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[38] Permeabilized cells
I381
PERMEABILIZEDCELLS
[381 P e r m e a b i l i z e d
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Cells
By ROBB E. MOSES Introduction Permeabilized cells allow measurement of authentic replicative DNA synthesis. They are notable for ease of preparation and a minimal disturbance of the replicative apparatus. The limitation is that there is no initiation of new cycles of DNA replication. Such systems serve well to monitor the effects of agents on DNA replication or repair since they allow ready definition of the category of synthesis. Bacterial Cells Several methods have been used to measure replicative DNA synthesis in bacteria. With E. coli as a model, plasmolyzed cells were studied extensively. This system was not satisfactory for many studies because the method of forming spheroplasts raised background levels of nonspecific D N A repair synthesis to unacceptable levels, l This is particularly a problem in cells containing D N A polymerase I. Several organic solvents have been tried. The best developed system consists of toluene treatment. Replication shows complete ATP dependence and a low background of nonspecific synthesis. The final product is of high molecular weight, made semiconservatively, and discontinuous DNA synthesis can be demonstrated. One advantageous aspect of the toluenetreated cell system is that cells containing DNA polymerase I can be used. This allows testing of strains in which the presence of a polA mutation would be lethal. Replicative synthesis is apparently elongation only, with sudden arrest in dnaB temperature-sensitive strains. The preparation of cells2 for assay is by the following steps. Escherichia coli cells are grown to a concentration of approximately 1 × 10 9 and harvested by centrifugation at 4°. The cell pellet is suspended in 50 mM, pH 7.4, KPO4 at one-tenth volume of the culture. The cell suspension is made in 1% (v/v) toluene and gently agitated for between 2 and 10 min at room temperature. Individual strains show different tolerance of this procedure and it is important to determine a time course for ATPdependent synthesis for each. G . B u t t i n a n d M. W r i g h t , Cold Spring Harbor Syrup. Quant. Biol. 33, 259 (1968). 2 R. E. M o s e s a n d C. C. R i c h a r d s o n , Natl. Acad. Sci. 67~ 6 7 4 (1970).
METHODS IN ENZYMOLOGY, VOL. 262
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
498
In Vitro REPLICATIONSYSTEMS
[38]
Following exposure, the cells are harvested and the cell pellet is washed thoroughly with the above buffer. Cells are resuspended to a final volume of 0.1 culture volume in 50 mM KPO4, pH 7.4. The cells may be divided into aliquots and quick frozen at - 8 0 °. Such cell suspensions are stable for extended periods of time for measuring replicative D N A synthesis. The assay for D N A replication contains the following in 0.3 ml: 66 mM potassium phosphate buffer, pH 7.4, 13 mM MgC12, 1.3 mM ATP, 33/xM each of dCTP, dGTP, ATP, dTTP, with a radioactive isotope in one of the triphosphates. Normally, 1 to 5 x 108 toluene-treated cells are used for measurement of replication. Reactions are terminated by the addition of 3 ml of cold 10% trichloroacetic acid-0.1 M sodium pyrophosphate. The reaction is allowed to stand for 5 min at 4 ° and then washed over a Whatman GF/C disk with vacuum aspiration. The filter is washed several times with 10% trichloroacetic acid-0.1 M sodium pyrophosphate, followed by 10 ml of cold 0.01 M HCI. The disks are dried and radioactivity determined. Replication is dependent on ATP, requires all 5'-deoxyribonucleoside triphosphates, and occurs at approximately the in vivo rate in this system. The system may be modified by the addition of Triton X-100, a nonionic detergent. The presence of 1% detergent does not alter the rate or extent of synthesis, nor does it change the semiconservative nature of the synthesis. However, the addition of the detergent allows accessibility to macromolecules. 3 With detergent, 10 mM 2-mercaptoethanol, final concentration, is added to the reaction.
Mammalian Cells Several methods have been used to permeabilize mammalian cells to allow assay of D N A replication. One system, which has the advantage of being reversible and allowing cell survival, uses lysolecithin for permeabilization. 4 Fibroblasts are grown on plates, trypsinized, and washed with 0.15 sucrose, 80 mM HCI, 5 mM MgCI2, and 35 mM HEPES buffer, pH 7.2, with protease inhibitor if wished. Cells are resuspended at 8 x 107/ml in the same components and 0.5 mg/ml lysolecithin is added. Synthesis is assayed in the above buffer system (1 to 5 x 107 cells/ml) in 0.4 ml with 1.25 mM ATP, 5.0 mM phosphoenol pyruvate (PEP), and 10 to 100/xM 3 R. E. Moses, J. Biol. Chem. 247, 6031 (1972). 4 M. R. Miller and D. N. Chinault, J. Biol. Chem. 257, 10,204 (1982).
