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5 Cellular and Viral-Induced Eukaryotic Polymerases
Cellular and Viral-Induced Eukaryotic Polymeruses A. WEISSBACH
I. Introduction and Perspective . . . . . . . . . 11. DNA Polymerase a . . . . . . . . . . . , . A. Purification and Properties. . . . . . . . . B. Biological Role . . . . . . . . . . . . . . 111. DNA Polymerase p . . . . . . . . . . . . . A. Purification and Properties . . . . . . . . . B. Biological Role . . . . . . . . . . . . . . IV. DNA Polymerase y . . . . . . . . . . . . . A. Purification and Properties . . . . . . . . . B. Biological Role . . . . . . . . . . . . . . V. Herpes Simplex Virus-Induced DNA Polymerase A. Purification and Properties . . . . . . . . . B. Biological Role . . . . . . . . . . . . . . VI. Vaccinia Virus-Induced DNA Polymerase . . . A. Purification and Properties . . . . . . . . . B. Biological Role . . . . . . . . . . . . . . VII. Conclusion . . . . . . . . . . . . . . . . .
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67 69 69 73 73 74 76 76 77 79 80 80 83 83 84 85
86
Introduction and Perspective
A nomenclature for the known eukaryotic DNA polymerases was proposed in 1975 (/). This classification, which recognized three major 1. A. Weissbach, D. Baltimore, F. J. Bollum, R. C. Gallo, and D. Korn, Scicizcc 190, 401 (1975).
67 THE ENZYMES, Vol. XIV
Copyright @ 1981 by Academic Press. Inc. All rights of reproduction in any form reserved
68
A. WEISSBACH
classes of cellular DNA polymerases--cY, /3, and y-has proved to be applicable to a wide range of species in the Animal Kingdom (2). In addition, the nomenclature scheme recognized the existence of a mitochondria1DNA polymerase and new viral-induced DNA polymerases that are produced in infected animal cells. This chapter limits itself to consideration of enzymes that have been well characterized, i.e., DNA polymerases a , P , and y from mammalian cells and the herpes simplex virus and vaccinia virus-induced DNA polymerases. A number of other reported DNA polymerase activities ( 2 4 ) , less well identified, are necessarily omitted. The three cellular DNA polymerases were named in the order of their discovery: DNA polymerase a was first isolated from calf thymus and characterized by Bollum (5, 6), and his work was an important starting point in the study of eukaryotic DNA polymerases. The enzyme has been identified in many species as a predominant activity, and has been purified from rat, murine, calf thymus, and human cells. In 1971 Weissbach et al. (7) reported a new, low molecular weight DNA polymerase in HeLa cell nuclei at the same time that Baril et al. (8)reported an enzyme with similar properties in rat liver nuclei. This enzyme has been designated as ppolymerase. Further identification and characterization of P-polymerase in calf thymus, rat liver, KB cells, and chick embryos followed shortly thereafter (9-11). In general, P-polymerase represents about 5% of the total DNA polymerase in growing, cultured cells, but is a major component of low DNA polymerase activity in quiescent cells. DNA polymerase y , first reported in 1972 by Fridlender et ul. (12), is a widely distributed enzyme (13) that shows a marked ability to copy ribohomopolymers, but a total inability to use natural RNA as a template. It thus has no relationship to the retrovirus reverse transcriptases. At first thought to be a minor activity in the cell, the DNA polymerase y level in 2. A. Weissbach, Annu. Rev. Biochem. 46, 25 (1977). 3. J . A. Kantor, X. H. Lee, J. G . Chirikjian, and W. G . Feller, Science 204, 511 (1979). 4. B. A. Brennessel, D. P. Buhrer, and A. Gottlieb,Anal. Biochem. 87, 411 (1978). 5. F. J. Bollurn, JBC 235, 2399 (1960). 6. M. Yoneda and F. J. Bollurn, JEC 240, 3385 (1965). 7. A. Weissbach, A. Schlabach, B. Fridlender, and A. Bolden,Notlrre New B i d . 231, 167 ( 197 1).
8. E. F. Baril, 0. E. Brown, M. D. Jenkins, and J. Laszlo, Binrhemistry 10, 1981 (1971). 9. L. M. S . Chang and F. J. Bollurn. JBC 246, 5835 (1971). 10. M. E. Haines, A . M. Holrnes, and I. R. Johnston, FEES (Fed.Eur. Biochem. SOC.) Lett. 17, 63 (1971). 11. H. Berger, Jr., R. C. Huang, and J. L. Irvin, JBC 246, 7275 (1971). 12. B. Fridlender, M. Fry, A. Bolden, and A . Weissbach, PNAS 69, 452 (1972). 13. A. Weissbach, Cell S, 101 (1975).
5. CELLULAR AND VIRAL-INDUCED DNA POLY MERASES
69
growing cultured cells is, in fact, equal to that of thep-polymerase, and in at least one case the y-polymerase is the major polymerase in the organism. Table I summarizes some relevant characteristics of the three cellular DNA polymerases obtained from various sources and also lists two wellcharacterized viral-induced DNA polymerases. It should be emphasized here that the properties, size, and behavior of the a-, P - , and y-polymerases can differ from those shown in Table I, depending on the source of the enzyme. The specific patterns shown by inhibitors, however, seem to be invariant. Each of these enzymes is considered in detail in the following sections. II. DNA Polymerase a
DNA polymerase a has been extensively purified from calf thymus (14, 1 5 ) , human cells (16), murine cells (171, and others (2). Because of the extensive heterogeneity of the enzyme in various species, isolation of a-polymerase in a pure form has been difficult. Nevertheless, a nearhomogeneous preparation of a-polymerase has been obtained from human cells by Fisher and Korn (161, and from mouse myeloma cells by Chen et al. (17). A. PURIFICATION AND PROPERTIES Table I1 summarizes the purification of a-polymerase from cultured human KB cells as described by Fisher and Korn (16). In this procedure all buffers contained 1 rnM P-mercaptoethanol and 1 m M EDTA and, after fraction V, 20% glycerol. A protease inhibitor, p-toluenesulfonyl fluoride, is present when the cells are broken. The second DEAE-cellulose step, which offers little purification per se, is apparently important for subsequent steps. The purified protein from human cells has a specific activity of 206,000 unitdmg, a unit being defined as the incorporation of 1 nrnol of dTMP in DNMhour at 37”. The enzyme, as isolated, exists either as a monomer of 140,000 daltons or as dimers of 265,000-280,000 daltons. It has an isoelectric point of 5.0-5.2 and can be resolved in denaturing polyacrylamide gel 14. 15. 16. 17.
F. J. Bollum, Progr. Nucleic Acid Res. Mol. Biol. 15, 109 (1975). K. McKune and A. M. Holmes, Nucleic Acids Res. 6, 3341 (1979). P. A. Fisher and D. Korn, JBC 252, 6528 (1977). Y. C. Chen, E. W. Bohn, S. R. Planck, and S. H. Wilson, JBC 254, 11678 (1979).
TABLE I
EUKARYOTIC DNA POLYMERASES DNA polymerases
Source Human cells
(KB)
Y
Novikoff hepatoma Chick embryo
Herpes simplexinduced Vacciniainduced
Infected HeLa cells Infected HeLa cells
Major cellular location Nucleus, cytoplasm Nucleus
Subunits (kilodaltons)
156
76, 66
3 I"
Nucleus, mitochondria
180
47
Nucleus
144
74,29
Cytoplasm
115 ~
a
Molecular weight (X l t 3 )
~
~~~
~~~~~~~~
DNA polymerase /3 from other cells has a reported molecular weight of 40,OOO-45,OOO.
Inhibitors Aphidicolin, N-ethylmaleimide, Ara-ATP Dideoxynucleoside triphosphates, iodoacetate Dideoxynucleoside triphosphates, N-ethylmakimide Phosphonoacetate, Ara-ATP, N-ethylmaleimide Phosphonoacetate, N-ethylmaleimide
TABLE I1 PURIFICATION OF Step Crude extract pH 5.5 precipitation Ultracentrifugation First DEAE-cellulose
2
Second DEAE-cellulose Phosphocellulose Hydroxylapatite DNA-ceUulose Gel electrophoresis'
Fraction I I1 (resolubilized precipitate) 11' (supernatant) 111 (supernatant) 111' (pellet) IV (adsorbed) IV' (flow-through) V VI VII VIII IX
DNA POLYMERASE a FROM KB CELLS" Velum$ (ml)
Proteid (mg)
Activit9 (units)
9.9 2.2
43 19
880 890
2.8
9
2.9
9 x 10-1
1.1
4 x 10-1 4 x 10-2 1 x 10-2 I x 1 r 3 1.6 x 10-4
3.8 x lo-' 6.6 x lo-* 6.5 x lo-*
60 790 50 490 100 280 240 130
33
Specific activity (unitdmg)
Yield (%)
20 47
(100) 108
88
97
550
67
700 6,000 13,000 33,000 206,000
32 28
I5 4
" As described by Fisher and Korn (16). Reaction mixes contained in 2 5 0 ~ 1 10 , mM Tris, pH 9.2,20 mM mercaptoethanol, bovine serum albumin 200 pglml, 10 m M MgCb, activated salmon sperm DNA, 800 pglrnl, dATP, dCTP, dGTP, and dTTP, 50 g C L M each, and PHIdlTP at a final specific activity of 0.04 Ci/mmol, and enzyme. A unit is the amount of enzyme that catalyzes the incorporation of 1 nmol of labeled dTMP into an acid-insoluble product in 1 hour at 37". Quantities are expressed per gram wet weight of KB Cells. Aliquots, 400 p l , of fraction VIII were used for nondenatunng gel electrophoresis. The protein value was derived by densitometry of a stained gel. Recovery of DNA polymerase activity by elution of slices of parallel unstained gels varied between 50 and 95%. The specific activity value is based on units of loaded activity.
