha
Bulk
77 94 64 90
70 91 51 55
TABLE 2 R E M O V A L OF N A - A A F - D N A A D D U C T S F R O M SYNCHRONIZED CULTURES Phase in cell cycle when cells were treated
G~ S phase Late S / G 2 Go (confluent)
Percentage of initial adducts remaining after 12 h Alpha
Bulk
73 97 73 82
70 98 71 62
cultures when compared to the bulk of the DNA (Table 1). No removal of adducts from either alpha or bulk DNA was observed when cultures were treated in S phase. An intermediate level of removal in both alpha and bulk DNA was found in cells treated in G 1. In cultures treated in late S p h a s e / G 2, AFB1 adducts were removed from bulk D N A at a similar rate as in G O cultures while adducts were removed from alpha DNA at a faster rate than observed in G O cultures. The interpretation of the loss of AFB~ adducts from cellular D N A is complicated by the multiple pathways by which it can occur, namely: (i) the spontaneous release of the adduct leaving an unaltered guanine; (ii) the spontaneous release of the A F B : g u a n i n e residue, which would lead to repair synthesis initiated by an apurinic endonuclease; (iii) the enzymatic recognition of the AFB~ adduct in DNA, initiating an excision repair pathway similar to that operating on pyrimidine dimers and other bulky adducts. Thus, changes in intracellular conditions during the cell cycle might be altering the spontaneous loss of adducts rather than affecting this enzymatic removal. To assess this possibility, we examined the removal of damage produced by NA-AAF, which also produces bulky DNA adducts, but whose adducts are more chemically stable than those produced by AFB1 (Howard et al., 1981; Leadon and Hanawalt, 1984). As with AFB~, a deficient removal of NA-AAF adducts from alpha DNA was observed in G O cultures (Table 2; Zolan et al., 1982; Leadon and Hanawalt, 1984). No repair was observed in either alpha or bulk DNA in cultures treated during S phase. Intermediate levels of adduct removal were found in cultures treated either in G1 or late S phase/G2.
76
Discussion Cultured African green monkey cells were synchronized by subculturing confluent dishes of cells. This produced an interval, termed G 1, of about 15 h before the onset of D N A synthesis. D N A synthesis lasted an additional 15 h and was followed by a period during which little or no D N A synthesis was detectable. These latter two periods were denoted as S phase and G2, respectively. Approximately 90% of the cell population was synchronized using this method. Replication of the alpha sequences was also measured in the synchronized cultures and was found to occur in late S phase as previously observed by Tobia et al. (1972). We have previously shown that in confluent cultures of monkey cells, removal of bulky chemical adducts from alpha D N A was deficient compared with that from bulk D N A (Zolan et al., 1982, 1984; Leadon et al., 1983; Leadon and Hanawalt, 1984). However, in cultures that have been synchronized by subculturing, proficient removal of AFB1 adducts occurred in alpha D N A during early S phase and late S p h a s e / G > The loss of these adducts from alpha D N A was probably not due to an increase in the spontaneous loss of AFBI adducts since the more chemically stable N A - A A F adducts were also proficiently removed during the same time periods in the cell cycle. Our working hypothesis is that some aspect of the chromatin structure of alpha D N A in confluent cultures inhibits the recognition and repair of bulky chemical adducts and that this has been altered in actively dividing cultures. These alterations are more likely associated with structural changes accompanying S phase, which occur in all regions of the genome, rather than with localized changes in specific genomic domains associated with the activation of cell-cycle-specific genes since the alpha sequences are not transcribed. However, chromatin structure alterations cannot provide an adequate explanation for the variations in repair of adducts during the cell cycle since no repair of damage was observed when the cells were treated during S phase when the chromatin is presumably more "relaxed". In addition, the events immediately preceding the replication of a region of the genome are not alone sufficient
to ensure the repair of damage in that region since damage is also not removed during S phase in alpha DNA, which is replicated 5-10 h later than the bulk of the genome. The efficiency of removal of alkylated bases from the genome overall, following treatment of 10T1/2 cells with N-methyl-N'nitro-N-nitrosoguanidine (MNNG), has also been found to decrease during S phase (G.J. Smith et al., 1980, 1981), while cytotoxicity to M N N G increased during G 1, became maximal during S phase and decreased again during late S phase (Grisham et al., 1980). The repair of D N A damage at various stages of the cell cycle may be dependent on additional factors, such as the cellular concentrations of repair enzymes and the superhelical tension in the genomic DNA. Variation in repair enzyme levels during the cell cycle has been suggested by Gupta and Sirover (1980, 1981) who found that uracil glycosylase activity and repair replication levels exhibited peaks just prior to S phase in synchronized cultures. At least as striking as the variability in repair over the cell cycle is the persistence of bulky chemical adducts seen at all phases of the cycle in both alpha and bulk DNA. Thus, well over half of the initial adducts remain 12 h after treatment. In view of the recent evidence for genomic heterogeneity in D N A repair in the dihydrofolate reductase gene of Chinese hamster ovary cells (Bohr et al., 1985) and in the E. coli gpt gene integrated into the monkey genome (Leadon, manuscript submitted), it will be important to determine which genomic regions are preferentially repaired during that initial 12-h period (Hanawalt, 1986).