I38]
PERMEABILIZEDCELLS
499
each of dATP, dTTP, dCTP, and d G T P with radioactive isotope label as needed. Reactions are terminated and determined as above. Applications Permeable cell systems are useful for defining the biochemical requirements and responses of D N A replication and repair. Access by substrates, DNA-damaging reagents, or inhibitors of synthesis, in combination with the use of mutants defective in known enzymatic function, has made cellular functions apparent. In either the prokaryotic or eukaryotic system, a density analog, BrdUTP, can be substituted for d T T P to differentiate replicative D N A synthesis from nonreplicative. Mutants showing temperature-sensitive D N A replication have also been useful. Each system has been shown to respond to D N A damage with repair synthesis. 5"6The E. c o l i system has been used to define the role of D N A polymerases in response to ultraviolet radiation or after DNA-damaging drugs, such as bleomycin. 6'7 Permeable cells also have been used to examine the role of D N A polymerases in response to hydrogen peroxide damage, showing that cells have more than one repair pathway in that response. 8,9 In human fibroblasts the use of inhibitors specific for different polymerases has allowed identification of the roles of these enzymes in response to damage secondary to mutagens. 5 Use of mutants with defined defciencies has, of course, been much less available in permeable human cells than in bacterial cells. In summary, permeable cell systems offer the advantage of testing the cellular response to low-molecular-weight agents in a situation representing minimal pertubation of the replisome.
5 R. A. Hammond, J. K. McClung, and M. R. Miller, Biochemistry 29, 286 (1990). 6D. Bowersock and R. E. Moses, J. Biol. Chem. 248, 7449 (1973). 7M. R. Miller and C. N. Chinault, Z Biol. Chem. 257, 46 (1982). M. E. Hagensee and R. E. Moses, J. Bacteriol. 168, 1059 (1986). 9M. E. Hagensee and R. E. Moses, J. Bacteriol. 171, 991 (1989).
PERMEABILIZEDCELLS
[381 P e r m e a b i l i z e d
497
Cells
By ROBB E. MOSES Introduction Permeabilized cells allow measurement of authentic replicative DNA synthesis. They are notable for ease of preparation and a minimal disturbance of the replicative apparatus. The limitation is that there is no initiation of new cycles of DNA replication. Such systems serve well to monitor the effects of agents on DNA replication or repair since they allow ready definition of the category of synthesis. Bacterial Cells Several methods have been used to measure replicative DNA synthesis in bacteria. With E. coli as a model, plasmolyzed cells were studied extensively. This system was not satisfactory for many studies because the method of forming spheroplasts raised background levels of nonspecific D N A repair synthesis to unacceptable levels, l This is particularly a problem in cells containing D N A polymerase I. Several organic solvents have been tried. The best developed system consists of toluene treatment. Replication shows complete ATP dependence and a low background of nonspecific synthesis. The final product is of high molecular weight, made semiconservatively, and discontinuous DNA synthesis can be demonstrated. One advantageous aspect of the toluenetreated cell system is that cells containing DNA polymerase I can be used. This allows testing of strains in which the presence of a polA mutation would be lethal. Replicative synthesis is apparently elongation only, with sudden arrest in dnaB temperature-sensitive strains. The preparation of cells2 for assay is by the following steps. Escherichia coli cells are grown to a concentration of approximately 1 × 10 9 and harvested by centrifugation at 4°. The cell pellet is suspended in 50 mM, pH 7.4, KPO4 at one-tenth volume of the culture. The cell suspension is made in 1% (v/v) toluene and gently agitated for between 2 and 10 min at room temperature. Individual strains show different tolerance of this procedure and it is important to determine a time course for ATPdependent synthesis for each. G . B u t t i n a n d M. W r i g h t , Cold Spring Harbor Syrup. Quant. Biol. 33, 259 (1968). 2 R. E. M o s e s a n d C. C. R i c h a r d s o n , Natl. Acad. Sci. 67~ 6 7 4 (1970).
