806
BIOCHEMICAL SOCIETY TRANSACTIONS
Craig, R. K. & Keir, H. M. (19756) Biochem. J. 145,225-232 Dunham, V. L. & Cherry, J. H. (1973) Biochem. Biophys. Res. Commun. 54,403-410 Holmes, A. M., Hesslewood, I. P. &Johnston, I. R. (1974) Eur. J. Biochem. 43, 487-499 Keir, H. M. & Craig, R. K. (1973) Biochem. SOC.Trans. 1, 1073-1077 McLennan, A. G. & Keir, H. M. (1975) Biochem. J. 151, 239-247 Robinson, N. E. & Bryant, J. A. (1975) Planta 127, 69-75 Srivastava, B. I. S. & Grace, J. T. (1974) Life Sci. 14, 1947-1954 Stevens, C., Jenns, S. M. & Bryant, J. A. (1975) Biochem. Soc. Trans. 3, 1126-1128
Deoxyribonucleic Acid Polymerases from Chlamydomonas reinhardii CAROLE A. ROSS and WILLIAM J. HARRIS Department of Biochemistry, University of Aberdeen, Marischal College, Aberdeen A 3 9 l A S , Scotland, U.K.
DNA-dependent DNA polymerases (EC 2.7.7.7) have been studied in detail in only a small number of protozoan species. The enzymes which have been most highly characterized have been those from Saccharomyces cerevisiae (Wintersberger & Wintersberger, 1970), Tetrahymena pyriformis (Crerar & Pearlman, 1974), the smut fungus Ustilago maydis (Banks et al., 1976) and Euglena gracilis (McLennan & Keir, 1975a). In all these organisms, multiple DNA polymerase activities have been detected. The major cellular activities have high, sometimes heterogeneous, molecular weights, which vary depending on the surrounding ionic environment. In this, these enzymes resemble the mammalian DNA polymerase (Chang, 1971; Holmes et al., 1974). Exonuclease activity has been found associated with protozoan DNA polymerases (Banks & Yarranton, 1976; McLennan & Keir, 1975b), although the mammalian enzymes are thought to be devoid of any deoxyribonuclease activities. Small amounts of DNA polymerase activity have previously been reported to be present in whole cell extracts of the unicellular alga Chlumydomonas reinhardii (Schonherr & Keir, 1972), extraordinarily high amounts of dexoyribonuclease activity in these extracts accounting for the low detectable activity. The present work describes the characterization of the major DNA polymerases of this organism, undertaken as part of a general study of DNA metabolism in the cell cycle of Chlumydomonas. At least three DNA polymerase activities have been isolated from extracts of Chlamydomonas reinhardii cells (strain wild type provided by the Culture Centre of Algae and Protozoa, Cambridge, U.K.) grown under photosynthesizing conditions (Kates &Jones, 1964). Of these, two enzyme activities have been purified 2000-fold with respect to protein content, and have been named DNA polymerases u and 6. These enzymes have been separated by ion-exchange and DNA-affinity chromatography. Both enzymes require MgZf and all four deoxyribonucleoside triphosphates for maximal activity. Calf thymus DNA, activated by a modification of the method of Loeb (1969), is the best natural DNA template. Both enzymes have a broad pH optimum in 5 0 m ~ Tris/HC1 buffer, DNA polymerase a having maximum activity at pH8.5, whereas maximum activity with DNA polymerase b is obtained at pH7.5. DNA polymerase a requires Mgz+at 2 m concentration, but DNA polymerase 6 maintains maximum activity with Mg2+ over the range 2-12mM. For both enzymes, maximum activity with Mn2+ is at a concentration of 0.1 mM of this ion, the ratio of maximum activity with Mn2+to Mg2+being 0.5 for DNA polymerase a but 0.2 for DNA polymerase b. The two enzymes differ markedly in their response to univalent cations, DNA polymerase a being rather insensitive to KCI concentrations below 1 2 0 m , and is inhibited thereafter. In contrast, DNA polymerase b is stimulated fivefold on raising the KCI concentration from 0 to 1 5 O m . DNA polymerase a seems to be a homogeneous species (in 1OOmMKC1/50m~-Tris/HCI,pH 7.8), of mol.wt. 90000, whereas DNA polymerase b has a broad molecular-weight distribution, with 75 % of the activity being of 200000mol.wt., and the remainder being of lower molecular weight. DNA polymerase b breaks down on storage at -50°C to lower-molecular-weight species.