Acknowledgements We thank D. Okumoto for technical help in the initial stages of these experiments and C.A. Smith for helpful discussions. This work was supported by a grant (NP161) from the American Cancer Society and by USPHS grant GM09901.
References Bohr, V.A., C.A. Smith, D.S. Okumoto and P.C. Hanawalt (1985) DNA repair in an active gene: removal of pyrimidine dimers from the D H F R gene of CHO cells is much more efficient than in the genome overall, Cell, 40, 359 369.
77 Chan, G.L., and J.B. Little (1979) Resistance of plateau-phase human and xeroderma pigmentosum fibroblasts to the cytotoxic effect of ultraviolet light, Mutation Res., 63, 401-412. Chan, G.L., and J.B. Little (1982) Further studies on the survival of nonproliferating human diploid fibroblasts irradiated with ultraviolet light, Int. J. Radiat. Biol., 41, 359-368. Enninga, I.C., R.T.L. Groenendijk, A.A. van Zeeland and J.W.I.M. Simmons (1985) Differential response of human fibroblasts to the cytotoxic and mutagenic effects of UV radiation in different phases of the cell cycle, Mutation Res., 148, 119-128. Grisham, J.W., D.S. Greenberg, D.G. Kaufman and G.J. Smith (1980) Cycle-related toxicity and transformation in 10T1/2 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine, Proc. Natl. Acad. Sci. (U.S.A.), 77, 4813-4817. Gupta, P.K., and M.A. Sirover (1980) Sequential stimulation of DNA repair and DNA replication on normal human cells, Mutation Res., 72, 273-284. Gupta, P.K., and M.A. Sirover (1981) Cell cycle regulation of DNA repair in normal and repair deficient cells, Chem.-Biol. Interact., 36, 19-31. Hanawalt, P.C. (1986) Intragenomic heterogeneity in DNA damage processing: potential implication for risk assessment, in: M. Simic, L Grossman and A. Upton (Eds.), Mechanisms of DNA Damage and Repair, Plenum, New York, in press. Hanawalt, P.C., P.K. Cooper, A.K. Ganesan and C.A. Smith (1979) DNA repair in bacterial and mammalian cells, Annu. Rev. Biochem., 48, 783-836. Howard, P.C., D.A. Casciano, F.A. Beland and J.G. Shaddock Jr. (1981) The binding of N-hydroxy-2-acetylaminofluorene to DNA and repair of the adducts in primary rat hepatocyte cultures, Carcinogenesis, 2, 97-102. Konze-Thomas, B., R.M. Hazard, V.M. Maher and J.J. McCormick (1982) Extent of excision repair before DNA synthesis determines the mutagenic but not the lethal effect of UV radiation, Mutation Res., 94, 421-434. Leadon, S.A., and P.C. Cerutti (1982) A rapid and mild procedure for the isolation of DNA from mammalian cells, Anal. Biochem., 120, 282-288. Leadon, S.A., and P.C. Hanawalt (1984) Ultraviolet irradiation of monkey cells enhances the repair of DNA adducts in alpha DNA, Carcinogenesis, 5, 1505-1510.