METHODS IN ENZYMOLOGY, VOL. 262
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
498
In Vitro REPLICATIONSYSTEMS
[38]
Following exposure, the cells are harvested and the cell pellet is washed thoroughly with the above buffer. Cells are resuspended to a final volume of 0.1 culture volume in 50 mM KPO4, pH 7.4. The cells may be divided into aliquots and quick frozen at - 8 0 °. Such cell suspensions are stable for extended periods of time for measuring replicative D N A synthesis. The assay for D N A replication contains the following in 0.3 ml: 66 mM potassium phosphate buffer, pH 7.4, 13 mM MgC12, 1.3 mM ATP, 33/xM each of dCTP, dGTP, ATP, dTTP, with a radioactive isotope in one of the triphosphates. Normally, 1 to 5 x 108 toluene-treated cells are used for measurement of replication. Reactions are terminated by the addition of 3 ml of cold 10% trichloroacetic acid-0.1 M sodium pyrophosphate. The reaction is allowed to stand for 5 min at 4 ° and then washed over a Whatman GF/C disk with vacuum aspiration. The filter is washed several times with 10% trichloroacetic acid-0.1 M sodium pyrophosphate, followed by 10 ml of cold 0.01 M HCI. The disks are dried and radioactivity determined. Replication is dependent on ATP, requires all 5'-deoxyribonucleoside triphosphates, and occurs at approximately the in vivo rate in this system. The system may be modified by the addition of Triton X-100, a nonionic detergent. The presence of 1% detergent does not alter the rate or extent of synthesis, nor does it change the semiconservative nature of the synthesis. However, the addition of the detergent allows accessibility to macromolecules. 3 With detergent, 10 mM 2-mercaptoethanol, final concentration, is added to the reaction.
Mammalian Cells Several methods have been used to permeabilize mammalian cells to allow assay of D N A replication. One system, which has the advantage of being reversible and allowing cell survival, uses lysolecithin for permeabilization. 4 Fibroblasts are grown on plates, trypsinized, and washed with 0.15 sucrose, 80 mM HCI, 5 mM MgCI2, and 35 mM HEPES buffer, pH 7.2, with protease inhibitor if wished. Cells are resuspended at 8 x 107/ml in the same components and 0.5 mg/ml lysolecithin is added. Synthesis is assayed in the above buffer system (1 to 5 x 107 cells/ml) in 0.4 ml with 1.25 mM ATP, 5.0 mM phosphoenol pyruvate (PEP), and 10 to 100/xM 3 R. E. Moses, J. Biol. Chem. 247, 6031 (1972). 4 M. R. Miller and D. N. Chinault, J. Biol. Chem. 257, 10,204 (1982).
I38]
PERMEABILIZEDCELLS
499
each of dATP, dTTP, dCTP, and d G T P with radioactive isotope label as needed. Reactions are terminated and determined as above. Applications Permeable cell systems are useful for defining the biochemical requirements and responses of D N A replication and repair. Access by substrates, DNA-damaging reagents, or inhibitors of synthesis, in combination with the use of mutants defective in known enzymatic function, has made cellular functions apparent. In either the prokaryotic or eukaryotic system, a density analog, BrdUTP, can be substituted for d T T P to differentiate replicative D N A synthesis from nonreplicative. Mutants showing temperature-sensitive D N A replication have also been useful. Each system has been shown to respond to D N A damage with repair synthesis. 5"6The E. c o l i system has been used to define the role of D N A polymerases in response to ultraviolet radiation or after DNA-damaging drugs, such as bleomycin. 6'7 Permeable cells also have been used to examine the role of D N A polymerases in response to hydrogen peroxide damage, showing that cells have more than one repair pathway in that response. 8,9 In human fibroblasts the use of inhibitors specific for different polymerases has allowed identification of the roles of these enzymes in response to damage secondary to mutagens. 5 Use of mutants with defined defciencies has, of course, been much less available in permeable human cells than in bacterial cells. In summary, permeable cell systems offer the advantage of testing the cellular response to low-molecular-weight agents in a situation representing minimal pertubation of the replisome.
5 R. A. Hammond, J. K. McClung, and M. R. Miller, Biochemistry 29, 286 (1990). 6D. Bowersock and R. E. Moses, J. Biol. Chem. 248, 7449 (1973). 7M. R. Miller and C. N. Chinault, Z Biol. Chem. 257, 46 (1982). M. E. Hagensee and R. E. Moses, J. Bacteriol. 168, 1059 (1986). 9M. E. Hagensee and R. E. Moses, J. Bacteriol. 171, 991 (1989).