72
A. WEISSBACH
electrophoresis into two subunits of 76,000 and 66,000 daltons. The purified enzyme has a half-life at 0" of 14 months if stored in a concentrated form in the presence of sucrose and potassium phosphate. Optimal reaction conditions include a pH between 7.5-8.5, and Mg'+ at 4-8 mM. Salt concentrations above 50 mM are inhibitory, with about 50% of the activity lost at 100 mM KCl. a-Polymerase is markedly inhibited by Caz+ and Li+. a-Polymerase is most reactive with duplex DNA templates containing gapped regions with available 3'-OH termini (activated DNA). A surprising property of the purified enzyme is its inability to catalyze the synthesis of long DNA chains. It is only slightly processive, synthesizing an 11 2 5 nucleotide length before coming off the template (18, 19). DNA polymerase a does not act at nicks or in short gaps below 20 nucleotides in length, and does not utilize a blunt-ended DNA template. The enzyme binds to single-stranded DNA that contains 3'-OH ends and can catalyze synthesis of hairpin molecules from such templates (20, 21). Synthetic DNA templates such as (dA), . (dT)12--IB are copied at 20% the rate of activated DNA, whereas the corresponding synthetic RNA template, (A), . dTlz, copies at only 3% the rate of activated DNA. However, murine DNA polymerase a copies (dT), . rA2, faster than any other template (22).Spermidine has been found to increase the apparent K , for M. Purified calf DNA (20). The K , for dNTPs is in the range of 1-4 x thymus DNA polymerase a catalyzes both pyrophosphorolysis and pyrophosphate exchange (14). As isolated, purified human a-polymerase has no detectable nuclease activity. There have been reports that bone marrow and calf thymus contain an a-polymerase-like enzyme that contains a 3' + 5' exonuclease, and has been called DNA polymerase 8 (23). Whether this represents a new enzyme or an association of a cellular 3' + 5' exonuclease (24) with the a-polymerase is still unclear. Chen et al. (17) obtained two nearhomogeneous preparations of a-polymerase from mouse myeloma. These 18. P. A. Fisher, T. S-F. Wang, and D. Korn,JBC 254, 6128 (1979). 19. K. McKune and A. M. Holmes, BBRC 90, 864 (1979). 20. P. A. Fisher and D . Korn, JBC 254, 11033 (1979). 21. P. A. Fisher and D. Korn,JBC 254, 11040 (1979). 22. S. H. Wilson, A. Matsukage, E. W. Bohn, Y. C. Chen, and M. Sivarajan, Nucleic Acids RPS.4, 3981 (1977). 23. M. Y. W. Tsang-Lee, C. K. Tan, A. G . So, and K. M. Downey,Biochemistry 19,20% (1980). 24. G. Villani, S. Spadari. S. Boiteux, M. Defais, P. Caillet-Fauquet, and M. Radman, Biochimie 60, 1145 (1978).
5 . CELLULAR AND VIRAL-INDUCED DNA POLYMERASES
73
large molecular weight enzymes (MW = 190,000) contain subunits of 47,000 and 54,000 daltons. One of the a-polymerase species contains both 3' + 5' and 5' + 3' exonucleases associated with it. a-Polymerase has also been isolated in highly purified form from calf thymus (15) and regenerating rat liver; in both cases a number of subunits ranging from 50,000 daltons to 70,000 daltons seem to be associated with a catalytic polypeptide whose molecular weight is about 150,000. Thus, a common denominator in many of these studies is the heterogeneity of DNA polymerase a, a feature which may have important implications in the control and function of this enzyme. B.
BIOLOGICAL ROLE
It is generally agreed that DNA polymerase a has a key role in the replication of nuclear DNA and in the synthesis of the DNA of the viruses SV40, polyoma, and adenovirus (14, 25). Understanding the role of a-polymerase has been aided by the availability of specific inhibitors such as aphidicolin, or the arabinose-containing nucleotides such as ara-ATP. The use of these inhibitors supports the concept that DNA polymerase a is the major replicative polymerase in mammalian cells (26-30). It represents 90-95% of the total DNA polymerase activity of cultured growing mammalian cells and drops to low levels in cells that have ceased nuclear DNA synthesis. 111.
DNA Polymerase /3
DNA polymerase /3 is the smallest of the known eukaryotic DNA polymerases and shows remarkable chemical stability under various conditions. As a result, and although it represents only about 5% of the total DNA polymerase in growing, cultured mammalian cells, it was the first eukaryotic DNA polymerase to be isolated in a homogeneous state. This has been accomplished from calf thymus (3I ), human KB cells (32), mouse 25. 26. 27. 28. 29. 30. 31. 32.
A. Weissbach, ABB 198, 386 (1979). H. J. Edenberg, S. Anderson, and M. L. DeParnphilis, JBC 253, 3273 (1978). M. A. Waqar, M. J . Evan, and J. A. Huberman, Nucleic Acids Res. 5, 1933 (1978). M. Ohashi, T. Taguchi, and S . Ikegami, BBRC 82, 1084 (1978). E. Wist and H. Prydz, Nucleic Acids Res. 6, 1583 (1979). E. Wist, BBA 562, 62 (1979). L. M. S. Chang, JBC 248, 3789 (1973). T. S-F. Wang, W. D. Sedwick, and D. Korn, JEC 250, 7040 (1975).
74
A. WEISSBACH
myeloma (33),Novikoff hepatoma (34) and chick embryos (35). The purification of DNA polymerase /3 from the latter is summarized in Section II1,A. A. PURIFICATION AND PROPERTIES The procedure used by Stalker et al. (34) is shown in Table 111, and yields a homogeneous enzyme after a 200,000-fold purification with a remarkable apparent yield of 46%. The starting material, Novikoff hepatoma, is an ascites tumor with a generation time of 12 hours when maintained in rats, so relatively large quantities of cells can be obtained conveniently. The purification relies on the sequential use of three chromatographic separations on DEAE-Sephadex, phosphocellulose, and hydroxylapatite. The final step in the purification procedure uses singlestranded DNA cellulose as an affinity column, which in a 25-fold enrichment step provides the pure enzyme. The enzyme is stable at 4" during the isolation procedure, and stabilization of the enzyme during purification is facilitated by the use of 10% glycerol in the elution buffers and, at the final step, by having bovine serum albumin (1 mg/ml) present. With this method, 200 pg of purified DNA polymerase P are obtained per kilogram of cells. The enzyme in whole cells is stable at - 20" for months, and the purified enzyme has been stored for 1 year at - 196" without loss of activity. The enzyme, as isolated, has a molecular weight of 31,000 although the calf thymus (31), KB cell (32), and chick (35) and mouse P-polymerases (33) have been reported to have molecular weights of 44,000, 43,000 and 40,000 daltons, respectively. Purified DNA polymerase /3 has no detectable nuclease activity. It shows an alkaline isoelectric point (8.51, a pH optimum of 8.4-9.2 and a K, for deoxynucleoside triphosphates of 7-8 p M . For maximal synthesis Mg2+ at 5-10 mM is required; MI?+ (1 mM) can also be used. The enzyme is stimulated twofold by 50 mM NaCl or by 100-200 mM KC1. The latter salt levels severely inhibit a-polymerase. Phosphate and pyrophosphate are inhibitory to /3-polymerase and should be avoided in reaction mixes. Neither the mouse nor human enzyme can catalyze pyrophosphate exchange, pyrophosphorolysis, or dNTP turnover (33, 36). 33. K. Tanabe, E. W. Bohn, and S. H. Wilson, Biochemistry 18, 3401 (1979). 34. D. M. Stalker, D. W. Mosbaugh, and R. R. Meyer, Biochemistry IS, 3114 (1976). 35. M. Yamaguchi, K. Tanabe, Y. N . Taguchi, M . Nishizawa, T. Takahashi, and A. Matsukage, JBC 255, 9942 (1980). 36. T. S-F. Wang, W. D. Sedwick, and D. Korn, JBC 249, 841 (1974).
75
5 . CELLULAR AND VIRAL-INDUCED DNA POLYMERASES
TABLE I11
PURIFICATION OF NOVIKOFF HEPATOMA DNA POLYMERASE P" Protein Fraction
Total unitsh
Specific (unitdmg)
Purification (-fold)
Yield
(mg)
1. Cell extract 11. Ammonium sulfate 111. DEAE-Sephadex IV. Phosphocellulose V. Hydroxylapatite VI. DNA-Cellulose
14,500 4,610 8,866 81.2 1.34 0.031
3,880 3,780 9,820 4,390 3,150 1,800
0.268 0.820 11.3
1.0 3.06 42.2 202 8,770 217,000
100 97.4 253
54.1
2,350 58,100'
(%)
113
81.2 46.4
' I From Stalker et al. (35).The reaction mixtures contained the following components in a final volume of 125 111: 25 mM Tris-HCI, pH 8.4; 5 mM 2mercaptoethanol; 7 mM magnesium acetate; 0.5 mM EDTA; 0.015 m M each of dATP, dCTP, dGTP, and PHIdTTP (specific activity 975 rnCi/mmol); 50 m M NaCI; 15% ( w h ) glycerol; 250 Fg/ml activated DNA; and 0.01-0.3 units of DNA polymerase fraction. Incubations were carried out for 1 hour at 37" and acid-insoluble radioactivity was determined. When incorporation was not linear for 1 hour, the data were extrapolated from a 30-minute incubation. * A unit is defined as the incorporation of 1 nmol of total nucleotide into DNA per hour at 37". ' With several different preparations, the specific activity varied from 32,000 to 62,000 unitslmg.
A reported characteristic of DNA polymerase /3 is its relative insensitivity to urea, acetone, and alcohol (14). The enzyme is stabilized by glycols and stimulated by spermidine (up to 10 mM) (34). Another general property of the P-polymerases is their relative resistance to N-ethylmaleimide (NEM), which is a powerful inhibitor of DNA polymerases a and y . At 4 mM, NEM shows a 28% inhibition of DNA polymerase @ from Novikoff hepatoma, a value that is slightly higher than previously reported for the human enzyme (37). The NEM partial inhibition is not unexpected since p-hydroxymercuribenzoate inhibits @-polymeraseat concentrations above 50 p M (14, 34). An important characteristic of P-polymerase is its ability to copy a synthetic ribohomopolymer such as (A), . dTlz as well as the corresponding deoxyribohomopolymer (dA), . dTlz or activated DNA (34).This is in contrast to a-polymerase, which utilizes the deoxyribohomopolymer (dA), . dT,,_,, eight times better than (A), . dTlz, which is, in fact, copied at only 3% the rate of activated DNA (18). Rat DNA polymerase 0 has been reported to have a uniquely high requirement for primers when 37. K. W. Knopf, M. Yamada, and A. Weissbach, Biochemistry 15, 4540 (1976).
76
A. WEISSBACH
copying poly(A) templates, and can thus be distinguished from y-polymerase or oncornavirus reverse transcriptase (38). Steady-state kinetic measurements suggest an ordered BiBi mechanism for polymerization and a scheme depicting two DNA binding sites on the enzyme has been advanced (33).Although the specific activity of the purified Novikoff hepatoma DNA polymerase p prepared by Stalkeret al. (S4)is 58,000 with activated DNA as a template, Ono et al. (38)reported rat ascites hepatoma DNA polymerase preparations with a specific activity of lo6 units/mg on an (A), * dTlz-ls template.
B. BIOLOGICAL ROLE The level of DNA polymerase L,3 in quiescent or growing cells or during the cell cycle has been reported to be relatively constant, leading to the suggestion that it may be involved in DNA repair synthesis (39). Hiibscher et al. (40) have shown that &polymerase can participate in the repair of UV-damaged DNA in neuronal nuclei, an organelle in which DNA polymerase p is the only detectable polymerase activity. The further role of this enzyme in other types of DNA synthesis is unknown at the present time. IV.