Leadon, S.A., M.E. Zolan and P.C. Hanawalt (1983) Restricted repair of aflatoxin BI induced damage in alpha DNA of monkey cells, Nucleic Acids Res., 11, 5675-5689. Maher, V.M., D.J. Dorney, A.L. Mendrala, B. Konze-Thomas and J.J. McCormick (1979) DNA excision repair processes in human cells can eliminate the cytotoxic and mutagenic consequences of ultraviolet irradiation, Mutation Res., 62, 311-323. Nagasawa, H., A.J. Fornace, M.A. Ritter and J.B. Little (1982) Relationship of enhanced survival during confluent holding recovery in ultraviolet-irradiated human and mouse cells to chromosome aberrations, sister chromatid exchanges, and DNA repair, Radiat. Res., 59, 483-496. Pardee, A.B., R. Dubrow, J.L. Hamlin and R.F. Kletzien (1978) Animal cell cycle, Annu. Rev, Biochem., 47, 715-750. Reeves, R. (1984) Transcriptionally active chromatin, Biochim. Biophys. Acta, 782, 343-393. Simmons, J.W.I.M. (1979) Development of a liquid-holding technique for the study of DNA repair in human diploid fibroblasts, Mutation Res., 59, 273-283. Smith, C.A., P.K. Cooper and P.C. Hanawalt (1981) in: E.C. Friedberg and P.C. Hanawalt (Eds.), DNA Repair, A Laboratory Manual of Research Procedures, Vol. 1, part B, Dekker, New York, pp. 289-305. Smith, G.J., D.G. Kaufman and J.W. Grisham (1980) Decreased excision of O6-methylguanine and N7-methylguanine during S phase in 10TI/2 cells, Biochem. Biophys. Res. Commun., 92, 787-794. Smith, G.J., J.W. Grisham and D.G. Kaufman (1981) Cycle-dependent removal of certain methylated bases from DNA of 10T1/2 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine, Cancer Res., 41, 1373-1378. Tobia, A.M,, E.H. Brown, R.J. Parker, C.L. Schildkraut and J.J. Maio (1972) DNA replication in synchronized cultured mammalian cells, IV. Replication of African green monkey component alpha and bulk DNA, Biochim. Biophys. Acta, 277, 256-268. Zolan, M.E., G.A. Cortopassi, C.A. Smith and P.C. Hanawalt (1982) Deficient repair of chemical adducts in alpha DNA of monkey cells, Cell, 28, 613-619. Zolan, M.E., C.A. Smith and P.C. Hanawalt (1984) Formation and repair of furocoumarin adducts in alpha deoxyribonucleic acid and bulk deoxyribonucleic acid of monkey cells, Biochemistry, 23, 63-69.
Mutation Research, 166 (1986) 71 77 DNA Repair Reports Elsevier MTR 06160
Cell-cycle-dependent repair of damage in alpha and bulk DNA of monkey cells Steven A. L e a d o n * a n d Philip C. H a n a w a l t Department of Biological Sciences, Stanford University, Stanford, CA 94305 (U.S.A.) (Received 21 October 1985) (Revision received 14 January 1986) (Accepted 14 January 1986)
Summary Excision repair of bulky chemical adducts in alpha DNA of confluent cultures of African green monkey cells has previously been shown to be deficient relative to that in the overall genome. We have compared the removal of adducts produced by treatment with aflatoxin B1 (AFB1) and N-acetoxy-2-acetylaminofluorene (NA-AAF) from alpha DNA sequences in synchronized and exponentially growing cultures of monkey cells. Proficient removal of AFB 1 adducts in alpha DNA was observed in exponentially growing cultures. However, as the cultures approached confluence, adduct removal from alpha DNA became deficient. Cells synchronized by subculturing confluent cultures exhibited proficient removal of adducts from both alpha and bulk DNA when treated in early G 1 or late S / G 2 while those cells treated in early S phase did not remove adducts from either alpha or bulk DNA. We conclude that the accessibility of chemical adducts to repair in alpha chromatin is influenced by the growth state and the cell cycle stage.
The covalent binding of chemical carcinogens to DNA in living cells elicits processes through which the alterations are recognized and the affected nucleotide sequences are replaced by repair replication using the undamaged DNA strand as template to restore the intact genome (Hanawalt et al., 1979). Such processes serve to protect the cells from the lethal, mutagenic and/or carcinogenic consequences of otherwise persisting DNA damage. However, the eukaryotic genome is comprised of a heterogeneous and dynamic population of domains with respect to both function and chromatin structure (Reeves, 1984). The biological consequences of DNA damage and the efficiency of its repair probably depend upon the state of the * Present address: Lawrence Berkeley Laboratory, Building 934, University of California, Berkeley, CA, U.S.A.
