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Nonhistone chromatin proteins from calf thymus and their role in DNA biosynthesis
ARCHIVES
OF
BIOCHEMISTRY
Nonhistone
AND
BIOPHYSICS
Chromatin
Depurlmenl,
Stale
Received
629-634
Proteins
Role
Biolq~/
122,
(1967)
from
Calf
in DNA
Biosynthesis’
TUNG-YUE
WANG
C’nivewit,y
May
oj 1Veu
20, 1967;
l’ork
accepted
al
Thymus
NuJdo,
Wuffuio,
Joule
23, 1967
and
.Veul
170~k
Their
14214
The nonhistone chromatin proteins have been isolated from calf thymes nuclei by dissociation of the OKA-protein complex. Chemical fractionation of these proteins separates them into seven fractions which are shown to be acidic by their amino acid compositions. One of t,he frlnctions of these chromatin acidic proteins is their participation in the biosynthesis of DNA. This has been shown by t)he partial prkfication of a DNA-polymerase from these chromatin acidic proteins. The partially ptlrified enzyme depends on a complete complement of all few deoxyribonllcleoside 5’-triphosuhates for its optimal activity in an in Q&O assay system. It preferentially cues heatdenattIred DIVA as the template.
In the mammalian chromosomes, DKA and histones represent the majority of the macromolecular constituents. Together with the nonhistone prot’eins, they constitute nearly the whole organized genetic framework. Although DKA and histones have been extensively studied, only passing interest has been shown to t,he nonhistone proteins in relation to chromosomal structure and gene function. The first demonstration of the chromosomal nonhistone proteins was reported by Mirsky and Pollister (l), who obtained an insoluble prot,ein fraction front 1 M NaCl extract of isolated cell nuclei and suggested that it represents the morphological backbone of chromosomes. When the 1 nI NaCl nuclear ext’ract is diluted to 0.14 M with respect to NaCl, D?;A and histones reassociate and precipitate, and the IWIIhistone chromatin proteins are released from the DNA-protein complex. The nonhist,one chromatin proteins have been isolated from rat liver (2) with t,his method, and have been shown to be acidic and to contain phosphoproteins, RNA, and some DXA. The rat
liver preparation is nlet)abolically active, forms complexes with histones and with DKA, and exhibit)s molecular het,erogeneit)y (2, a. We have reported previously (4) that in calf t’hymus, the soluble fraction of the 1 nr KaCl nuclear extract aft’er precipitation of the D,I’A-histones contains significant DXApolymerase activity. P’rom the point of view of DNA replication, the close association of enzymes involved in the replicative process with tJhe DNA primer is a logical expectation. In the light of these considerations, investigations were undert’aken to study the nonhistone chromat’in proteins from calf thymus with respect to t)heir heterogeneity and DKA-synt’hesizing activity. This report presents a procedure for the fractionation of the calf t,hymus nonhistone chromatin proteins. The fractionation is assessed by the differences in the amino acid composition of the various fractions. Furthermore, in order to show the relationship of the nonhistone proteins with gene activity, the partial purification of a DKA-polymerase (DNAnucleotidyltransferase) from the chromatin acidic proteins is described:
1 This invest,igation was supported by grants from the American Cancer Society (E-444) and ihe U.S. Public Health Service (GM-11698-05). 629
MATERIALS
AND
METHODS