DNA Polymerase y
DNA polymerase y exists in at least two forms and is found in the nucleus, cytoplasm, and mitochondria (41-43). Like a-polymerase, DNA polymerase y readily undergoes reversible aggregations that, in v i m at least, are salt-dependent. It comprises about 5% of the total DNA polymerase activity in the growing, cultured mammalian cell, and therefore is about equal to the &polymerase level. In developing chick embryos, y-polymerase represents 45% of the total DNA polymerase activity and is present in larger amounts than either 0- or P-polymerases (44). It is clear that the so-called “mitochondrial” DNA polymerase is one species 38. K. Ono, A. Ohashi, K. Tanabe, A. Matsukage, M. Nishizawa, and T. Takahashi, Nucleic Acids Res. 7, 715 (1979). 39. G. Pedrali Noy, L. Dalpra’, M. A. Pedrini, G. Ciarrocchi, E. Gidotto, F.Nuzzo, and A. Falaschi, Nucleic Acids Res. 1, 1183 (1974). 40. U. Hiibscher, C. C. Kuenzle, and S. Spadari, PNAS 76, 2316 (1979). 41. S. Spadari and A. Weissbach, JBC 249, 5809 (1974). 42. G. Pedrali Noy and A. Weissbach, BBA 477, 70 (1977). 43. A. Bolden, G. Pedrali Noy, and A. Weissbach, JBC 252, 3351 (1977). 44. M. Yamaguchi, A. Matsukage, and T. Takahashi, JBC 255, 7002 (1980).
5 . CELLULAR AND VIRAL-INDUCED DNA POLY MERASES
77
of the y-polymerase class, and that the nuclear species of DNA polymerase y can be distinguished from it (43). Despite extensive efforts, the enzyme has not been prepared in pure form from mammalian tissues (45), perhaps due, in part, to its heterogeneity: but it has been purified to near-homogeneity from chick embryos (44). A.
PURIFICATION AND PROPERTIES
An outline of the purification of the y-polymerase from chick embryos as described by Yamaguchi et al. (44) is shown in Table IV. In this procedure, frozen 11-day-old embryos are minced and sonicated in a buffer containing 0.5 M KCl, 10% glycerol, and eventually, 0.5% Triton X-100. The purification scheme uses two phosphocellulose column chromatographic steps, a Sephadex G-200 gel filtration, and hydroxylapatite adsorption chromatography. Following the second phosphocellulose column, the enzymatic activity separates into two gel components, a 180,000- and a 280,000-dalton species, during gel filtration on a Sephadex G-200 column, and each species is further purified separately. The final separation step, which gives a 1000-foldenrichment, involves affinity chromatography on a double-stranded DNA cellulose column, and can be compared to the single-stranded DNA cellulose columns used in the purification of a- and P-polymerases. Attempts to purify DNA polymerase y by d n i t y chromatography on poly(rA)-Sepharose columns leads to inactivation of the enzyme. The purified enzyme can be stored at -80" but loses 50% of its activity in one freeze-thaw cycle. The purified enzyme sediments at 7.5 S and the molecular weight is estimated to be 180,000. SDS polyacrylamide gel electrophoresis shows a prominent polypeptide at 47,000 daltons, so the native enzyme appears to be a tetramer of this subunit. Based on this, the specific activity of the purified enzyme is calculated to be 660,000 unitslmg on a poly(A) template. With (A), dTlz-ls as a primer-template, the K , value for dTTP is about 1 p M, the optimal pH 8.5-9.0, and the optimal KCI concentration is 220 p M. In the presence of increasing levels of potassium phosphate, the optimal KCI concentration drops proportionately (45); and Mn2+ at 0.5-0.6 mM is fivefold more effective than the optimum Mg2+concentration of 12 mM. The structure of native DNA polymerase y in mammalian cells will probably differ somewhat from the avian enzyme. Rat liver DNA polymerase y can be obtained as a 4 S species (60,000 daltons) (43), whereas the smallest species of the chick native enzyme sediments at 7.5 45. K-W. Knopf, M. Yamada, and
A. Weissbach, Biochemistry
15, 4540 (1976).
TABLE IV
PURIFICATION O F DNA POLYMERASE y FROM CHICK
Step Crude extract First phosphocellulose and ammonium sulfate fractionation Second phosphocellulose Sephadex G-200 Hydroxylapatite Double-stranded DNA cellulose
Fraction I
I1
EMBRYOS~
Protein
Activityb units
(mg)
(%)
0,38 3s
80 4.8 4.1 x 10-' 1.0 x lo-'
I11 IV- 1 IV-2 v-1 v-2 VI- 1
9.0 x
4.1(14) 5.0(17) 3.6(12)
(VI-1-dT VI-2
(1.3 x 10-7 1.3 x lCV
(4 4.5( 15)
9.0 x 1C2 8.4 x 10-3 8.1 x 10-3
specific activity (unitdmg)
2x77) 8.4(28) 9.0(30)
56 84 100 490 620 400.000
Purification (-fold) 1 9.2 150
220 260 1,300 1,600 1,100,000 (1,500,000) 920,000
From Yamaguchi et al. (44). The assay mixture contained in 25pl,50 mM Tns, pH 8.5, 1 mM dithiothreitol, 0.5 mM MnC&.,80 pglml poly (rA) 16 pg/mI dT,,-,,, 0.1 mM [3H]dTTP(60cpdpmol), 15% glycerol, 400 pg/ml bovine serum albumin, 100-120 mM KCI, 20-40 mM potassium phosphate (pH 8.5) and enzyme. A unit is the amount of enzyme that catalyzes the polymerization of 1 nmol of dTMP in 60 minutes.
* Quantities are expressed per gram wet weight of
11-day-old chick embryos.
' Fractions in the peak of DNA polymerase activity (see Fig. 5A).
S. CELLULAR A N D VIRAL-INDUCED DNA POLY MERASES
79
S (150,000-180,000 daltons). By contrast, HeLa cell DNA polymerase y can be separated into two species on phosphocellulose chromatography, both with the same or similar apparent molecular weight of 110,000 (do), and human lymphoblast DNA polymerase y has a reported molecular weight of 120,000 (46). The interspecies difference is further illustrated by the report that sea urchin DNA polymerase y has a sedimentation value of 3.3 s (47). A salient feature of y-polymerases is their ability to copy ribohomopolymers at a rate greater than activated DNA. Under proper conditions the HeLa cell DNA polymerase y will utilize (A), * dT12-18five to ten times more efficiently than activated DNA (44). This is in contrast to the template characteristics of a-polymerase, which utilizes this synthetic template at 3% the rate at which it uses activated DNA. In addition, y-polymerase is active at potassium phosphate concentrations (50 mM) that are inhibitory to DNA polymerase /3(14,45), an enzyme that is known to copy (A), . dTlz-18 at about the same efficiency it copies activated
DNA.
B. BIOLOGICAL ROLE Of the three cellular DNA polymerases, only DNA polymerase y is capable of synthesizing continuous long DNA chains in a processive manner (48). DNA polymerase a , by comparison, is highly discontinuous, polymerizing 10-15 nucleotides at a time and then leaving the template (18,49).,&Polymerase also shows discontinuous synthesis when copying a poly(A) template (49). The ability of the y-polymerase to carry out a processive and continuous synthesis of a DNA chain may explain its known physiological roles. One of the forms of the enzyme is responsible for mitochondrial DNA synthesis (43, 50, 51) and another, the nuclear y-polymerase, is involved in the replication of adenovirus DNA (52, 53). The synthesis of adenovirus DNA and mitochondria1 DNA share a strand-displacement step in their replication process; this has led to the 46. M. Robert-Guroff, A. W. Schrecker, B. J . Brinkman, and R. C. Gallo, Biochemistry
16, 2866 (1977).
47. A. Habara, H. Nagano, and Y. Mano, BBA 561, 17 (1979). 48. M. Yamaguchi, A. Matsukage, and T. Takahashi, Nature (London) 285, 45 (1980). 49. A. Matsukage, M. Nihizawa, T. Takahashi, and T. Hozumi, J . Eiochern. (Tokyo),in press (1980). 50. U. Hubscher, C. C. Kuenzle, and S . Spadari, PNAS 76, 2316 (1979). 51. W. timmemann, S-M. Chen, A. Bolden, and A. Weissbach,JBC 255, 11847 (1980). 52. P. C. Van der Vliet and M. M. Kwant, Nature (London) 276, 532 (1978). 53. H. Krokan, P. SchaEer, and M. L. DePamphilis, Biochemistry, 18, 4431 (1979).
80
A. WEISSBACH
suggestion that y-polymerase has a unique role in strand-displacement syntheses (52, 25). Since both mitochondria1 DNA and adenovirus DNA are synthesized in a continuous mode without the apparent formation of short intermediates, such as Okazaki fragments, the processive character demonstrated by y-polymerase in vitro is also reflected in vivo . However, it is apparent that the basic physiological role of DNA polymerase y in the nucleus of the cell remains unknown. V.
Herpes Simplex Virus-Induced
DNA
Polymerase
The recognition in 1963 that herpes simplex virus (HSV) induced a new DNA polymerase in infected cells (54, 5 5 ) followed shortly after the discovery of DNA polymerase a , and predates the identification of DNA polymerases p and y . The HSV-induced DNA polymerase is therefore one of the earliest eukaryotic DNA polymerases studied. The altered properties of the enzyme were recognized by Keir et al. ( 5 3 , and the enzyme was partially purified and characterized by Weissbach er a!. (56). Highly purified, near-homogeneous preparations of the HSV polymerase have been prepared from HSV-1-infected HEp-2 cells (57) and from African green monkey cells (58). Purification of the viral-induced enzyme is facilitated by the large amounts of virus that are produced in the infected cell (59). Thus, the amount of HSV-1 DNA polymerase in HSV-1-infected HeLa cells can rise to four times the combined level of all the host cell DNA polymerases. A. PURIFICATION AND PROPERTIES As described by Knopf (58), African green monkey cells (RC-37; Italdiagnostic Products) grown in monolayers were infected at 5 pfdcell with HSV-1 (Angelotti) that had previously been passed through RC-37 five times. Six hours after infection the cells were collected, disrupted by sonication in 0.25 M potassium phosphate, pH 7.5, containing 0.5% Triton X-100. All the subsequent purification steps shown in Table V were per54. H. M. Keir, J. Hay, J. M. Momson, and J. Subak-Shape, Nature (London)210, 369 (1966). 55. H. M. Keir, J. Subak-Shape, W. I. H . Shedden, D. H. Watson, and P. Wildy, Virology 30, 154 (1966). 56. A. Weissbach, S-C. L. Hong, J. Aucker, and R. Muller, JBC 248, 6270 (1973). 57. K . L. Powell and D. J . M. hrifoy, J . Vlrol. 24, 616 (1977). 58. K. Knopf, EJB 98, 231 (1979). 59. M. Yamada, G. Brun, and A. Weissbach, J . Virol. 26, 281 (1978).