chromatin in the respective domains. In actively dividing cells, the chromatin structure undergoes a number of changes to facilitate gene expression and prepare for DNA synthesis which might affect the processing of DNA damage (Pardee et al., 1978; Reeves, 1984). When human fibroblasts are temporarily prevented from entering S phase, the UV-induced mutation frequency is decreased (Maher et al., 1979; Enninga et al., 1985) while cell survival is enhanced (Simmons, 1979; Chan and Little, 1979; Nagasawa et al., 1982). Konze-Thomas et al. (1982) reported that the UV-induced mutation frequency was strongly influenced by the length of the period between irradiation and the beginning of S phase. UV survival, however, has not been found to be dependent on the stage in the cell cycle in which the cells were irradiated (Konze-Thomas et al., 1982; Chan
0167-8817/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
72 and Little, 1982; Enninga et al., 1985). A deficiency in the excision repair of bulky chemical adducts in alpha DNA, a 172-base-pair (bp) highly repeated D N A sequence, was observed in confluent cultures of African green monkey cells (Zolan et al., 1982; Leadon et al., 1983). The repair of furocoumarin photoadducts in alpha D N A was only about 30% of that in the bulk DNA, even though the initial levels of damage, the time course of excision repair and the repair patch size were indistinguishable for the two D N A classes. In addition, D N A interstrand crosslink formation was restricted in alpha D N A (Zolan et al., 1984), In contrast, damage produced by 254-nm UV was repaired equally well in alpha and bulk DNA. These results suggested that some feature of the alpha D N A conformation a n d / o r its chromatin structure inhibited the recognition and repair of furocoumarin damage but not UV damage. We have also reported a much slower loss of aflatoxin B~ (AFB1) adducts from alpha D N A than from bulk DNA, representing a deficiency in the active removal of the primary initial adduct. 2,3-dihyro-2-( N 7-deoxyguanosyl)-3-hydroxyaflatoxin B 1 (Leadon et al., 1983). However, the repair of aguaninic sites produced by the spontaneous loss of A F B ~ - D N A adducts appeared normal. A slightly higher level of N-acetoxy-2-acetylaminofluorene (NA-AAF)-induced D N A adducts was found in alpha D N A than in the bulk of the genome (Leadon and Hanawalk 1984). This was due to an increase in the acetylated C8 adduct of guanine in alpha DNA, Although both the acetylated and deacetylated adducts were removed from the two D N A species, the level of repair was greater in the bulk DNA. Furthermore, the treatment of the cells with UV in addition to either AFB~ or N A - A A F resulted in the active removal of the carcinogen-induced D N A adducts from alpha D N A (Leadon et al., 1983; Leadon and Hanawalt, 1984). Thus, either the UV photoproducts or some step(s) in their repair evidently facilitate access for repair enzymes to bulky chemical adducts in alpha chromatin. It is important to note that all of the previously reported studies on repair in alpha D N A have been carried out on confluent cultures. We have now extended our study to examine the repair of bulky chemical adducts produced by AFB 1 and
NA-AAF in alpha and bulk D N A in exponentially growing and synchronized cultures of monkey cells. We find little difference in the initial rate of removal of D N A adducts from alpha and bulk D N A in exponentially growing and synchronized cultures. In addition, no removal of D N A adducts was detected during S phase in either class of DNA. Materials and methods Chemicals
[3H]AFB1 (20 C i / m m o l e ) was purchased from Moravek Biochemicals. [3H]N-acetoxy-2-acetylaminofluorene (9.8 C i / m m o l e ; Midwest Research Institute) was generously provided by T.D. Tlsty, Stanford University. After evaporation of the methylene chloride solvent, the sample was dissolved in anhydrous dimethyl sulfoxide (Pierce Chemical) to a final concentration of 1.5 raM. The $9 microsomal fraction from rat liver was generously provided by A.D. Burrell (General Products Division, I.B.M. Corp., San Jose, CA). Cell cuhure conditions and treatment
BS-C-1 African green monkey cells (ATCC CCL 26) were cultured as previously described (Zolan et al.. 1982), resulting in a cell-doubling time of approximately 30 h. For labeling of cellular DNA, the cultures were split at a ratio of 1 : 1 0 and grown for 7 days in either 4 /~Ci/ml carrier-free [~2P]orthophosphate (New England Nuclear) or 0.05 /~Ci/ml [14C]thymidine (50.5 m C i / m m o l e : New England Nuclear). For synchronization, cells from confluent plates were removed by trypsinization, pooled and counted with a hemocytometer and approximately 1 × 10 6 cells were seeded into 100-mm petri dishes (Falcon). Exponentially growing cells were prepared from cultures split at a ratio of 1 : 10 and grown for 4 days in 2 ~ C i / m l carrier-free [32p]orthophosphate, trypsinized. pooled and replated at a density of 0.5 × 106 cells/100-mm petri dish. These cultures were then incubated for an additional 2 days before exposure to AFB 1. Cultures were exposed to 0.25 /~M [3H]AFB1 in the presence of an activation buffer containing $9 microsomes for 30 min (Leadon et al., 1983) or to 0.15 /xM [3H]NA-AAF for 20 rain at 37°C (Leadon and Hanawalt, 1984).