Preparation of the nonhistone chromatin proteins jrom isolated calf thymus nuclei. Thymus glands from 3- to 8week-old calves were obtained from a local packing house immediately after the kill and were shipped to the laboratory in packed ice. All operations were performed in a cold room at 4”. The glands were briefly rinsed with cold physiological saline and trimmed free from fat and connective tissue. Cell nuclei were isolated from the thymus gland according to the procedure of Allfrey et al. (5). The isolated cell nuclei were repeatedly extracted with 0.14 M NaCl-0.05 M TrisHCl, pH 7.5, by blending in a Lourdes omnimixer at 50 V for 3 minutes, followed by stirring in the cold for 1 hour and centrifuging to remove the soluble extract. The extracted nuclei were cut into small pieces and homogenized in 1 M NaCl with a loose-fitting Dounce homogenizer (6). More 1 M NaCl was added to about 1 liter/50 gm of the original tissue weight. The suspension was stirred slowly overnight, and then centrifuged at 20,000 rpm in a Spinco 20 rotor for 1 hour. The clear sttpernatant, solution from t,his centrifugation was carefully collected and dialyzed against 6 volumes of cold distilled water for 8 hours. After dialysis, the suspension was centrifuged and the supernatant fluid was saved. The precipitate, which is mostly re-associated DNA-histones, was reextracted with 1 M NaCl, and the extract was again dialyzed against 6 volumes of water and centrifuged, and the supernatant solmion was collected. Supernatant, solutions from both precipitations were combined and concentrated by ultrafiltration to about 5 mg prcJtein/d. This solution represents the total nonhistone chromatin acidic proteins used for the studies reported here. Fractionation of the nonhistone chromatin acidic proteins. The fractionation of the calf thymus chromatin acidic proteins was carried out as described previously (2). The total nonhistone chromat,in proteins were centrifuged at 105,000 y in a Spinco 40 rotor for 1 hour. Pellets obtained from this centrifugat.ion were collected and dispersed carefully in 0.05 M Tris-HCl, pH 8.5. This fraction is referred t,o here as the pellet fraction. The supernatant fluid was acidified with 1 N acetic acid to pH 5.9, and yielded a pH 6 percipitate. The supernatant fluid from the pH 6 precipitation was further acidified to pH 4.8, and yielded a pH 5 precipitate. Finally, the pH 5 supernatant fraction was made to 80% saturation with respect to ammonium sulfate by slow addition of 51.6 gm of the ammonium sulfate solid to each 100 ml of protein solut,ion. The mixture was allowed to stand for 10 minutes, and the precipitate was collected by centrifugation. This ammonium sulfate
precipitate is designated as t,he ammonium sulfate fraction. For further fractionation of the ammonium sulfate fraction, the original pH 5 supernatant fraction was used. The pH 5 supernatant solution was salted-out srtccessively with 30, 40, 50, and 6080% saturated ammonium sulfate; each precipitate was collected by centrifugation, and an appropriate amount of ammonium sulfate was added to the supernat,ant fluid. Four fractions were thus obtained from the pH 5 supernatant fraction, and these are referred to as the ammonium sulfate subfractions. All fractions were dialyzed with 0.05 M Tris-HCl, pH 8.5, before use. Partial puriJication of DxA-polymerase from the pH 6 fraction. The pH 6 fraction was thoroughly dialyzed overnight against 0.005 M Tris-phosphate buffer, pH 8.0, containing 0.001 M 2-mercaptoethanol. The dialyzed pH 6 fraction was absorbed onto a 2.9 X 28 cm DEAE-cellulose (DE-32, Reeve Angel) column which had been equilibrated previously with the same buffer. The DEAE-column with its absorbed proteins was first washed with the buffer and then eluted with buffered 0.14 M NaCl. To the total 0.14 M NaCl eluate solid ammonium sulfate was added to a 40yc saturation and the precipitate was discarded. More ammonium sulfate was added to the solution to a final 607, saturation of ammonium sulfate. After standing in the cold for 10 minutes, the precipitate was collected by centrifugation and dissolved in and dialyzed against 0.02 M Tris-HCl, pH 8.0, containing 0.001 M 2-mercaptoethanol. The protein concent,ration was usually adjusted to about 5 mg/ml for use. DNA-polymerase assay. The assay of DNApolymerase was performed essentially