TABLE V PURIFICATION OF
HSV-1-DNA POLYMERASE FROM INFECTED RC-37 CELLP ~~
Purification
Volume (ml)
Total protein (mg)
Total activity (units)
Specific activity (unitdmg protein)
Purification
(%)
Cell extract dialysate DEAE-cellulose Phosphocellulose DNA-cellulose DNA-cellulose peak (fraction 37)
370 575 247 30 0.9
1191.4 217.8 28.5 1.38 0.033
125,280 150,480 95,168 3 1,570 1,575
105.2 690.9 3339.2 22876.8 47727.3
1 6.6 31.7 217.5 453.7
100 120 76 25
~~
Total recovery
~
From Knoff (58). Reaction mixtures contained in lOOpl50 mM Tns-HCI (pH KO), 7.5 mM MgCl,, 100 m M ammonium sulfate, 5 0 p g bovine serum albumin, 0.5 mM dithiothreitol, 0.1 mM each of dATP, dCTP, dGTP, and PHld'lTP (0.4 Ci/mmol), and 25 pg of activated salmon sperm DNA prepared as described by Pedrali Noy and Weissbach (42).A unit is the amount of enzyme that catalyzes the polymerization of 1 nmol of nucleotide in 60 minutes under standard assay conditions. a
82
A. WEISSBACH
formed with buffers containing 0.5 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride. The purification is relatively simple and involves three chromatographic separations on DEAE-cellulose, phosphocellulose, and double-stranded DNA cellulose, which yield a highly purified preparation after only a 450-fold purification. Using similar steps with DEAE-cellulose, phosphocellulose, and single-stranded DNA-cellulose separations, Powell and Purifoy (2 years prior to Knopf's report) purified the HSV-induced polymerase from HEp-2 cells almost 1700-fold with almost a 50% recovery (57). The purified enzyme, stored in 50 mM TrisHC1, 1 mM EDTA in 50% glycerol is stable at - 20 or -70". As isolated by Knopf, the purified enzyme shows a major polypeptide of 144,000 daltons on SDS polyacrylamide gel electrophoresis, which is in agreement with the 150,000-dalton species found by Powell and Purifoy (57). The enzyme isolated from RC-37 cells also shows the presence of two other polypeptides of 74,000 and 29,000 daltons, which were not observed by Powell and Purifoy and which may represent impurities. A prominent feature of HSV-DNA polymerase, and one that facilitates its identification, is its activity at high salt concentrations. The presence of 150 mM KCI or 100 mM (N&)2S04 leads to a two- to threefold enhancement of the enzymatic activity, whereas the cellular a-polymerase is inhibited nearly 90% at these salt concentrations. The HSV-1-induced DNA polymerase has a pH optimum of 8-8.5, and a M$+ optimum of 3 mM (in the presence of activated DNA template). Dithiothreitol ( 5 mM) also stimulates the enzyme threefold. The enzyme is inhibited by Zn2+, N-ethylmaleimide, and the pyrophosphate analogs, phosphonoacetic acid, or phosphoformate (60-62). Inorganic pyrophosphate does not inhibit the enzyme, which is able to catalyze pyrophosphate exchange into dNTPs. Aphidicolin, a powerful inhibitor of DNA polymerase a , also inhibits the HSV-induced DNA polymerase as well as the vaccinia-induced DNA polymerase described in Section VI (63). It has been observed that any inhibitor of DNA polymerase a also inhibits the HSV-induced DNA polymerase and the vaccinia-induced DNA polymerase, and vice versa (21). Since there is no known relationship or structural similarity between these viral-induced enzymes and DNA polymerase a , it will be of consid60. A. Bolden, J. Aucker, and A. Weissbach, J . Virol. 16, 1584 (1975). 61. S . Leinbach, J. M. Reno, L. Lee, A . F. Isbell, and J. A. Baezi, Biochtrnistry 15, 426
(1976). 62. B. Eriksson, A. L&rsmn,E. He!gstrand, N. G. Johansson, and B. Oberg, BBA607,53 (1980). 63. G . Pedrali Noy and S . Spadari, J . Virology 36, 457 (1980).
5 . CELLULAR AND VIRAL-INDUCED DNA POLYMERASES
83
erable interest to elucidate the active sites of these enzymes and compare them. The HSV-DNA polymerase contains a 3' + 5' exonuclease activity that copurifies with the enzyme and is apparently an intrinsic activity. This is in contrast to the purified host-cell DNA polymerases, which are devoid of nuclease activity in their most purified form, although preparations of DNA polymerase (Y with nuclease activity have been reported (22, 2 3 ) . Whether the exonuclease serves as a "proof-reading'' activity, as has been postulated for E. coli DNA polymerase I (64) and T4-DNA polymerase ( 6 3 , remains to be determined.
B. BIOLOGICAL ROLE Herpes virus contains a relatively large genome of about 10' daltons. A genome of this size would be expected to code for 100-150 proteins, and it would not be surprising if one of these proteins might be a new DNA polymerase. Genetic evidence for this exists since certain viral DNA negative mutations are located at the chromosomal site that determines the DNA polymerase expression (66, 67). It thus appears self-evident that HSV-induced DNA polymerase is required for synthesis of the viral DNA. In addition, almost all other members of the herpes group seem to induce a new DNA polymerase in host cells after infection (25).
VI.
Vaccinia Virus-Induced DNA Polymerase
The pox viruses, of which vaccinia virus is a member, are among the largest viruses and contain DNA genomes of 1.2-2 x 10' daltons. Jungwirth and Joklik (68) and Magee and Miller (69) suggested in the 1960's that vaccinia virus could induce a new DNA polymerase in infected cells. This viral-induced DNA polymerase was partially purified by Berns er al. (70), and later clearly separated from the host DNA polymerases by 64. M. P. Deutscher and A. Kornberg, JBC 244, 3019 (1969). 65. M. S. Hershfield and N . G. Nossal, JBC 247, 3393 (1972). 66. P. Chartrand, C. S . Crumpacker, P. S . Schaffer, and N. M. Wilkie, Virology 103,311 (1980). 67. L . E. Schnipper and C. S. Crumpacker, PNAS 77, 2270 (1980). 68. C. Jungwirth and W. K. Joklik, Virology 27, 80 (1965). 69. W. E. Magee and 0. V. Miller, Virology 31, 64 (1967). 70. K. I. Berns, C. Silverman, and A. Weissbach, J . Virol. 4, I5 (1969).
84
A. WEISSBACH TABLE VI
PURIFICATION OF
VACCINIAVIRUSDNA POLYMERASE"
Fraction
Activity (units x lo-'])
Protein (mg)
Specific activity (unitdmg)
55 28 6.6 3.9 2.8 1.7
1,495 268 10.9 1.3 0.52 0.089
36 104 610 2,800 5,400 19,000
I. Extract' 11. DEAE-cellulose'
111. IV. V. VI.
DNA-agarose Phosphocellulose Hydroxylapatite Glycerol gradient
From Challberg and Englund (72). A unit is the amount of enzyme that catalyzes the incorporation of 1 nmol of total nucleotide into an acid insoluble form in 30 minutes at 37". ' Activity in Fractions I and I1 includes both vaccinia and host polymerases.
Citarellaet al. (71). It has been purified to near homogeneity from infected HeLa cells by Challberg and Englund (72). A. PURIFICATION AND PROPERTIES Because of the relatively large amount of viral-induced DNA polymerase formed in the infected cell ( 7 / ) , Challberg and Englund (72) were able to isolate 100 p g of purified enzyme from 27 g of vacciniainfected HeLa cells. Vaccinia-infected HeLa cells, obtained 53 hours after infection, and stored at -2W, were broken by Dounce homogenization in 10 volumes of 10 mM NaC1, 2 mM Tris, pH 7.6, 0.1 mM benzamidine. The lysate was clarified by centrifugation at 15,OOOg, and the supernatant fluid containing 13% glycerol and 4 m M diisopropyl fluorophosphate (DFP) was incubated 1 hour at 0" and applied to a DEAE-cellulose column. The outline of the further purification procedure is shown in Table VI, and is unique in that the DNA affinity column step is performed before the phosphocellulose and hydroxylapatite steps. Elution of the enzyme activity in each chromatographic separation utilizes buffers containing 10% glycerol and yields about a 50% recovery of enzymatic activity in each step. The final step of the preparation yields an enzymatic activity that is at least 500-fold purified from the crude cytoplasmic fraction and is 95% homogeneous. 71. R. V. Citarella, R. Muller, A. Schlabach, and A. Weissbach, J. Virol. 10, 721 (1972). 72. M. D. Challberg and P. T. Englund, JBC 254, 7812 (1979).
5. CELLULAR AND VIRAL-INDUCED DNA POLYMERASES
85
The vaccinia DNA polymerase activity in the infected cells is stable for one month at -20” and is stable for 24 hours at 0” in the cytoplasmic extract (fraction I). The most purified preparations (fractions IV and VI) are stable for months at -20”. In the absence of protease inhibitors, such as DFP and benzamidine, proteolysis of the enzyme during purification occurs even at the phosphocellulose step. Native vaccinia-DNA polymerase is a single polypeptide with a molecular weight of 110,000-1 15,000. It is maximally active in the presence of 5 mM MgC1, and shows a pH optimum in 50 mM potassium phosphate, at 8-9. Its activity in Tris-HC1 at the same pH is 10% that shown in potassium phosphate buffers. The enzyme requires the presence of SH groups and is inhibited by 10 mM N-ethylamaleimide or 30 pM p chloromercuribenzoate. In contrast to the herpes simplex-induced DNA polymerase, the vaccinia-DNA polymerase is inhibited by salt (50% at 200 mM NaCl). The vaccinia DNA polymerase shows maximal activity in an activated DNA template, but will neither nick-translate nor strand-displace a nicked 4x174 DNA template. The enzyme seems sensitive to the secondary structure of the template since in copying 4x174 templates it pauses at regions that contain potential hairpin structures (73). As previously reported (71), the purified polymerase contains a strong exonuclease activity that is apparently part of the DNA polymerase polypeptide since both the polymerase and nuclease activity show the same kinetics of heat inactivation at 45”. The intrinsic nuclease activity is a 3’ + 5’ exonuclease that produces 5’-mononucleotides. Although the pH optimum of the exonuclease, 8-9, is similar to the pH optimum of the polymerase activity, the nuclease activity is twice as active in Mn2’ (50 pM MnC1,) as in the optimum MgCl, concentration (10 mM). In addition, the exonuclease is twice as active in Tris-HC1 as in potassium phosphate and is inhibited 50% by 50 mM NaCl. The polymerase-associated exonuclease hydrolyzes single-stranded DNA somewhat faster than the equivalent duplex DNA. This preference for single-stranded DNA increases as the size of the DNA piece becomes smaller.
B. BIOLOGICAL ROLE The vaccinia-induced DNA polymerase is assumed to be required for the synthesis of the viral DNA, although this remains unproved. Since the genetic loci for this enzyme on the vaccinia chromosome has not been 73. M. D. Challberg and P. T. Englund, J B t 254, 7XLU (IYIY). 74. A. Kornberg, “DNA Replication.” Freeman, San Francisco, 1980.
86
A. WEISSBACH
determined, genetic analysis of the components of DNA replication, as was done for the herpes virus, remains to be investigated. VII.
Conclusion
The mechanism(s) of DNA replication in the cell’s nucleus remain unknown. Further understanding of the physiological role of each of the cellular DNA polymerases will parallel the unraveling of the complex events that accompany and control the synthesis of nuclear DNA. The smaller viral chromosomes, which should be more vulnerable to genetic manipulation and analysis, would seem to offer a promising avenue of research, in parallel perhaps, to the extraordinary detail emerging from the studies of E. coli and its phages (74). The present lack of knowledge portends that our perception of the types and nature of eukaryotic DNA polymerases, as well as their roles, may change within the next few years.