+,
1976
563rd MEETING, LONDON
807
Exonuclease activity has been measured in preparations of both enzymes. The activity associated with DNA polymerase a requires denatured DNA, Mg2+and is ATP-dependent. The polymerase b-associated nuclease requires denatured DNA and Mg2+or Caz+. Studies of the distribution of these enzymes during the cell cycle have shown that extracts of cells harvested at the time of nuclear DNA synthesis (Chiang & Sueoka, 1967) have DNA polymerase b as the predominant DNA-synthesizing activity, whereas DNA polymerase a is the major activity in cells harvested at a resting phase of growth. The two major DNA polymerases of Chlamydomonas reinhardii therefore seem to differ in physical and enzyme properties and in the time of appearance during the vegetative cell cycle. We thank the Medical Research Council for the award of a studentship to C. A. R. Banks, G. R. & Yarranton, G. T. (1976) Eur. J. Biochem. 62, 143-150 Banks, G. R., Holloman, W. K., Kairis, M. V., Spanos, A. & Yarranton, G. T. (1976) Eur. J. Biochcm. 62, I3 1- I42 Chang, L. M. S. (1971) Biochem. Biophys. Res. Commun. 44, 124-131 Chiang, K. S. & Sueoka, N. (1967) J. Cell. Physiol. 70, Suppl. 1, 89-112 Crerar, M. & Pearlman, R. (1974) J. Biol. Chem. 249, 3123-3131 Holrnes, A. H., Hesslewood, 1. P. &Johnston, I. R. (1974) Eur. J. Biochem. 43, 487-499 Kates, J. R. & Jones, R. F. (1964) J. Cell. Comp. Physiol. 63, 157-164 Loeb, L. A. (1969) J. Biol. Chent. 244, 1672-1681 McLennan, A. G. & Keir, H. M. (1975~)Biochem. J. 151, 227-238 McLennan, A. G. & Keir, H. M. (19756) Biochern. J. 151, 239-247 Schonherr, 0. Th. & Keir, H. M. (1972) Biochem. J. 129, 285-290 Wintersberger, U. & Wintersberger, E. (1970) Eur. J. Biochem. 13, 20-27
Role of Deoxyribonucleic Acid Polymerase p in Nuclear Deoxyribonucleic Acid Synthesis TAUSEEF R. BUTT, WILLIAM M. WOOD* and ROGER L. P. ADAMS Department of Biochemistry, Uniwrsity of Glasgow, Glasgow GI 2 SQQ, Scotland, U.K.
Homogenization of mouse L929 cells in hypo-osmotic buffer releases most of the DNA polymerase a into the supernatant fractions. However, nuclei isolated under these conditions still contain both DNA polymerase a and DNA polymerase B ( A d a m et al., 1973). DNA polymerase a shows an increase in activity as cells leave a resting phase and enter into S phase (Lindsay et al., 1970; Chang et al., 1973), but the activity of DNA polymerase fl is independent of the state of growth of the cells. Although this points to a possible rolc for DNA polymerase Bin repair of DNA, it does not exclude some role in DNA replication. It has been reported ( A d a m &Wood, 1973) that removal of up to 50 % of the total nuclear DNA polymerase has little effect on the ability of isolated nuclei to synthesize DNA. Here we present some evidence that DNA polymerase /?alone is sufficient to catalyse this nuclear DNA synthesis in uitro. L929 cells were grown as described by Lindsay et al. (1970), and nuclei were prepared by homogenizing the cells in 0.25 M-sucrose containing 20m~-Tris/HCI,pH7.5, and 5m-2-mercaptoethanoI. DNA synthesis was assayed in nuclei, washed twice with the same buffer, by incubation in the reaction mixture of Hershey et al. (1973). This contains 1OmM-MgCI,, 4Om~-Tris/HCl, pH 7.8, 100m~-NaC1,0.5 m - E D T A , 4 m~-2-mercaptoethanol, SrnM-ATP, 0.1 mM each of dATP, dCTP, dGTP and r3H]dTTP (specific radioactivity 5OpCi/pmol and 2mCi/pmol). To extract DNA polymerase a differentially, nuclei from S-phase cells were suspended gently inTris/sucrose buffer containing02~~KC1, left for 15minand sedimentedat 8OOg.