according to Mantsavinos (7). The assay mixture contained, in a total volume of 0.5 ml, the following: 2Opmoles of glycine buffer, pH 8.0; 0.5 pmole of 2-mercaptoethanol; 5 rmoles of MgClz; 20 ,ug of enzyme; 100 fig of calf thymus DNA (heat-denatured); 40 mpmoles each of dATP, dCTP, and dGTP (Sigma Chemical Co.); and 16 mpmoles of 3H-dTTP (Schwartz Bioresearch, Inc.). The mixture was incubated at 37” for 30 minutes. Bt’ the end of the incubation, the mixture was chilled in ice-water, and after adding 0.2 mg of carrier bovine serum albumin, was precipitated with cold 5y0 trichloroacetic acid. The suspension was filtered through a Millipore filter (0.45,~ pore size), washed with five portions of 5 ml each of 5y0 trichloroacetic acid, t)aken up in 15 ml of Bray scintillation fluid (8), and counted in a Packard liquid scintillation spectrometer. Amino acid analyses. The protein sample to be analyzed was extracted twice with 5% trichloroacetic acid at 90” for 30 minutes. The nucleic
NONHISTONE
CHR.OMATIN
acid-free protein residue was then washed twice each with ethanol-ether (1:2), and ether, dried in air, and stored desiccated. A weighed sample was hydrolyzed iu constant, boiling HCl under reduced pressure at 110” for 18 hours. After hydrolysis, the hydrolyzate was evaporated of HCl, and desiccated over NaOH pellets. ,4 Spinco model 120C alltomatic amino acid analyzer was used for the analysis of amino acids (9). Other methods. Protein concentration was determined either by the Folk procedllre (lo), or, when P-mercaptoet,hanol was present, estimated from absorbancies at 280 rnr and 260 rnp (II). Ribonucleic acid and DNA were det,ermined by orcinol reaction (12) and Burton’s method (13), respectively. Calf thymus DNA was prepared according to Thomas et al. (14). Heat-denaturation of DNA was achieved by heating DNA in boiling water for 10 minutes and rapidly cooling it in ice-water. RESULTS
Composition of the nonhistone chromatin proteins. The total nonhist,one chromatin prot’eins have on the average 94.2 % protein, 3.2% DNA, and 2.6% RNA. Table I shows the diskibution of protein and nucleic acids among t’he fractions. The largest fractions are the pH 6 and the ammonium sulfate fractions, which account for about threefourths of the total nonhistone chromatin proteins. It’ is also seen that all four fractions contain some RKA and DNA. Amino acid compositions of the nonhistone proteins are shown in Tables II and III. Except for the pellet fract’ion, which has only a slightly higher acidic than basic amino acid residues, all other three fractions are TABLE
I
DISTRIBUTION AND I)NA-POLYMERSSE OF THE NONHIST~NE CHROMATIN PROTEIN FRXTIONS
ACTIVITY
Fractions
-____ Pellet fraction PH 6 PH 5 A.8. fractions S mpmoles 30 minutes.
1.2
13.0
30.7
37.1
0.74
4.0 1.3 2.7
43.4 14.5 29.1
28.7 23.1 17.9
24.3 22.8 15.8
0.55 0.17 0.07
3H-dTTP
incorporated/mg
enzyme/
631
PROTEINS TABLE
II
AMINO ARID COMPOSITION OF CHHOM~TIN ACIDK PROTEINSOFC.~LFTHYMUS(MOLESAMINOACIDS PER 100 MOLES AMINO ACIDS I~ECOVERED) Amino
acid
I RRNP
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine !$-Cystine Valine Methionine Isoleucine Lerlcine Tyrosine Phenylaline (Asp + Glu) (Lys + His + Arg)
6
PH 5
AS. raction
7.75 2.52 8.07 9.25 5.47 6.12 10.7 3.86 7.13 8.35 0.48 6.09 2.29 4.80 9.57 3.82 4.11
7.30 2.34 6.24 9.02 4.81 5.40 13.2 4.88 G.60 7.30 0.88 6.29 4.00 4.93 9.91 3.14 3.80
6.89 2.00 5.66 9.78 4.60 5.36 15.7 4.48 6.60 9.19 0.47 5.95 3.42 4.36 9.31 2.65 3.59
6.94 1.74 4.25 9.78 5.76 5.79 14.4 6.38 7.65 7.65 0.54 6.39 3.74 4.70 8.23 2.20 3.66
1.09
1.40
1.75
1.87
TABLE AMIXO
PH
III
ACID COMPOSITION OF AMMONIUM SULFATE SUBFR.~WIONS FROM THE CHROM~TIN ACIDIC PROTEINS Amino
acid
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine >$-Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine (Asp + Glu) (Lys + IIis + Arg)
-
AS-30
AS-40
AS-50
i1%60-80
4.64 1.67 4.06 8.92 5.71 5.70 14.0 8.84 8.06 8.36 0.44 6.38 4.30 4.60 8.60 2.15 3.51