73 Measurement of A F B l and N A - A A F damage DNA from cells treated with [3H]AFB1 was isolated on 47-mm polycarbonate filters (Nucleopore) (Leadon and Cerutti, 1982). The solutions used for D N A isolation, restriction endonuclease digestion and electrophoresis were adjusted to pH 6.6-6.9 to minimize spontaneous decomposition of the A F B 1 - D N A adducts. DNA isolated on the filters was released by incubation with 8 U Hind III endonuclease (Bethesda Research Laboratories) per t~g DNA for 6 h at 37°C in 20 mM phosphate buffer, 7 mM MgC12, 60 mM NaC1 pH 6.9. The DNA digests were fractionated by electrophoresis on 2% agarose gels in 25 mM histidine, 30 mM MOPS pH 6.6 at 4°C (Leadon et al., 1983). Cells treated with [3H]NA-AAF were lysed, treated with proteinase K (Boehringer Mannhem) and the DNA was isolated by centrifugation in CsC1 gradients (C.A. Smith et al., 1981). The fractions containing the DNA were then dialyzed against 10 mM Tris, 0.1 mM EDTA pH 8, treated with RNAase A at a concentration of 250 btg/ml for 1 h at 37°C and extracted twice with chloroformisoamyl alcohol (24:1) followed by precipitation of the DNA with ethanol. Purified DNA was digested with Hind III for 16 h at 37°C in the buffer accompanying the endonuclease and subjected to electrophoresis on 2% agarose gels in Tris-acetate-NaC1 buffer. The DNA bands were visualized by staining with ethidium bromide and the 172-bp alpha monomer band and the bulk DNA (the brightly fluorescent region near the top of the gel) were excised, removed from the gel fragments by electroelution, and assayed for radioactivity (Zolan et al., 1982). Total adduct frequencies were determined from the 32P-specific activity of the DNA from each sample and the 3H-specific activity of the [3H]AFBl and [3H]NA-AAF preparations. Measurement of rate of DNA synthesis and proportion of DNA repficated For measurement of the rate of total DNA synthesis and the proportion of cells synchronized, [14C]thymidine_labeled cultures were subcultured as described above in the presence of 5 ~M FdUrd and 50/~M BrdUrd. At 3-h intervals, beginning at 6 h after splitting, the cell cultures were pulsed with 10 ttCi/ml [3H]thymidine (80.1 Ci/mmole;
New England Nuclear) for 30 rain. The cells were lysed, treated with proteinase K and the parentaldensity DNA was resolved from hybrid-density DNA (synthesized by semi-conservative replication) by buoyant equilibrium centrifugation with CsC1 as described by C.A. Smith et al. (1981). The rate of DNA synthesis was determined from the amount of [3H]thymidine incorporated into hybrid-density DNA that was acid precipitable. The proportion of DNA that had replicated was determined from the relative amount of 14C-labeled DNA in the hybrid-density peak. For measurements of the rate of thymidine incorporation into alpha DNA, 32p-labeled cells were subcultured and pulsed with 2 /~Ci/ml [3H]thymidine at various times for 30 min at 37°C. The DNA was isolated as described above from CsCI gradients and electrophoresed on 2% agarose gels in Tris-acetate-NaC1 buffer. The DNA bands were visualized by staining with ethidium bromide and the 172-bp alpha monomer and the bulk of the DNA were excised, melted in 0.1 ml 1 N HC1 and assayed for radioactivity. Results
Adduct removal in exponentially growing and confluent cultures Exponentially growing cultures were obtained by subculturing cells after a 4-day growth period for an additional 2 days. At the time of treatment, the cultures were showing an exponential increase with time in the incorporation of [3H]thymidine into their DNA (data not shown). These cultures were exposed to [3H]AFB1 and the removal of adducts from alpha and bulk DNA was measured at various times after treatment. An initial rapid removal of AFB~-DNA adducts from both alpha and bulk DNA was observed in the exponentially growing cultures (Fig. 1). The initial rate of removal of damage from alpha DNA was faster in exponentially growing cultures than in confluent cultures. However, the initial rate of adduct removal from bulk DNA was slower in exponentially growing cultures than in confluent cultures. By 24 h after treatment, a difference in the removal of damage from alpha DNA compared with bulk DNA in exponentially growing cultures was observed while by 48 h post treatment, the rate of
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Fig. 1. Kinetics of the disappearance of total AFBn adducts from alpha D N A and bulk D N A in confluent and in exponentially growing cultures. The cells were prelabeled with 32p and treated with activated [3H]AFB1 (0.25 /xM). Total AFB 1 adducts bound to D N A were determined from the 3 H / 3 2 p ratio: alpha D N A (e) and bulk D N A ( O ) from confluent cultures; alpha D N A (a) and bulk D N A (A) from exponentially growing cultures.
adduct removal from alpha and bulk D N A was similar to .that found in confluent cultures. The similarity in the removal of AFB1 adducts in "exponentially growing" and confluent cultures by 48 h post treatment is most likely due to the reduced rate of D N A synthesis in the exponential cultures which occurs by 4 days after subculturing at the cell densities used in these experiments.
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Fig. 2. Kinetics of D N A replication in BS-C-1 cells. The cells were prelabeled with either [14C]thymidine or 32p, grown to confluence and then subcultured. At various times, the cell cultures were pulsed with [3H]thymidine for 30 min. (A) -~H incoporation into total D N A (e) or into D N A that was resolved on agarose gels as alpha D N A (a) or bulk D N A (A). (B) lac-labeled cells were subcuhured in the presence of 50 /~M BrdUrd and 5 /zM FrUrd. Parental-density D N A was resolved from hybrid-density (newly replicated) D N A by centrifugation in CsC1 gradients. The percentage of D N A that had replicated was determined from the relative amount of 14C-labeled D N A in the hybrid-density peak.