I. Introduction and Perspective . . . . . . . . . 11. DNA Polymerase a . . . . . . . . . . . , . A. Purification and Properties. . . . . . . . . B. Biological Role . . . . . . . . . . . . . . 111. DNA Polymerase p . . . . . . . . . . . . . A. Purification and Properties . . . . . . . . . B. Biological Role . . . . . . . . . . . . . . IV. DNA Polymerase y . . . . . . . . . . . . . A. Purification and Properties . . . . . . . . . B. Biological Role . . . . . . . . . . . . . . V. Herpes Simplex Virus-Induced DNA Polymerase A. Purification and Properties . . . . . . . . . B. Biological Role . . . . . . . . . . . . . . VI. Vaccinia Virus-Induced DNA Polymerase . . . A. Purification and Properties . . . . . . . . . B. Biological Role . . . . . . . . . . . . . . VII. Conclusion . . . . . . . . . . . . . . . . .
1.
. . . . . . . . . . . . . . . . . . . .
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. . . . . . . . . . . . . . . . . .
. . . . . . , . . . . . . . . . . . . . .
. . . . . . . . . . . .
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. .
. . . . . . . . . . . . . . . . . . . . . . . .
, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
67 69 69 73 73 74 76 76 77 79 80 80 83 83 84 85
86
Introduction and Perspective
A nomenclature for the known eukaryotic DNA polymerases was proposed in 1975 (/). This classification, which recognized three major 1. A. Weissbach, D. Baltimore, F. J. Bollum, R. C. Gallo, and D. Korn, Scicizcc 190, 401 (1975).
67 THE ENZYMES, Vol. XIV
Copyright @ 1981 by Academic Press. Inc. All rights of reproduction in any form reserved
68
A. WEISSBACH
classes of cellular DNA polymerases--cY, /3, and y-has proved to be applicable to a wide range of species in the Animal Kingdom (2). In addition, the nomenclature scheme recognized the existence of a mitochondria1DNA polymerase and new viral-induced DNA polymerases that are produced in infected animal cells. This chapter limits itself to consideration of enzymes that have been well characterized, i.e., DNA polymerases a , P , and y from mammalian cells and the herpes simplex virus and vaccinia virus-induced DNA polymerases. A number of other reported DNA polymerase activities ( 2 4 ) , less well identified, are necessarily omitted. The three cellular DNA polymerases were named in the order of their discovery: DNA polymerase a was first isolated from calf thymus and characterized by Bollum (5, 6), and his work was an important starting point in the study of eukaryotic DNA polymerases. The enzyme has been identified in many species as a predominant activity, and has been purified from rat, murine, calf thymus, and human cells. In 1971 Weissbach et al. (7) reported a new, low molecular weight DNA polymerase in HeLa cell nuclei at the same time that Baril et al. (8)reported an enzyme with similar properties in rat liver nuclei. This enzyme has been designated as ppolymerase. Further identification and characterization of P-polymerase in calf thymus, rat liver, KB cells, and chick embryos followed shortly thereafter (9-11). In general, P-polymerase represents about 5% of the total DNA polymerase in growing, cultured cells, but is a major component of low DNA polymerase activity in quiescent cells. DNA polymerase y , first reported in 1972 by Fridlender et ul. (12), is a widely distributed enzyme (13) that shows a marked ability to copy ribohomopolymers, but a total inability to use natural RNA as a template. It thus has no relationship to the retrovirus reverse transcriptases. At first thought to be a minor activity in the cell, the DNA polymerase y level in 2. A. Weissbach, Annu. Rev. Biochem. 46, 25 (1977). 3. J . A. Kantor, X. H. Lee, J. G . Chirikjian, and W. G . Feller, Science 204, 511 (1979). 4. B. A. Brennessel, D. P. Buhrer, and A. Gottlieb,Anal. Biochem. 87, 411 (1978). 5. F. J. Bollurn, JBC 235, 2399 (1960). 6. M. Yoneda and F. J. Bollurn, JEC 240, 3385 (1965). 7. A. Weissbach, A. Schlabach, B. Fridlender, and A. Bolden,Notlrre New B i d . 231, 167 ( 197 1).
8. E. F. Baril, 0. E. Brown, M. D. Jenkins, and J. Laszlo, Binrhemistry 10, 1981 (1971). 9. L. M. S . Chang and F. J. Bollurn. JBC 246, 5835 (1971). 10. M. E. Haines, A . M. Holrnes, and I. R. Johnston, FEES (Fed.Eur. Biochem. SOC.) Lett. 17, 63 (1971). 11. H. Berger, Jr., R. C. Huang, and J. L. Irvin, JBC 246, 7275 (1971). 12. B. Fridlender, M. Fry, A. Bolden, and A . Weissbach, PNAS 69, 452 (1972). 13. A. Weissbach, Cell S, 101 (1975).
5. CELLULAR AND VIRAL-INDUCED DNA POLY MERASES
69
growing cultured cells is, in fact, equal to that of thep-polymerase, and in at least one case the y-polymerase is the major polymerase in the organism. Table I summarizes some relevant characteristics of the three cellular DNA polymerases obtained from various sources and also lists two wellcharacterized viral-induced DNA polymerases. It should be emphasized here that the properties, size, and behavior of the a-, P - , and y-polymerases can differ from those shown in Table I, depending on the source of the enzyme. The specific patterns shown by inhibitors, however, seem to be invariant. Each of these enzymes is considered in detail in the following sections. II. DNA Polymerase a
DNA polymerase a has been extensively purified from calf thymus (14, 1 5 ) , human cells (16), murine cells (171, and others (2). Because of the extensive heterogeneity of the enzyme in various species, isolation of a-polymerase in a pure form has been difficult. Nevertheless, a nearhomogeneous preparation of a-polymerase has been obtained from human cells by Fisher and Korn (161, and from mouse myeloma cells by Chen et al. (17). A. PURIFICATION AND PROPERTIES Table I1 summarizes the purification of a-polymerase from cultured human KB cells as described by Fisher and Korn (16). In this procedure all buffers contained 1 rnM P-mercaptoethanol and 1 m M EDTA and, after fraction V, 20% glycerol. A protease inhibitor, p-toluenesulfonyl fluoride, is present when the cells are broken. The second DEAE-cellulose step, which offers little purification per se, is apparently important for subsequent steps. The purified protein from human cells has a specific activity of 206,000 unitdmg, a unit being defined as the incorporation of 1 nrnol of dTMP in DNMhour at 37”. The enzyme, as isolated, exists either as a monomer of 140,000 daltons or as dimers of 265,000-280,000 daltons. It has an isoelectric point of 5.0-5.2 and can be resolved in denaturing polyacrylamide gel 14. 15. 16. 17.
F. J. Bollum, Progr. Nucleic Acid Res. Mol. Biol. 15, 109 (1975). K. McKune and A. M. Holmes, Nucleic Acids Res. 6, 3341 (1979). P. A. Fisher and D. Korn, JBC 252, 6528 (1977). Y. C. Chen, E. W. Bohn, S. R. Planck, and S. H. Wilson, JBC 254, 11678 (1979).
TABLE I
EUKARYOTIC DNA POLYMERASES DNA polymerases
Source Human cells
(KB)
Y
Novikoff hepatoma Chick embryo
Herpes simplexinduced Vacciniainduced
Infected HeLa cells Infected HeLa cells
Major cellular location Nucleus, cytoplasm Nucleus
Subunits (kilodaltons)
156
76, 66
3 I"
Nucleus, mitochondria
180
47
Nucleus
144
74,29
Cytoplasm
115 ~
a
Molecular weight (X l t 3 )
~
~~~
~~~~~~~~
DNA polymerase /3 from other cells has a reported molecular weight of 40,OOO-45,OOO.
Inhibitors Aphidicolin, N-ethylmaleimide, Ara-ATP Dideoxynucleoside triphosphates, iodoacetate Dideoxynucleoside triphosphates, N-ethylmakimide Phosphonoacetate, Ara-ATP, N-ethylmaleimide Phosphonoacetate, N-ethylmaleimide
TABLE I1 PURIFICATION OF Step Crude extract pH 5.5 precipitation Ultracentrifugation First DEAE-cellulose
2
Second DEAE-cellulose Phosphocellulose Hydroxylapatite DNA-ceUulose Gel electrophoresis'
Fraction I I1 (resolubilized precipitate) 11' (supernatant) 111 (supernatant) 111' (pellet) IV (adsorbed) IV' (flow-through) V VI VII VIII IX
DNA POLYMERASE a FROM KB CELLS" Velum$ (ml)
Proteid (mg)
Activit9 (units)
9.9 2.2
43 19
880 890
2.8
9
2.9
9 x 10-1
1.1
4 x 10-1 4 x 10-2 1 x 10-2 I x 1 r 3 1.6 x 10-4
3.8 x lo-' 6.6 x lo-* 6.5 x lo-*
60 790 50 490 100 280 240 130
33
Specific activity (unitdmg)
Yield (%)
20 47
(100) 108
88
97
550
67
700 6,000 13,000 33,000 206,000
32 28
I5 4
" As described by Fisher and Korn (16). Reaction mixes contained in 2 5 0 ~ 1 10 , mM Tris, pH 9.2,20 mM mercaptoethanol, bovine serum albumin 200 pglml, 10 m M MgCb, activated salmon sperm DNA, 800 pglrnl, dATP, dCTP, dGTP, and dTTP, 50 g C L M each, and PHIdlTP at a final specific activity of 0.04 Ci/mmol, and enzyme. A unit is the amount of enzyme that catalyzes the incorporation of 1 nmol of labeled dTMP into an acid-insoluble product in 1 hour at 37". Quantities are expressed per gram wet weight of KB Cells. Aliquots, 400 p l , of fraction VIII were used for nondenatunng gel electrophoresis. The protein value was derived by densitometry of a stained gel. Recovery of DNA polymerase activity by elution of slices of parallel unstained gels varied between 50 and 95%. The specific activity value is based on units of loaded activity.