6.63 2.01 4.71 10.1 5.20 5.54 14.3 6.50 7.69 7.55 0.67 6.63 3.67 4.82 8.19 2.23 3.49
8.11 1.76 3.78 10.4 5.42 6.06 14.7 5.11 7.69 7.15 0.50 6.30 3.95 4.84 7.98 2.34 3.96
10.1 1.47 5.38 9.71 8.13 5.48 14.7 3.81 6.58 7.74 0.72 G.33 1.87 4.42 8.19 1.87 3.44
2.21
1.83
1.85
1.44
-
632
WANG TABLE
PURIFICATION CHROM~TIN
OF
IV
DNA-POLYMERASE ACIDIC
CALF
PHOTEINS
OF
THYMUS Total protein
Fraction
FROM
b%)
Total activitya
Specific
activity
(mf.anoles/ w)
1. Total chromatin acidic proteins
800
102.0
0.13
2. pH 6 3. DEAE
314 40
172.7 68.6
0.55 1.51
4. Ammonium
sulfate
8.8
25.9
2.94
a mrmoles of 3H-dTTP incorporated/30 minutes lmder conditions as described in Mat,erials and Methods.
characterized by high glutamic acid content. In general, amino acid analyses show differences among the four chromatin acidic protein fractions mainly by their relative acidic and basic amino acid contents. Amino acid analyses of the ammonium sulfate subfractions show that, they are also distinctly different in acidic to basic amino acids ratios as well as in other amino acids. Notable differences are manifested by their lysine and arginine contents. This indicates that the differences in acidic character is due to variances in basic amino acids. Other differences in amino acid residues are observed in their proline, glycine, and methionine contents. Properties of the partially purijied DNApolymeruse. Of the four nonhistone acidic protein fractions, only the pellet and the pH 6 fractions show substantial DNA-polymerase activity (Table I). While the pellet fraction activity,
has a higher its total activity
specific is only
enzymic
25% that of the pH 6 fraction. When the pH 6 fraction is fractionated 011 DEAE-cellulose, most of the DNA-polymerase activity is eluted between 0.01 and 0.14 M NaCI. The DEAE-purification step gives a threefold increase in enzymic activity. Precipitation of the DEAE-fraction with 40-60% ammonium sulfate results in an overall 22-fold purification of the enzyme from the whole nonhistone proteins. The partial purification steps of the DNA-polymerase are tabulated in Table IV. Table V summarizes some of the charac-
teristics of the partially purified DNApolymerase. The calf thymus preparation is dependent on the presence of all four deoxyribonucleoside triphosphates for optimal activity. It preferentially uses heatdenatured DNA as the template. The incorporation of 3H-dTTP into DNA in the presence of all four deoxynucleoside triphosphates is inhibited by actinomycin D and pyrophosphate. The presence of a thiol appears to be essential, for in the absenceof 2mercaptoethano1, incorporation of 3HdTTP is reduced to 50%. DISCUSSION
The amino acid composition of the calf thymus chromatin acidic protein fractions in general showsa close similarity to that of the corresponding rat liver fractions (2) with the exception of the pellet fraction. The calf thymus pellet fraction has a higher arginine content (8%) and lower glutamic acid content than the other three fractions and the rat liver fractions. The pellet fraction also shows relatively lower proline and methionine content. Since the pellet fraction was obtained by 105,000 g centrifugation, any histone contamination would be likely in this fraction. The difference in relative amounts of arginine and glutamic acid in the calf thymus pellet fract’ion and its comparatively high DiYA content suggest that, if histone is the contaminant, it perhaps exists as the arginine-rich histone in association with DNA. TABLE
V
OF 3H-dTTP INTO DNA BY CALF CHEOM~TIN DNA-POLYMERASE’
INCORPORATION THYMUS
m~moles 3H-dTTP
System
1. Complete
incorporated
system
0.30 0.010 0.094 0.16 0.27 0.070 0.022 0.38 0.14
-DNA -dATP, -dCTP, -dGTP -2-mercaptoethanol 2. Complete system +50 pg actinomycin D
+15 rmoles Na-pyrophosphate 3. Complete -DNA,
+
system native
a In all three enzyme (Fraction tions was used.