The more rapid removal of AFB 1 adducts from alpha D N A in exponentially growing cultures compared with that in confluent cultures indicated that regions of the genome which are inaccessible to repair enzymes when the cells are in a quiescent phase might be altered during division to then allow repair to occur. Since the exponentially growing cultures include cells at all stages of the cell cycle, we examined the removal of D N A damage in synchronized cultures in order to test this hypothesis. The rate of [3H]thymidine incorporation was used to determine the onset and duration of S phase in cells in which the D N A had been prelabeled with [14C]thymidine. An increase
in [3H]thymidine incorporation was observed in cells 12 h after subculturing (Fig. 2A). The rate of [3H]thymidine incorporation into the total D N A increased steadily until a peak value was reached 21 h after subculturing. The rate of incorporaton decreased from this point as the cells progressed into G 2 and mitosis. The length of time during which the cells were synthesizing D N A was approximately 15 h and was termed S phase. Little or no synchrony in [3H]thymidine incorporation was observed in the subsequent 40 72 h after subculturing. Using this technique, we found that approximately 90% of the D N A in the cell popula-
75
tion had been replicated by the end of S phase (Fig. 2B). In order to determine when alpha DNA was replicated in relation to the bulk of the genomic DNA, the incorporation of [3 H]thymidine into the 172-bp alpha D N A monomer was monitored in cells in which the DNA had been prelabeled with 32p (Fig. 2A). The bulk of the DNA, which was isolated as a high-molecular-weight band at the top of the gel and is depleted of alpha sequences, showed a pattern of D N A replication similar to that of total DNA. In contrast, alpha DNA was found to replicate late in S phase in agreement with a previous report (Tobia et al., 1972). Adduct removal in synchronized cell cultures The pattern of synchronization of cells observed in Fig. 2 following subculturing was arbitrarily divided into 3 successive 12-h periods: 3 h through 15 h (designated G1/early S phase), 15 h through 27 h (S phase) and 27 h through 39 h (late S phase/G2). At the beginning of each time interval, the cultures were exposed to [3H]AFB1 (0.25 /~M) in the presence of a microsomal activation system. The initial frequency of AFB~-DNA adducts and the amount remaining after 12 h were then measured in alpha DNA and in the bulk of the DNA and compared to the values obtained for confluent cultures (Go). The initial frequencies of D N A adducts for all 3 time periods in synchronized cultures were 1.3-1.8 AFB1 adducts/108 dalton DNA in both alpha and bulk DNA. Similar levels were also observed in confluent cultures. As previously observed, little or no removal of AFB 1 adducts from alpha D N A was observed in G O
TABLE 1 R E M O V A L OF A F B 1 - D N A CHRONIZED CULTURES Phase in cell cycle when cells were treated
G1 S phase Late S / G 2 G o (confluent)
ADDUCTS
FROM
SYN-
Percentage of initial adducts remaining after 12 h Alpha
Bulk
77 94 64 90
70 91 51 55
TABLE 2 R E M O V A L OF N A - A A F - D N A A D D U C T S F R O M SYNCHRONIZED CULTURES Phase in cell cycle when cells were treated
G~ S phase Late S / G 2 Go (confluent)
Percentage of initial adducts remaining after 12 h Alpha
Bulk
73 97 73 82
70 98 71 62
cultures when compared to the bulk of the DNA (Table 1). No removal of adducts from either alpha or bulk DNA was observed when cultures were treated in S phase. An intermediate level of removal in both alpha and bulk DNA was found in cells treated in G 1. In cultures treated in late S p h a s e / G 2, AFB1 adducts were removed from bulk D N A at a similar rate as in G O cultures while adducts were removed from alpha DNA at a faster rate than observed in G O cultures. The interpretation of the loss of AFB~ adducts from cellular D N A is complicated by the multiple pathways by which it can occur, namely: (i) the spontaneous release of the adduct leaving an unaltered guanine; (ii) the spontaneous release of the A F B : g u a n i n e residue, which would lead to repair synthesis initiated by an apurinic endonuclease; (iii) the enzymatic recognition of the AFB~ adduct in DNA, initiating an excision repair pathway similar to that operating on pyrimidine dimers and other bulky adducts. Thus, changes in intracellular conditions during the cell cycle might be altering the spontaneous loss of adducts rather than affecting this enzymatic removal. To assess this possibility, we examined the removal of damage produced by NA-AAF, which also produces bulky DNA adducts, but whose adducts are more chemically stable than those produced by AFB1 (Howard et al., 1981; Leadon and Hanawalt, 1984). As with AFB~, a deficient removal of NA-AAF adducts from alpha DNA was observed in G O cultures (Table 2; Zolan et al., 1982; Leadon and Hanawalt, 1984). No repair was observed in either alpha or bulk DNA in cultures treated during S phase. Intermediate levels of adduct removal were found in cultures treated either in G1 or late S phase/G2.