72
A. WEISSBACH
electrophoresis into two subunits of 76,000 and 66,000 daltons. The purified enzyme has a half-life at 0" of 14 months if stored in a concentrated form in the presence of sucrose and potassium phosphate. Optimal reaction conditions include a pH between 7.5-8.5, and Mg'+ at 4-8 mM. Salt concentrations above 50 mM are inhibitory, with about 50% of the activity lost at 100 mM KCl. a-Polymerase is markedly inhibited by Caz+ and Li+. a-Polymerase is most reactive with duplex DNA templates containing gapped regions with available 3'-OH termini (activated DNA). A surprising property of the purified enzyme is its inability to catalyze the synthesis of long DNA chains. It is only slightly processive, synthesizing an 11 2 5 nucleotide length before coming off the template (18, 19). DNA polymerase a does not act at nicks or in short gaps below 20 nucleotides in length, and does not utilize a blunt-ended DNA template. The enzyme binds to single-stranded DNA that contains 3'-OH ends and can catalyze synthesis of hairpin molecules from such templates (20, 21). Synthetic DNA templates such as (dA), . (dT)12--IB are copied at 20% the rate of activated DNA, whereas the corresponding synthetic RNA template, (A), . dTlz, copies at only 3% the rate of activated DNA. However, murine DNA polymerase a copies (dT), . rA2, faster than any other template (22).Spermidine has been found to increase the apparent K , for M. Purified calf DNA (20). The K , for dNTPs is in the range of 1-4 x thymus DNA polymerase a catalyzes both pyrophosphorolysis and pyrophosphate exchange (14). As isolated, purified human a-polymerase has no detectable nuclease activity. There have been reports that bone marrow and calf thymus contain an a-polymerase-like enzyme that contains a 3' + 5' exonuclease, and has been called DNA polymerase 8 (23). Whether this represents a new enzyme or an association of a cellular 3' + 5' exonuclease (24) with the a-polymerase is still unclear. Chen et al. (17) obtained two nearhomogeneous preparations of a-polymerase from mouse myeloma. These 18. P. A. Fisher, T. S-F. Wang, and D. Korn,JBC 254, 6128 (1979). 19. K. McKune and A. M. Holmes, BBRC 90, 864 (1979). 20. P. A. Fisher and D . Korn, JBC 254, 11033 (1979). 21. P. A. Fisher and D. Korn,JBC 254, 11040 (1979). 22. S. H. Wilson, A. Matsukage, E. W. Bohn, Y. C. Chen, and M. Sivarajan, Nucleic Acids RPS.4, 3981 (1977). 23. M. Y. W. Tsang-Lee, C. K. Tan, A. G . So, and K. M. Downey,Biochemistry 19,20% (1980). 24. G. Villani, S. Spadari. S. Boiteux, M. Defais, P. Caillet-Fauquet, and M. Radman, Biochimie 60, 1145 (1978).
5 . CELLULAR AND VIRAL-INDUCED DNA POLYMERASES
73
large molecular weight enzymes (MW = 190,000) contain subunits of 47,000 and 54,000 daltons. One of the a-polymerase species contains both 3' + 5' and 5' + 3' exonucleases associated with it. a-Polymerase has also been isolated in highly purified form from calf thymus (15) and regenerating rat liver; in both cases a number of subunits ranging from 50,000 daltons to 70,000 daltons seem to be associated with a catalytic polypeptide whose molecular weight is about 150,000. Thus, a common denominator in many of these studies is the heterogeneity of DNA polymerase a, a feature which may have important implications in the control and function of this enzyme. B.
BIOLOGICAL ROLE
It is generally agreed that DNA polymerase a has a key role in the replication of nuclear DNA and in the synthesis of the DNA of the viruses SV40, polyoma, and adenovirus (14, 25). Understanding the role of a-polymerase has been aided by the availability of specific inhibitors such as aphidicolin, or the arabinose-containing nucleotides such as ara-ATP. The use of these inhibitors supports the concept that DNA polymerase a is the major replicative polymerase in mammalian cells (26-30). It represents 90-95% of the total DNA polymerase activity of cultured growing mammalian cells and drops to low levels in cells that have ceased nuclear DNA synthesis. 111.
DNA Polymerase /3
DNA polymerase /3 is the smallest of the known eukaryotic DNA polymerases and shows remarkable chemical stability under various conditions. As a result, and although it represents only about 5% of the total DNA polymerase in growing, cultured mammalian cells, it was the first eukaryotic DNA polymerase to be isolated in a homogeneous state. This has been accomplished from calf thymus (3I ), human KB cells (32), mouse 25. 26. 27. 28. 29. 30. 31. 32.
A. Weissbach, ABB 198, 386 (1979). H. J. Edenberg, S. Anderson, and M. L. DeParnphilis, JBC 253, 3273 (1978). M. A. Waqar, M. J . Evan, and J. A. Huberman, Nucleic Acids Res. 5, 1933 (1978). M. Ohashi, T. Taguchi, and S . Ikegami, BBRC 82, 1084 (1978). E. Wist and H. Prydz, Nucleic Acids Res. 6, 1583 (1979). E. Wist, BBA 562, 62 (1979). L. M. S. Chang, JBC 248, 3789 (1973). T. S-F. Wang, W. D. Sedwick, and D. Korn, JEC 250, 7040 (1975).
74
A. WEISSBACH
myeloma (33),Novikoff hepatoma (34) and chick embryos (35). The purification of DNA polymerase /3 from the latter is summarized in Section II1,A. A. PURIFICATION AND PROPERTIES The procedure used by Stalker et al. (34) is shown in Table 111, and yields a homogeneous enzyme after a 200,000-fold purification with a remarkable apparent yield of 46%. The starting material, Novikoff hepatoma, is an ascites tumor with a generation time of 12 hours when maintained in rats, so relatively large quantities of cells can be obtained conveniently. The purification relies on the sequential use of three chromatographic separations on DEAE-Sephadex, phosphocellulose, and hydroxylapatite. The final step in the purification procedure uses singlestranded DNA cellulose as an affinity column, which in a 25-fold enrichment step provides the pure enzyme. The enzyme is stable at 4" during the isolation procedure, and stabilization of the enzyme during purification is facilitated by the use of 10% glycerol in the elution buffers and, at the final step, by having bovine serum albumin (1 mg/ml) present. With this method, 200 pg of purified DNA polymerase P are obtained per kilogram of cells. The enzyme in whole cells is stable at - 20" for months, and the purified enzyme has been stored for 1 year at - 196" without loss of activity. The enzyme, as isolated, has a molecular weight of 31,000 although the calf thymus (31), KB cell (32), and chick (35) and mouse P-polymerases (33) have been reported to have molecular weights of 44,000, 43,000 and 40,000 daltons, respectively. Purified DNA polymerase /3 has no detectable nuclease activity. It shows an alkaline isoelectric point (8.51, a pH optimum of 8.4-9.2 and a K, for deoxynucleoside triphosphates of 7-8 p M . For maximal synthesis Mg2+ at 5-10 mM is required; MI?+ (1 mM) can also be used. The enzyme is stimulated twofold by 50 mM NaCl or by 100-200 mM KC1. The latter salt levels severely inhibit a-polymerase. Phosphate and pyrophosphate are inhibitory to /3-polymerase and should be avoided in reaction mixes. Neither the mouse nor human enzyme can catalyze pyrophosphate exchange, pyrophosphorolysis, or dNTP turnover (33, 36). 33. K. Tanabe, E. W. Bohn, and S. H. Wilson, Biochemistry 18, 3401 (1979). 34. D. M. Stalker, D. W. Mosbaugh, and R. R. Meyer, Biochemistry IS, 3114 (1976). 35. M. Yamaguchi, K. Tanabe, Y. N . Taguchi, M . Nishizawa, T. Takahashi, and A. Matsukage, JBC 255, 9942 (1980). 36. T. S-F. Wang, W. D. Sedwick, and D. Korn, JBC 249, 841 (1974).
75
5 . CELLULAR AND VIRAL-INDUCED DNA POLYMERASES
TABLE I11
PURIFICATION OF NOVIKOFF HEPATOMA DNA POLYMERASE P" Protein Fraction
Total unitsh
Specific (unitdmg)
Purification (-fold)
Yield
(mg)
1. Cell extract 11. Ammonium sulfate 111. DEAE-Sephadex IV. Phosphocellulose V. Hydroxylapatite VI. DNA-Cellulose
14,500 4,610 8,866 81.2 1.34 0.031
3,880 3,780 9,820 4,390 3,150 1,800
0.268 0.820 11.3
1.0 3.06 42.2 202 8,770 217,000
100 97.4 253
54.1
2,350 58,100'
(%)
113
81.2 46.4
' I From Stalker et al. (35).The reaction mixtures contained the following components in a final volume of 125 111: 25 mM Tris-HCI, pH 8.4; 5 mM 2mercaptoethanol; 7 mM magnesium acetate; 0.5 mM EDTA; 0.015 m M each of dATP, dCTP, dGTP, and PHIdTTP (specific activity 975 rnCi/mmol); 50 m M NaCI; 15% ( w h ) glycerol; 250 Fg/ml activated DNA; and 0.01-0.3 units of DNA polymerase fraction. Incubations were carried out for 1 hour at 37" and acid-insoluble radioactivity was determined. When incorporation was not linear for 1 hour, the data were extrapolated from a 30-minute incubation. * A unit is defined as the incorporation of 1 nmol of total nucleotide into DNA per hour at 37". ' With several different preparations, the specific activity varied from 32,000 to 62,000 unitslmg.
A reported characteristic of DNA polymerase /3 is its relative insensitivity to urea, acetone, and alcohol (14). The enzyme is stabilized by glycols and stimulated by spermidine (up to 10 mM) (34). Another general property of the P-polymerases is their relative resistance to N-ethylmaleimide (NEM), which is a powerful inhibitor of DNA polymerases a and y . At 4 mM, NEM shows a 28% inhibition of DNA polymerase @ from Novikoff hepatoma, a value that is slightly higher than previously reported for the human enzyme (37). The NEM partial inhibition is not unexpected since p-hydroxymercuribenzoate inhibits @-polymeraseat concentrations above 50 p M (14, 34). An important characteristic of P-polymerase is its ability to copy a synthetic ribohomopolymer such as (A), . dTlz as well as the corresponding deoxyribohomopolymer (dA), . dTlz or activated DNA (34).This is in contrast to a-polymerase, which utilizes the deoxyribohomopolymer (dA), . dT,,_,, eight times better than (A), . dTlz, which is, in fact, copied at only 3% the rate of activated DNA (18). Rat DNA polymerase 0 has been reported to have a uniquely high requirement for primers when 37. K. W. Knopf, M. Yamada, and A. Weissbach, Biochemistry 15, 4540 (1976).
76
A. WEISSBACH
copying poly(A) templates, and can thus be distinguished from y-polymerase or oncornavirus reverse transcriptase (38). Steady-state kinetic measurements suggest an ordered BiBi mechanism for polymerization and a scheme depicting two DNA binding sites on the enzyme has been advanced (33).Although the specific activity of the purified Novikoff hepatoma DNA polymerase p prepared by Stalkeret al. (S4)is 58,000 with activated DNA as a template, Ono et al. (38)reported rat ascites hepatoma DNA polymerase preparations with a specific activity of lo6 units/mg on an (A), * dTlz-ls template.
B. BIOLOGICAL ROLE The level of DNA polymerase L,3 in quiescent or growing cells or during the cell cycle has been reported to be relatively constant, leading to the suggestion that it may be involved in DNA repair synthesis (39). Hiibscher et al. (40) have shown that &polymerase can participate in the repair of UV-damaged DNA in neuronal nuclei, an organelle in which DNA polymerase p is the only detectable polymerase activity. The further role of this enzyme in other types of DNA synthesis is unknown at the present time. IV.