DNA
experiments, 4) from three
about different
100 pg of prepara-
NONHISTONE
CFIROMATIN
Among the four nlajor chromat#in acidic protein fractions, the ammonium sulfate fraction has the highest proportion of acidic amino acids relative t’o basic amino acids, indicating strong acidity. Differential salting-out of this ammonium sulfate fraction into subfractions shows that the nlain difference among these subfractions resides in t*heir variance in lysine content, ranging from 4.64 to 10.1 moles per cent, and to a lesser extent in proline, glycine, and methionine. The amount of glutamic acid among these subfractions remains practically COINstant. In spite of the present preliminary fractionation procedure, the differences in amino acid compositions among t,he many fractions demonstrate their distinction as well as t,heir heterogeneity. An early sedimentation st’udy of t,he calf thymus chromatin a,cidic prot,eins (15) has indeed shown a polydisperse pattern similar to that of the dilute saline nuclear ext’ract. These observations t,hus indicate the complex nature and the high heterogeneity of the chromatin acidic proteins. Calf t hynlus DSA-polymerase has generally been prepared from soluble extract of tissue (16) or nuclei (17). A preliminary study on the intranuclear fractions of calf thpmus, hwever, also suggests a chromosomal origin for this enzyme (4). Mazia and Hinegardner (18) have correlated DNApolymerase activity of sea urchin embryos with t,he number of chromosomal sets. Since t,he acidic proteins can only be obtained by dissociation of the DNA-protein complex, the partial purification of a D?JApolymerase from the chromatin acidic proteins thus demonstrates the chromosomal location of this enzyme. The association of DSA-polymerase with chromatin is logical, for D1\JA is almost exclusively present in the chromat,in, a nat,ural sit’e for DNA svnthesis. - The chromnt,in DNA-polymerase of calf t,hymus described here does not exhibit an absolute dependence on the complete supplement of all four deoxyribonucleoside triphosphates. In the absence of dATP, dCTP, and dGTP, incorporation of 3HdTTP int)o DSA is about 30 5%of that in the presence of the other three unlabeled
633
PROTEINS
deoxyribonucleosidc triphosphates. Addition of actinomycin D inhibits 3H-dTTP incorporation to about t)he same level. These results suggest that, most of the enzymic act(ivit,y of the calf t8hymus chromatin DKA-polymerase is of the replicative type, which uses DNA as template. DXAnucleot~idyltransferase catalyzing the t,erminal addition of single deoxyribonucleoside triphosphate has been isolated from calf t’hymus by Krakow et al. (19), Yoneda and Bollum (20), and Keir and Snmh (21). The lack of absolute dependence on a complete supplement’ of four deoxynucleoside triphosphates of t’he chromatin enzyme perhaps indicat’es the presence in the chromatin preparat~ion of two such enzymes possessing different, specificities. Like the calf t,hymus soluble DNApolymerase (22), t,he chromat,in enzyme of calf t,hymus uses heat-denatured DNA as t,hc template. Similar template preference has also been shown with DNA-polymerase prepared from Landshutz ascites-tumor (23). However, DKA-polymerases purified from rat liver soluble prot,eins (7) and chromatin acidic: proteins (24) exhibit a preferential use of na.tive DXA as the template. Thus, t,he specificity of template requirement’ in DNA synthesis appears to be related t,o specific: t#issue or cells rat,her than to the intracellular fract,ions. ACKNOWLEDGMENT The author wishes to thank Mrs. I<. Bogdan for her technical assistance in this work. REFERENCES A. I&, .iNI) POLIJSTER, A. W., J. Gem Physiol. 