* Present address :Department of Physiology and Biophysics, University of Illinois at UrbanaChampaign, IL 61801, U.S.A. Vol. 4
BIOCHEMICAL SOCIETY TRANSACTIONS
Craig, R. K. & Keir, H. M. (19756) Biochem. J. 145,225-232 Dunham, V. L. & Cherry, J. H. (1973) Biochem. Biophys. Res. Commun. 54,403-410 Holmes, A. M., Hesslewood, I. P. &Johnston, I. R. (1974) Eur. J. Biochem. 43, 487-499 Keir, H. M. & Craig, R. K. (1973) Biochem. SOC.Trans. 1, 1073-1077 McLennan, A. G. & Keir, H. M. (1975) Biochem. J. 151, 239-247 Robinson, N. E. & Bryant, J. A. (1975) Planta 127, 69-75 Srivastava, B. I. S. & Grace, J. T. (1974) Life Sci. 14, 1947-1954 Stevens, C., Jenns, S. M. & Bryant, J. A. (1975) Biochem. Soc. Trans. 3, 1126-1128
Deoxyribonucleic Acid Polymerases from Chlamydomonas reinhardii CAROLE A. ROSS and WILLIAM J. HARRIS Department of Biochemistry, University of Aberdeen, Marischal College, Aberdeen A 3 9 l A S , Scotland, U.K.
DNA-dependent DNA polymerases (EC 2.7.7.7) have been studied in detail in only a small number of protozoan species. The enzymes which have been most highly characterized have been those from Saccharomyces cerevisiae (Wintersberger & Wintersberger, 1970), Tetrahymena pyriformis (Crerar & Pearlman, 1974), the smut fungus Ustilago maydis (Banks et al., 1976) and Euglena gracilis (McLennan & Keir, 1975a). In all these organisms, multiple DNA polymerase activities have been detected. The major cellular activities have high, sometimes heterogeneous, molecular weights, which vary depending on the surrounding ionic environment. In this, these enzymes resemble the mammalian DNA polymerase (Chang, 1971; Holmes et al., 1974). Exonuclease activity has been found associated with protozoan DNA polymerases (Banks & Yarranton, 1976; McLennan & Keir, 1975b), although the mammalian enzymes are thought to be devoid of any deoxyribonuclease activities. Small amounts of DNA polymerase activity have previously been reported to be present in whole cell extracts of the unicellular alga Chlumydomonas reinhardii (Schonherr & Keir, 1972), extraordinarily high amounts of dexoyribonuclease activity in these extracts accounting for the low detectable activity. The present work describes the characterization of the major DNA polymerases of this organism, undertaken as part of a general study of DNA metabolism in the cell cycle of Chlumydomonas. At least three DNA polymerase activities have been isolated from extracts of Chlamydomonas reinhardii cells (strain wild type provided by the Culture Centre of Algae and Protozoa, Cambridge, U.K.) grown under photosynthesizing conditions (Kates &Jones, 1964). Of these, two enzyme activities have been purified 2000-fold with respect to protein content, and have been named DNA polymerases u and 6. These enzymes have been separated by ion-exchange and DNA-affinity chromatography. Both enzymes require MgZf and all four deoxyribonucleoside triphosphates for maximal activity. Calf thymus DNA, activated by a modification of the method of Loeb (1969), is the best natural DNA template. Both enzymes have a broad pH optimum in 5 0 m ~ Tris/HC1 buffer, DNA polymerase a having maximum activity at pH8.5, whereas maximum activity with DNA polymerase b is obtained at pH7.5. DNA polymerase a requires Mgz+at 2 m concentration, but DNA polymerase 6 maintains maximum activity with Mg2+ over the range 2-12mM. For both enzymes, maximum activity with Mn2+ is at a concentration of 0.1 mM of this ion, the ratio of maximum activity with Mn2+to Mg2+being 0.5 for DNA polymerase a but 0.2 for DNA polymerase b. The two enzymes differ markedly in their response to univalent cations, DNA polymerase a being rather insensitive to KCI concentrations below 1 2 0 m , and is inhibited thereafter. In contrast, DNA polymerase b is stimulated fivefold on raising the KCI concentration from 0 to 1 5 O m . DNA polymerase a seems to be a homogeneous species (in 1OOmMKC1/50m~-Tris/HCI,pH 7.8), of mol.wt. 90000, whereas DNA polymerase b has a broad molecular-weight distribution, with 75 % of the activity being of 200000mol.wt., and the remainder being of lower molecular weight. DNA polymerase b breaks down on storage at -50°C to lower-molecular-weight species.