DNA Polymerase y
DNA polymerase y exists in at least two forms and is found in the nucleus, cytoplasm, and mitochondria (41-43). Like a-polymerase, DNA polymerase y readily undergoes reversible aggregations that, in v i m at least, are salt-dependent. It comprises about 5% of the total DNA polymerase activity in the growing, cultured mammalian cell, and therefore is about equal to the &polymerase level. In developing chick embryos, y-polymerase represents 45% of the total DNA polymerase activity and is present in larger amounts than either 0- or P-polymerases (44). It is clear that the so-called “mitochondrial” DNA polymerase is one species 38. K. Ono, A. Ohashi, K. Tanabe, A. Matsukage, M. Nishizawa, and T. Takahashi, Nucleic Acids Res. 7, 715 (1979). 39. G. Pedrali Noy, L. Dalpra’, M. A. Pedrini, G. Ciarrocchi, E. Gidotto, F.Nuzzo, and A. Falaschi, Nucleic Acids Res. 1, 1183 (1974). 40. U. Hiibscher, C. C. Kuenzle, and S. Spadari, PNAS 76, 2316 (1979). 41. S. Spadari and A. Weissbach, JBC 249, 5809 (1974). 42. G. Pedrali Noy and A. Weissbach, BBA 477, 70 (1977). 43. A. Bolden, G. Pedrali Noy, and A. Weissbach, JBC 252, 3351 (1977). 44. M. Yamaguchi, A. Matsukage, and T. Takahashi, JBC 255, 7002 (1980).
5 . CELLULAR AND VIRAL-INDUCED DNA POLY MERASES
77
of the y-polymerase class, and that the nuclear species of DNA polymerase y can be distinguished from it (43). Despite extensive efforts, the enzyme has not been prepared in pure form from mammalian tissues (45), perhaps due, in part, to its heterogeneity: but it has been purified to near-homogeneity from chick embryos (44). A.
PURIFICATION AND PROPERTIES
An outline of the purification of the y-polymerase from chick embryos as described by Yamaguchi et al. (44) is shown in Table IV. In this procedure, frozen 11-day-old embryos are minced and sonicated in a buffer containing 0.5 M KCl, 10% glycerol, and eventually, 0.5% Triton X-100. The purification scheme uses two phosphocellulose column chromatographic steps, a Sephadex G-200 gel filtration, and hydroxylapatite adsorption chromatography. Following the second phosphocellulose column, the enzymatic activity separates into two gel components, a 180,000- and a 280,000-dalton species, during gel filtration on a Sephadex G-200 column, and each species is further purified separately. The final separation step, which gives a 1000-foldenrichment, involves affinity chromatography on a double-stranded DNA cellulose column, and can be compared to the single-stranded DNA cellulose columns used in the purification of a- and P-polymerases. Attempts to purify DNA polymerase y by d n i t y chromatography on poly(rA)-Sepharose columns leads to inactivation of the enzyme. The purified enzyme can be stored at -80" but loses 50% of its activity in one freeze-thaw cycle. The purified enzyme sediments at 7.5 S and the molecular weight is estimated to be 180,000. SDS polyacrylamide gel electrophoresis shows a prominent polypeptide at 47,000 daltons, so the native enzyme appears to be a tetramer of this subunit. Based on this, the specific activity of the purified enzyme is calculated to be 660,000 unitslmg on a poly(A) template. With (A), dTlz-ls as a primer-template, the K , value for dTTP is about 1 p M, the optimal pH 8.5-9.0, and the optimal KCI concentration is 220 p M. In the presence of increasing levels of potassium phosphate, the optimal KCI concentration drops proportionately (45); and Mn2+ at 0.5-0.6 mM is fivefold more effective than the optimum Mg2+concentration of 12 mM. The structure of native DNA polymerase y in mammalian cells will probably differ somewhat from the avian enzyme. Rat liver DNA polymerase y can be obtained as a 4 S species (60,000 daltons) (43), whereas the smallest species of the chick native enzyme sediments at 7.5 45. K-W. Knopf, M. Yamada, and
A. Weissbach, Biochemistry
15, 4540 (1976).
TABLE IV
PURIFICATION O F DNA POLYMERASE y FROM CHICK
Step Crude extract First phosphocellulose and ammonium sulfate fractionation Second phosphocellulose Sephadex G-200 Hydroxylapatite Double-stranded DNA cellulose
Fraction I
I1
EMBRYOS~
Protein
Activityb units
(mg)
(%)
0,38 3s
80 4.8 4.1 x 10-' 1.0 x lo-'
I11 IV- 1 IV-2 v-1 v-2 VI- 1
9.0 x
4.1(14) 5.0(17) 3.6(12)
(VI-1-dT VI-2
(1.3 x 10-7 1.3 x lCV
(4 4.5( 15)
9.0 x 1C2 8.4 x 10-3 8.1 x 10-3
specific activity (unitdmg)
2x77) 8.4(28) 9.0(30)
56 84 100 490 620 400.000
Purification (-fold) 1 9.2 150
220 260 1,300 1,600 1,100,000 (1,500,000) 920,000
From Yamaguchi et al. (44). The assay mixture contained in 25pl,50 mM Tns, pH 8.5, 1 mM dithiothreitol, 0.5 mM MnC&.,80 pglml poly (rA) 16 pg/mI dT,,-,,, 0.1 mM [3H]dTTP(60cpdpmol), 15% glycerol, 400 pg/ml bovine serum albumin, 100-120 mM KCI, 20-40 mM potassium phosphate (pH 8.5) and enzyme. A unit is the amount of enzyme that catalyzes the polymerization of 1 nmol of dTMP in 60 minutes.
* Quantities are expressed per gram wet weight of
11-day-old chick embryos.
' Fractions in the peak of DNA polymerase activity (see Fig. 5A).
S. CELLULAR A N D VIRAL-INDUCED DNA POLY MERASES
79
S (150,000-180,000 daltons). By contrast, HeLa cell DNA polymerase y can be separated into two species on phosphocellulose chromatography, both with the same or similar apparent molecular weight of 110,000 (do), and human lymphoblast DNA polymerase y has a reported molecular weight of 120,000 (46). The interspecies difference is further illustrated by the report that sea urchin DNA polymerase y has a sedimentation value of 3.3 s (47). A salient feature of y-polymerases is their ability to copy ribohomopolymers at a rate greater than activated DNA. Under proper conditions the HeLa cell DNA polymerase y will utilize (A), * dT12-18five to ten times more efficiently than activated DNA (44). This is in contrast to the template characteristics of a-polymerase, which utilizes this synthetic template at 3% the rate at which it uses activated DNA. In addition, y-polymerase is active at potassium phosphate concentrations (50 mM) that are inhibitory to DNA polymerase /3(14,45), an enzyme that is known to copy (A), . dTlz-18 at about the same efficiency it copies activated
DNA.
B. BIOLOGICAL ROLE Of the three cellular DNA polymerases, only DNA polymerase y is capable of synthesizing continuous long DNA chains in a processive manner (48). DNA polymerase a , by comparison, is highly discontinuous, polymerizing 10-15 nucleotides at a time and then leaving the template (18,49).,&Polymerase also shows discontinuous synthesis when copying a poly(A) template (49). The ability of the y-polymerase to carry out a processive and continuous synthesis of a DNA chain may explain its known physiological roles. One of the forms of the enzyme is responsible for mitochondrial DNA synthesis (43, 50, 51) and another, the nuclear y-polymerase, is involved in the replication of adenovirus DNA (52, 53). The synthesis of adenovirus DNA and mitochondria1 DNA share a strand-displacement step in their replication process; this has led to the 46. M. Robert-Guroff, A. W. Schrecker, B. J . Brinkman, and R. C. Gallo, Biochemistry
16, 2866 (1977).
47. A. Habara, H. Nagano, and Y. Mano, BBA 561, 17 (1979). 48. M. Yamaguchi, A. Matsukage, and T. Takahashi, Nature (London) 285, 45 (1980). 49. A. Matsukage, M. Nihizawa, T. Takahashi, and T. Hozumi, J . Eiochern. (Tokyo),in press (1980). 50. U. Hubscher, C. C. Kuenzle, and S . Spadari, PNAS 76, 2316 (1979). 51. W. timmemann, S-M. Chen, A. Bolden, and A. Weissbach,JBC 255, 11847 (1980). 52. P. C. Van der Vliet and M. M. Kwant, Nature (London) 276, 532 (1978). 53. H. Krokan, P. SchaEer, and M. L. DePamphilis, Biochemistry, 18, 4431 (1979).
80
A. WEISSBACH
suggestion that y-polymerase has a unique role in strand-displacement syntheses (52, 25). Since both mitochondria1 DNA and adenovirus DNA are synthesized in a continuous mode without the apparent formation of short intermediates, such as Okazaki fragments, the processive character demonstrated by y-polymerase in vitro is also reflected in vivo . However, it is apparent that the basic physiological role of DNA polymerase y in the nucleus of the cell remains unknown. V.
Herpes Simplex Virus-Induced
DNA
Polymerase
The recognition in 1963 that herpes simplex virus (HSV) induced a new DNA polymerase in infected cells (54, 5 5 ) followed shortly after the discovery of DNA polymerase a , and predates the identification of DNA polymerases p and y . The HSV-induced DNA polymerase is therefore one of the earliest eukaryotic DNA polymerases studied. The altered properties of the enzyme were recognized by Keir et al. ( 5 3 , and the enzyme was partially purified and characterized by Weissbach er a!. (56). Highly purified, near-homogeneous preparations of the HSV polymerase have been prepared from HSV-1-infected HEp-2 cells (57) and from African green monkey cells (58). Purification of the viral-induced enzyme is facilitated by the large amounts of virus that are produced in the infected cell (59). Thus, the amount of HSV-1 DNA polymerase in HSV-1-infected HeLa cells can rise to four times the combined level of all the host cell DNA polymerases. A. PURIFICATION AND PROPERTIES As described by Knopf (58), African green monkey cells (RC-37; Italdiagnostic Products) grown in monolayers were infected at 5 pfdcell with HSV-1 (Angelotti) that had previously been passed through RC-37 five times. Six hours after infection the cells were collected, disrupted by sonication in 0.25 M potassium phosphate, pH 7.5, containing 0.5% Triton X-100. All the subsequent purification steps shown in Table V were per54. H. M. Keir, J. Hay, J. M. Momson, and J. Subak-Shape, Nature (London)210, 369 (1966). 55. H. M. Keir, J. Subak-Shape, W. I. H . Shedden, D. H. Watson, and P. Wildy, Virology 30, 154 (1966). 56. A. Weissbach, S-C. L. Hong, J. Aucker, and R. Muller, JBC 248, 6270 (1973). 57. K . L. Powell and D. J . M. hrifoy, J . Vlrol. 24, 616 (1977). 58. K. Knopf, EJB 98, 231 (1979). 59. M. Yamada, G. Brun, and A. Weissbach, J . Virol. 26, 281 (1978).