30, 117 (1946). 2. WANG, T. Y., J. Biol. Chem., 242, 1220 (1967). 3. WaNG, T. Y. in “The Cell Nllclells: Metabolism and I~adioserlsitivit.4,” p. 243. Taylor and Francis, Ltd., l,ondon (1966). 4. Waxc, T. Y., .txu P.\‘L’EI,, (+., I,ije Sri. 4, 1893 (lYG5). 5. ALLFREY, V. G., MIILHKY, A. E., .AND Oxawa, S., J. Gen. Ph.!ysioZ. 40, 451 (1957). 6. UOUXCE, A. L., WITTER, lL F.,MoNTY,K. J., PhT'E, s., AND COTTOXE, M. A., J. Biophys. Bioch,em. Cytol. 1, 139 (1955). 7. MANTUVINOS. R.., 1. Viol. Chem. 239, 3431 (1964). 8. BUY, A. A., Anal. Hiochem. 1, 279 (1960). 1. MIRSKY,
634
WANG
9. MOORE, s., SPACKMAN, D. H., AND STEIN, W. H., Anal. Chem. 30, 1185 (1958). 10. LOIVRY, 0. IS., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, 13. J., J. Biol. Chem. 193, 265 (1951). 11. LAYNE, E., Methods Enzymol. 3, 457 (1957). 12. LUSEN.~, C. V., Can. J. Biochem. Physiol. 29, 107 (1951). 13. BURTON, K., Biochem. J. 62, 315 (1956). 14. THOMAS, C. A., JR., BERNS, K. I., AND KELLY, T. J., JR., in “Procedures in Nucleic Acid Research” (G. L. Cantoni and It. Davies, eds.), p. 535. Harper, New York (1966). 15. WANG, T. Y., Biochim. Biophys. Acta 49, 239 (1961). 16. BOLLUM, F. J., J. Biol. Chem. 236, 2399 (1960).
17. SMITH, M. J., AND KIER, H. M., Biochim. Biophys. dcta 68, 578 (1903). 18. MAZIA, D., AND HINEGARDNER, R. T., Proc. ‘l;d. ;Icad. sci. u. s. 60, 148 (1963). 19. KRIKOJV, J., COUTSOGEORGOPOULOS, C., AND CANELLAKIS, E. H., Biochim. Biophys. dcta 66, G39 (1962). 20. YOXEDA, M., AND BOLLUM, F. J., J. Biol. Chem. 240, 3385 (1965). 21. HEIR, H. M., AND SMITH, M. J., Biochim. Biophys. Acta 68, 589 (1963). 22. BOLLUM, F. J., J. Biol. Chem. 234, 2733 (1959). 23. KEIR, H. M., BINNIE, B., AND SMELLIE, R. M. S., Biochem. J. 82, 493 (1962). 24. P.&TEL, C., HOWK, It., AND WANG, T. Y., Nature, in press.
OF
BIOCHEMISTRY
Nonhistone
AND
BIOPHYSICS
Chromatin
Depurlmenl,
Stale
Received
629-634
Proteins
Role
Biolq~/
122,
(1967)
from
Calf
in DNA
Biosynthesis’
TUNG-YUE
WANG
C’nivewit,y
May
oj 1Veu
20, 1967;
l’ork
accepted
al
Thymus
NuJdo,
Wuffuio,
Joule
23, 1967
and
.Veul
170~k
Their
14214
The nonhistone chromatin proteins have been isolated from calf thymes nuclei by dissociation of the OKA-protein complex. Chemical fractionation of these proteins separates them into seven fractions which are shown to be acidic by their amino acid compositions. One of t,he frlnctions of these chromatin acidic proteins is their participation in the biosynthesis of DNA. This has been shown by t)he partial prkfication of a DNA-polymerase from these chromatin acidic proteins. The partially ptlrified enzyme depends on a complete complement of all few deoxyribonllcleoside 5’-triphosuhates for its optimal activity in an in Q&O assay system. It preferentially cues heatdenattIred DIVA as the template.