+,
1976
563rd MEETING, LONDON
807
Exonuclease activity has been measured in preparations of both enzymes. The activity associated with DNA polymerase a requires denatured DNA, Mg2+and is ATP-dependent. The polymerase b-associated nuclease requires denatured DNA and Mg2+or Caz+. Studies of the distribution of these enzymes during the cell cycle have shown that extracts of cells harvested at the time of nuclear DNA synthesis (Chiang & Sueoka, 1967) have DNA polymerase b as the predominant DNA-synthesizing activity, whereas DNA polymerase a is the major activity in cells harvested at a resting phase of growth. The two major DNA polymerases of Chlamydomonas reinhardii therefore seem to differ in physical and enzyme properties and in the time of appearance during the vegetative cell cycle. We thank the Medical Research Council for the award of a studentship to C. A. R. Banks, G. R. & Yarranton, G. T. (1976) Eur. J. Biochem. 62, 143-150 Banks, G. R., Holloman, W. K., Kairis, M. V., Spanos, A. & Yarranton, G. T. (1976) Eur. J. Biochcm. 62, I3 1- I42 Chang, L. M. S. (1971) Biochem. Biophys. Res. Commun. 44, 124-131 Chiang, K. S. & Sueoka, N. (1967) J. Cell. Physiol. 70, Suppl. 1, 89-112 Crerar, M. & Pearlman, R. (1974) J. Biol. Chem. 249, 3123-3131 Holrnes, A. H., Hesslewood, 1. P. &Johnston, I. R. (1974) Eur. J. Biochem. 43, 487-499 Kates, J. R. & Jones, R. F. (1964) J. Cell. Comp. Physiol. 63, 157-164 Loeb, L. A. (1969) J. Biol. Chent. 244, 1672-1681 McLennan, A. G. & Keir, H. M. (1975~)Biochem. J. 151, 227-238 McLennan, A. G. & Keir, H. M. (19756) Biochern. J. 151, 239-247 Schonherr, 0. Th. & Keir, H. M. (1972) Biochem. J. 129, 285-290 Wintersberger, U. & Wintersberger, E. (1970) Eur. J. Biochem. 13, 20-27
Role of Deoxyribonucleic Acid Polymerase p in Nuclear Deoxyribonucleic Acid Synthesis TAUSEEF R. BUTT, WILLIAM M. WOOD* and ROGER L. P. ADAMS Department of Biochemistry, Uniwrsity of Glasgow, Glasgow GI 2 SQQ, Scotland, U.K.
Homogenization of mouse L929 cells in hypo-osmotic buffer releases most of the DNA polymerase a into the supernatant fractions. However, nuclei isolated under these conditions still contain both DNA polymerase a and DNA polymerase B ( A d a m et al., 1973). DNA polymerase a shows an increase in activity as cells leave a resting phase and enter into S phase (Lindsay et al., 1970; Chang et al., 1973), but the activity of DNA polymerase fl is independent of the state of growth of the cells. Although this points to a possible rolc for DNA polymerase Bin repair of DNA, it does not exclude some role in DNA replication. It has been reported ( A d a m &Wood, 1973) that removal of up to 50 % of the total nuclear DNA polymerase has little effect on the ability of isolated nuclei to synthesize DNA. Here we present some evidence that DNA polymerase /?alone is sufficient to catalyse this nuclear DNA synthesis in uitro. L929 cells were grown as described by Lindsay et al. (1970), and nuclei were prepared by homogenizing the cells in 0.25 M-sucrose containing 20m~-Tris/HCI,pH7.5, and 5m-2-mercaptoethanoI. DNA synthesis was assayed in nuclei, washed twice with the same buffer, by incubation in the reaction mixture of Hershey et al. (1973). This contains 1OmM-MgCI,, 4Om~-Tris/HCl, pH 7.8, 100m~-NaC1,0.5 m - E D T A , 4 m~-2-mercaptoethanol, SrnM-ATP, 0.1 mM each of dATP, dCTP, dGTP and r3H]dTTP (specific radioactivity 5OpCi/pmol and 2mCi/pmol). To extract DNA polymerase a differentially, nuclei from S-phase cells were suspended gently inTris/sucrose buffer containing02~~KC1, left for 15minand sedimentedat 8OOg.
* Present address :Department of Physiology and Biophysics, University of Illinois at UrbanaChampaign, IL 61801, U.S.A. Vol. 4