TABLE V PURIFICATION OF
HSV-1-DNA POLYMERASE FROM INFECTED RC-37 CELLP ~~
Purification
Volume (ml)
Total protein (mg)
Total activity (units)
Specific activity (unitdmg protein)
Purification
(%)
Cell extract dialysate DEAE-cellulose Phosphocellulose DNA-cellulose DNA-cellulose peak (fraction 37)
370 575 247 30 0.9
1191.4 217.8 28.5 1.38 0.033
125,280 150,480 95,168 3 1,570 1,575
105.2 690.9 3339.2 22876.8 47727.3
1 6.6 31.7 217.5 453.7
100 120 76 25
~~
Total recovery
~
From Knoff (58). Reaction mixtures contained in lOOpl50 mM Tns-HCI (pH KO), 7.5 mM MgCl,, 100 m M ammonium sulfate, 5 0 p g bovine serum albumin, 0.5 mM dithiothreitol, 0.1 mM each of dATP, dCTP, dGTP, and PHld'lTP (0.4 Ci/mmol), and 25 pg of activated salmon sperm DNA prepared as described by Pedrali Noy and Weissbach (42).A unit is the amount of enzyme that catalyzes the polymerization of 1 nmol of nucleotide in 60 minutes under standard assay conditions. a
82
A. WEISSBACH
formed with buffers containing 0.5 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride. The purification is relatively simple and involves three chromatographic separations on DEAE-cellulose, phosphocellulose, and double-stranded DNA cellulose, which yield a highly purified preparation after only a 450-fold purification. Using similar steps with DEAE-cellulose, phosphocellulose, and single-stranded DNA-cellulose separations, Powell and Purifoy (2 years prior to Knopf's report) purified the HSV-induced polymerase from HEp-2 cells almost 1700-fold with almost a 50% recovery (57). The purified enzyme, stored in 50 mM TrisHC1, 1 mM EDTA in 50% glycerol is stable at - 20 or -70". As isolated by Knopf, the purified enzyme shows a major polypeptide of 144,000 daltons on SDS polyacrylamide gel electrophoresis, which is in agreement with the 150,000-dalton species found by Powell and Purifoy (57). The enzyme isolated from RC-37 cells also shows the presence of two other polypeptides of 74,000 and 29,000 daltons, which were not observed by Powell and Purifoy and which may represent impurities. A prominent feature of HSV-DNA polymerase, and one that facilitates its identification, is its activity at high salt concentrations. The presence of 150 mM KCI or 100 mM (N&)2S04 leads to a two- to threefold enhancement of the enzymatic activity, whereas the cellular a-polymerase is inhibited nearly 90% at these salt concentrations. The HSV-1-induced DNA polymerase has a pH optimum of 8-8.5, and a M$+ optimum of 3 mM (in the presence of activated DNA template). Dithiothreitol ( 5 mM) also stimulates the enzyme threefold. The enzyme is inhibited by Zn2+, N-ethylmaleimide, and the pyrophosphate analogs, phosphonoacetic acid, or phosphoformate (60-62). Inorganic pyrophosphate does not inhibit the enzyme, which is able to catalyze pyrophosphate exchange into dNTPs. Aphidicolin, a powerful inhibitor of DNA polymerase a , also inhibits the HSV-induced DNA polymerase as well as the vaccinia-induced DNA polymerase described in Section VI (63). It has been observed that any inhibitor of DNA polymerase a also inhibits the HSV-induced DNA polymerase and the vaccinia-induced DNA polymerase, and vice versa (21). Since there is no known relationship or structural similarity between these viral-induced enzymes and DNA polymerase a , it will be of consid60. A. Bolden, J. Aucker, and A. Weissbach, J . Virol. 16, 1584 (1975). 61. S . Leinbach, J. M. Reno, L. Lee, A . F. Isbell, and J. A. Baezi, Biochtrnistry 15, 426
(1976). 62. B. Eriksson, A. L&rsmn,E. He!gstrand, N. G. Johansson, and B. Oberg, BBA607,53 (1980). 63. G . Pedrali Noy and S . Spadari, J . Virology 36, 457 (1980).
5 . CELLULAR AND VIRAL-INDUCED DNA POLYMERASES
83
erable interest to elucidate the active sites of these enzymes and compare them. The HSV-DNA polymerase contains a 3' + 5' exonuclease activity that copurifies with the enzyme and is apparently an intrinsic activity. This is in contrast to the purified host-cell DNA polymerases, which are devoid of nuclease activity in their most purified form, although preparations of DNA polymerase (Y with nuclease activity have been reported (22, 2 3 ) . Whether the exonuclease serves as a "proof-reading'' activity, as has been postulated for E. coli DNA polymerase I (64) and T4-DNA polymerase ( 6 3 , remains to be determined.
B. BIOLOGICAL ROLE Herpes virus contains a relatively large genome of about 10' daltons. A genome of this size would be expected to code for 100-150 proteins, and it would not be surprising if one of these proteins might be a new DNA polymerase. Genetic evidence for this exists since certain viral DNA negative mutations are located at the chromosomal site that determines the DNA polymerase expression (66, 67). It thus appears self-evident that HSV-induced DNA polymerase is required for synthesis of the viral DNA. In addition, almost all other members of the herpes group seem to induce a new DNA polymerase in host cells after infection (25).
VI.
Vaccinia Virus-Induced DNA Polymerase
The pox viruses, of which vaccinia virus is a member, are among the largest viruses and contain DNA genomes of 1.2-2 x 10' daltons. Jungwirth and Joklik (68) and Magee and Miller (69) suggested in the 1960's that vaccinia virus could induce a new DNA polymerase in infected cells. This viral-induced DNA polymerase was partially purified by Berns er al. (70), and later clearly separated from the host DNA polymerases by 64. M. P. Deutscher and A. Kornberg, JBC 244, 3019 (1969). 65. M. S. Hershfield and N . G. Nossal, JBC 247, 3393 (1972). 66. P. Chartrand, C. S . Crumpacker, P. S . Schaffer, and N. M. Wilkie, Virology 103,311 (1980). 67. L . E. Schnipper and C. S. Crumpacker, PNAS 77, 2270 (1980). 68. C. Jungwirth and W. K. Joklik, Virology 27, 80 (1965). 69. W. E. Magee and 0. V. Miller, Virology 31, 64 (1967). 70. K. I. Berns, C. Silverman, and A. Weissbach, J . Virol. 4, I5 (1969).
84
A. WEISSBACH TABLE VI
PURIFICATION OF
VACCINIAVIRUSDNA POLYMERASE"
Fraction
Activity (units x lo-'])
Protein (mg)
Specific activity (unitdmg)
55 28 6.6 3.9 2.8 1.7
1,495 268 10.9 1.3 0.52 0.089
36 104 610 2,800 5,400 19,000
I. Extract' 11. DEAE-cellulose'
111. IV. V. VI.
DNA-agarose Phosphocellulose Hydroxylapatite Glycerol gradient
From Challberg and Englund (72). A unit is the amount of enzyme that catalyzes the incorporation of 1 nmol of total nucleotide into an acid insoluble form in 30 minutes at 37". ' Activity in Fractions I and I1 includes both vaccinia and host polymerases.
Citarellaet al. (71). It has been purified to near homogeneity from infected HeLa cells by Challberg and Englund (72). A. PURIFICATION AND PROPERTIES Because of the relatively large amount of viral-induced DNA polymerase formed in the infected cell ( 7 / ) , Challberg and Englund (72) were able to isolate 100 p g of purified enzyme from 27 g of vacciniainfected HeLa cells. Vaccinia-infected HeLa cells, obtained 53 hours after infection, and stored at -2W, were broken by Dounce homogenization in 10 volumes of 10 mM NaC1, 2 mM Tris, pH 7.6, 0.1 mM benzamidine. The lysate was clarified by centrifugation at 15,OOOg, and the supernatant fluid containing 13% glycerol and 4 m M diisopropyl fluorophosphate (DFP) was incubated 1 hour at 0" and applied to a DEAE-cellulose column. The outline of the further purification procedure is shown in Table VI, and is unique in that the DNA affinity column step is performed before the phosphocellulose and hydroxylapatite steps. Elution of the enzyme activity in each chromatographic separation utilizes buffers containing 10% glycerol and yields about a 50% recovery of enzymatic activity in each step. The final step of the preparation yields an enzymatic activity that is at least 500-fold purified from the crude cytoplasmic fraction and is 95% homogeneous. 71. R. V. Citarella, R. Muller, A. Schlabach, and A. Weissbach, J. Virol. 10, 721 (1972). 72. M. D. Challberg and P. T. Englund, JBC 254, 7812 (1979).
5. CELLULAR AND VIRAL-INDUCED DNA POLYMERASES
85
The vaccinia DNA polymerase activity in the infected cells is stable for one month at -20” and is stable for 24 hours at 0” in the cytoplasmic extract (fraction I). The most purified preparations (fractions IV and VI) are stable for months at -20”. In the absence of protease inhibitors, such as DFP and benzamidine, proteolysis of the enzyme during purification occurs even at the phosphocellulose step. Native vaccinia-DNA polymerase is a single polypeptide with a molecular weight of 110,000-1 15,000. It is maximally active in the presence of 5 mM MgC1, and shows a pH optimum in 50 mM potassium phosphate, at 8-9. Its activity in Tris-HC1 at the same pH is 10% that shown in potassium phosphate buffers. The enzyme requires the presence of SH groups and is inhibited by 10 mM N-ethylamaleimide or 30 pM p chloromercuribenzoate. In contrast to the herpes simplex-induced DNA polymerase, the vaccinia-DNA polymerase is inhibited by salt (50% at 200 mM NaCl). The vaccinia DNA polymerase shows maximal activity in an activated DNA template, but will neither nick-translate nor strand-displace a nicked 4x174 DNA template. The enzyme seems sensitive to the secondary structure of the template since in copying 4x174 templates it pauses at regions that contain potential hairpin structures (73). As previously reported (71), the purified polymerase contains a strong exonuclease activity that is apparently part of the DNA polymerase polypeptide since both the polymerase and nuclease activity show the same kinetics of heat inactivation at 45”. The intrinsic nuclease activity is a 3’ + 5’ exonuclease that produces 5’-mononucleotides. Although the pH optimum of the exonuclease, 8-9, is similar to the pH optimum of the polymerase activity, the nuclease activity is twice as active in Mn2’ (50 pM MnC1,) as in the optimum MgCl, concentration (10 mM). In addition, the exonuclease is twice as active in Tris-HC1 as in potassium phosphate and is inhibited 50% by 50 mM NaCl. The polymerase-associated exonuclease hydrolyzes single-stranded DNA somewhat faster than the equivalent duplex DNA. This preference for single-stranded DNA increases as the size of the DNA piece becomes smaller.
B. BIOLOGICAL ROLE The vaccinia-induced DNA polymerase is assumed to be required for the synthesis of the viral DNA, although this remains unproved. Since the genetic loci for this enzyme on the vaccinia chromosome has not been 73. M. D. Challberg and P. T. Englund, J B t 254, 7XLU (IYIY). 74. A. Kornberg, “DNA Replication.” Freeman, San Francisco, 1980.
86
A. WEISSBACH
determined, genetic analysis of the components of DNA replication, as was done for the herpes virus, remains to be investigated. VII.
Conclusion
The mechanism(s) of DNA replication in the cell’s nucleus remain unknown. Further understanding of the physiological role of each of the cellular DNA polymerases will parallel the unraveling of the complex events that accompany and control the synthesis of nuclear DNA. The smaller viral chromosomes, which should be more vulnerable to genetic manipulation and analysis, would seem to offer a promising avenue of research, in parallel perhaps, to the extraordinary detail emerging from the studies of E. coli and its phages (74). The present lack of knowledge portends that our perception of the types and nature of eukaryotic DNA polymerases, as well as their roles, may change within the next few years.