76
Discussion Cultured African green monkey cells were synchronized by subculturing confluent dishes of cells. This produced an interval, termed G 1, of about 15 h before the onset of D N A synthesis. D N A synthesis lasted an additional 15 h and was followed by a period during which little or no D N A synthesis was detectable. These latter two periods were denoted as S phase and G2, respectively. Approximately 90% of the cell population was synchronized using this method. Replication of the alpha sequences was also measured in the synchronized cultures and was found to occur in late S phase as previously observed by Tobia et al. (1972). We have previously shown that in confluent cultures of monkey cells, removal of bulky chemical adducts from alpha D N A was deficient compared with that from bulk D N A (Zolan et al., 1982, 1984; Leadon et al., 1983; Leadon and Hanawalt, 1984). However, in cultures that have been synchronized by subculturing, proficient removal of AFB1 adducts occurred in alpha D N A during early S phase and late S p h a s e / G > The loss of these adducts from alpha D N A was probably not due to an increase in the spontaneous loss of AFBI adducts since the more chemically stable N A - A A F adducts were also proficiently removed during the same time periods in the cell cycle. Our working hypothesis is that some aspect of the chromatin structure of alpha D N A in confluent cultures inhibits the recognition and repair of bulky chemical adducts and that this has been altered in actively dividing cultures. These alterations are more likely associated with structural changes accompanying S phase, which occur in all regions of the genome, rather than with localized changes in specific genomic domains associated with the activation of cell-cycle-specific genes since the alpha sequences are not transcribed. However, chromatin structure alterations cannot provide an adequate explanation for the variations in repair of adducts during the cell cycle since no repair of damage was observed when the cells were treated during S phase when the chromatin is presumably more "relaxed". In addition, the events immediately preceding the replication of a region of the genome are not alone sufficient
to ensure the repair of damage in that region since damage is also not removed during S phase in alpha DNA, which is replicated 5-10 h later than the bulk of the genome. The efficiency of removal of alkylated bases from the genome overall, following treatment of 10T1/2 cells with N-methyl-N'nitro-N-nitrosoguanidine (MNNG), has also been found to decrease during S phase (G.J. Smith et al., 1980, 1981), while cytotoxicity to M N N G increased during G 1, became maximal during S phase and decreased again during late S phase (Grisham et al., 1980). The repair of D N A damage at various stages of the cell cycle may be dependent on additional factors, such as the cellular concentrations of repair enzymes and the superhelical tension in the genomic DNA. Variation in repair enzyme levels during the cell cycle has been suggested by Gupta and Sirover (1980, 1981) who found that uracil glycosylase activity and repair replication levels exhibited peaks just prior to S phase in synchronized cultures. At least as striking as the variability in repair over the cell cycle is the persistence of bulky chemical adducts seen at all phases of the cycle in both alpha and bulk DNA. Thus, well over half of the initial adducts remain 12 h after treatment. In view of the recent evidence for genomic heterogeneity in D N A repair in the dihydrofolate reductase gene of Chinese hamster ovary cells (Bohr et al., 1985) and in the E. coli gpt gene integrated into the monkey genome (Leadon, manuscript submitted), it will be important to determine which genomic regions are preferentially repaired during that initial 12-h period (Hanawalt, 1986).
Acknowledgements We thank D. Okumoto for technical help in the initial stages of these experiments and C.A. Smith for helpful discussions. This work was supported by a grant (NP161) from the American Cancer Society and by USPHS grant GM09901.
References Bohr, V.A., C.A. Smith, D.S. Okumoto and P.C. Hanawalt (1985) DNA repair in an active gene: removal of pyrimidine dimers from the D H F R gene of CHO cells is much more efficient than in the genome overall, Cell, 40, 359 369.