In the mammalian chromosomes, DKA and histones represent the majority of the macromolecular constituents. Together with the nonhistone prot’eins, they constitute nearly the whole organized genetic framework. Although DKA and histones have been extensively studied, only passing interest has been shown to t,he nonhistone proteins in relation to chromosomal structure and gene function. The first demonstration of the chromosomal nonhistone proteins was reported by Mirsky and Pollister (l), who obtained an insoluble prot,ein fraction front 1 M NaCl extract of isolated cell nuclei and suggested that it represents the morphological backbone of chromosomes. When the 1 nI NaCl nuclear ext’ract is diluted to 0.14 M with respect to NaCl, D?;A and histones reassociate and precipitate, and the IWIIhistone chromatin proteins are released from the DNA-protein complex. The nonhist,one chromatin proteins have been isolated from rat liver (2) with t,his method, and have been shown to be acidic and to contain phosphoproteins, RNA, and some DXA. The rat
liver preparation is nlet)abolically active, forms complexes with histones and with DKA, and exhibit)s molecular het,erogeneit)y (2, a. We have reported previously (4) that in calf t’hymus, the soluble fraction of the 1 nr KaCl nuclear extract aft’er precipitation of the D,I’A-histones contains significant DXApolymerase activity. P’rom the point of view of DNA replication, the close association of enzymes involved in the replicative process with tJhe DNA primer is a logical expectation. In the light of these considerations, investigations were undert’aken to study the nonhistone chromat’in proteins from calf thymus with respect to t)heir heterogeneity and DKA-synt’hesizing activity. This report presents a procedure for the fractionation of the calf t,hymus nonhistone chromatin proteins. The fractionation is assessed by the differences in the amino acid composition of the various fractions. Furthermore, in order to show the relationship of the nonhistone proteins with gene activity, the partial purification of a DKA-polymerase (DNAnucleotidyltransferase) from the chromatin acidic proteins is described:
1 This invest,igation was supported by grants from the American Cancer Society (E-444) and ihe U.S. Public Health Service (GM-11698-05). 629
MATERIALS
AND
METHODS
Preparation of the nonhistone chromatin proteins jrom isolated calf thymus nuclei. Thymus glands from 3- to 8week-old calves were obtained from a local packing house immediately after the kill and were shipped to the laboratory in packed ice. All operations were performed in a cold room at 4”. The glands were briefly rinsed with cold physiological saline and trimmed free from fat and connective tissue. Cell nuclei were isolated from the thymus gland according to the procedure of Allfrey et al. (5). The isolated cell nuclei were repeatedly extracted with 0.14 M NaCl-0.05 M TrisHCl, pH 7.5, by blending in a Lourdes omnimixer at 50 V for 3 minutes, followed by stirring in the cold for 1 hour and centrifuging to remove the soluble extract. The extracted nuclei were cut into small pieces and homogenized in 1 M NaCl with a loose-fitting Dounce homogenizer (6). More 1 M NaCl was added to about 1 liter/50 gm of the original tissue weight. The suspension was stirred slowly overnight, and then centrifuged at 20,000 rpm in a Spinco 20 rotor for 1 hour. The clear sttpernatant, solution from t,his centrifugation was carefully collected and dialyzed against 6 volumes of cold distilled water for 8 hours. After dialysis, the suspension was centrifuged and the supernatant fluid was saved. The precipitate, which is mostly re-associated DNA-histones, was reextracted with 1 M NaCl, and the extract was again dialyzed against 6 volumes of water and centrifuged, and the supernatant solmion was collected. Supernatant, solutions from both precipitations were combined and concentrated by ultrafiltration to about 5 mg prcJtein/d. This solution represents the total nonhistone chromatin acidic proteins used for the studies reported here. Fractionation of the nonhistone chromatin acidic proteins. The