77 Chan, G.L., and J.B. Little (1979) Resistance of plateau-phase human and xeroderma pigmentosum fibroblasts to the cytotoxic effect of ultraviolet light, Mutation Res., 63, 401-412. Chan, G.L., and J.B. Little (1982) Further studies on the survival of nonproliferating human diploid fibroblasts irradiated with ultraviolet light, Int. J. Radiat. Biol., 41, 359-368. Enninga, I.C., R.T.L. Groenendijk, A.A. van Zeeland and J.W.I.M. Simmons (1985) Differential response of human fibroblasts to the cytotoxic and mutagenic effects of UV radiation in different phases of the cell cycle, Mutation Res., 148, 119-128. Grisham, J.W., D.S. Greenberg, D.G. Kaufman and G.J. Smith (1980) Cycle-related toxicity and transformation in 10T1/2 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine, Proc. Natl. Acad. Sci. (U.S.A.), 77, 4813-4817. Gupta, P.K., and M.A. Sirover (1980) Sequential stimulation of DNA repair and DNA replication on normal human cells, Mutation Res., 72, 273-284. Gupta, P.K., and M.A. Sirover (1981) Cell cycle regulation of DNA repair in normal and repair deficient cells, Chem.-Biol. Interact., 36, 19-31. Hanawalt, P.C. (1986) Intragenomic heterogeneity in DNA damage processing: potential implication for risk assessment, in: M. Simic, L Grossman and A. Upton (Eds.), Mechanisms of DNA Damage and Repair, Plenum, New York, in press. Hanawalt, P.C., P.K. Cooper, A.K. Ganesan and C.A. Smith (1979) DNA repair in bacterial and mammalian cells, Annu. Rev. Biochem., 48, 783-836. Howard, P.C., D.A. Casciano, F.A. Beland and J.G. Shaddock Jr. (1981) The binding of N-hydroxy-2-acetylaminofluorene to DNA and repair of the adducts in primary rat hepatocyte cultures, Carcinogenesis, 2, 97-102. Konze-Thomas, B., R.M. Hazard, V.M. Maher and J.J. McCormick (1982) Extent of excision repair before DNA synthesis determines the mutagenic but not the lethal effect of UV radiation, Mutation Res., 94, 421-434. Leadon, S.A., and P.C. Cerutti (1982) A rapid and mild procedure for the isolation of DNA from mammalian cells, Anal. Biochem., 120, 282-288. Leadon, S.A., and P.C. Hanawalt (1984) Ultraviolet irradiation of monkey cells enhances the repair of DNA adducts in alpha DNA, Carcinogenesis, 5, 1505-1510.
Leadon, S.A., M.E. Zolan and P.C. Hanawalt (1983) Restricted repair of aflatoxin BI induced damage in alpha DNA of monkey cells, Nucleic Acids Res., 11, 5675-5689. Maher, V.M., D.J. Dorney, A.L. Mendrala, B. Konze-Thomas and J.J. McCormick (1979) DNA excision repair processes in human cells can eliminate the cytotoxic and mutagenic consequences of ultraviolet irradiation, Mutation Res., 62, 311-323. Nagasawa, H., A.J. Fornace, M.A. Ritter and J.B. Little (1982) Relationship of enhanced survival during confluent holding recovery in ultraviolet-irradiated human and mouse cells to chromosome aberrations, sister chromatid exchanges, and DNA repair, Radiat. Res., 59, 483-496. Pardee, A.B., R. Dubrow, J.L. Hamlin and R.F. Kletzien (1978) Animal cell cycle, Annu. Rev, Biochem., 47, 715-750. Reeves, R. (1984) Transcriptionally active chromatin, Biochim. Biophys. Acta, 782, 343-393. Simmons, J.W.I.M. (1979) Development of a liquid-holding technique for the study of DNA repair in human diploid fibroblasts, Mutation Res., 59, 273-283. Smith, C.A., P.K. Cooper and P.C. Hanawalt (1981) in: E.C. Friedberg and P.C. Hanawalt (Eds.), DNA Repair, A Laboratory Manual of Research Procedures, Vol. 1, part B, Dekker, New York, pp. 289-305. Smith, G.J., D.G. Kaufman and J.W. Grisham (1980) Decreased excision of O6-methylguanine and N7-methylguanine during S phase in 10TI/2 cells, Biochem. Biophys. Res. Commun., 92, 787-794. Smith, G.J., J.W. Grisham and D.G. Kaufman (1981) Cycle-dependent removal of certain methylated bases from DNA of 10T1/2 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine, Cancer Res., 41, 1373-1378. Tobia, A.M,, E.H. Brown, R.J. Parker, C.L. Schildkraut and J.J. Maio (1972) DNA replication in synchronized cultured mammalian cells, IV. Replication of African green monkey component alpha and bulk DNA, Biochim. Biophys. Acta, 277, 256-268. Zolan, M.E., G.A. Cortopassi, C.A. Smith and P.C. Hanawalt (1982) Deficient repair of chemical adducts in alpha DNA of monkey cells, Cell, 28, 613-619. Zolan, M.E., C.A. Smith and P.C. Hanawalt (1984) Formation and repair of furocoumarin adducts in alpha deoxyribonucleic acid and bulk deoxyribonucleic acid of monkey cells, Biochemistry, 23, 63-69.