fractionation of the calf thymus chromatin acidic proteins was carried out as described previously (2). The total nonhistone chromat,in proteins were centrifuged at 105,000 y in a Spinco 40 rotor for 1 hour. Pellets obtained from this centrifugat.ion were collected and dispersed carefully in 0.05 M Tris-HCl, pH 8.5. This fraction is referred t,o here as the pellet fraction. The supernatant fluid was acidified with 1 N acetic acid to pH 5.9, and yielded a pH 6 percipitate. The supernatant fluid from the pH 6 precipitation was further acidified to pH 4.8, and yielded a pH 5 precipitate. Finally, the pH 5 supernatant fraction was made to 80% saturation with respect to ammonium sulfate by slow addition of 51.6 gm of the ammonium sulfate solid to each 100 ml of protein solut,ion. The mixture was allowed to stand for 10 minutes, and the precipitate was collected by centrifugation. This ammonium sulfate
precipitate is designated as t,he ammonium sulfate fraction. For further fractionation of the ammonium sulfate fraction, the original pH 5 supernatant fraction was used. The pH 5 supernatant solution was salted-out srtccessively with 30, 40, 50, and 6080% saturated ammonium sulfate; each precipitate was collected by centrifugation, and an appropriate amount of ammonium sulfate was added to the supernat,ant fluid. Four fractions were thus obtained from the pH 5 supernatant fraction, and these are referred to as the ammonium sulfate subfractions. All fractions were dialyzed with 0.05 M Tris-HCl, pH 8.5, before use. Partial puriJication of DxA-polymerase from the pH 6 fraction. The pH 6 fraction was thoroughly dialyzed overnight against 0.005 M Tris-phosphate buffer, pH 8.0, containing 0.001 M 2-mercaptoethanol. The dialyzed pH 6 fraction was absorbed onto a 2.9 X 28 cm DEAE-cellulose (DE-32, Reeve Angel) column which had been equilibrated previously with the same buffer. The DEAE-column with its absorbed proteins was first washed with the buffer and then eluted with buffered 0.14 M NaCl. To the total 0.14 M NaCl eluate solid ammonium sulfate was added to a 40yc saturation and the precipitate was discarded. More ammonium sulfate was added to the solution to a final 607, saturation of ammonium sulfate. After standing in the cold for 10 minutes, the precipitate was collected by centrifugation and dissolved in and dialyzed against 0.02 M Tris-HCl, pH 8.0, containing 0.001 M 2-mercaptoethanol. The protein concent,ration was usually adjusted to about 5 mg/ml for use. DNA-polymerase assay. The assay of DNApolymerase was performed essentially according to Mantsavinos (7). The assay mixture contained, in a total volume of 0.5 ml, the following: 2Opmoles of glycine buffer, pH 8.0; 0.5 pmole of 2-mercaptoethanol; 5 rmoles of MgClz; 20 ,ug of enzyme; 100 fig of calf thymus DNA (heat-denatured); 40 mpmoles each of dATP, dCTP, and dGTP (Sigma Chemical Co.); and 16 mpmoles of 3H-dTTP (Schwartz Bioresearch, Inc.). The mixture was incubated at 37” for 30 minutes. Bt’ the end of the incubation, the mixture was chilled in ice-water, and after adding 0.2 mg of carrier bovine serum albumin, was precipitated with cold 5y0 trichloroacetic acid. The suspension was filtered through a Millipore filter (0.45,~ pore size), washed with five portions of 5 ml each of 5y0 trichloroacetic acid, t)aken up in 15 ml of Bray scintillation fluid (8), and counted in a Packard liquid scintillation spectrometer. Amino acid analyses. The protein sample to be analyzed was extracted twice with 5% trichloroacetic acid at 90” for 30 minutes. The nucleic
NONHISTONE
CHR.OMATIN
acid-free protein residue was then washed twice each with ethanol-ether (1:2), and ether, dried in air, and stored desiccated. A weighed sample was hydrolyzed iu constant, boiling HCl under reduced pressure at 110” for 18 hours. After hydrolysis, the hydrolyzate was evaporated of HCl, and desiccated over NaOH pellets. ,4 Spinco model 120C alltomatic amino acid analyzer was used for the analysis of amino acids (9). Other methods. Protein concentration was determined either by the Folk procedllre (lo), or, when P-mercaptoet,hanol was present, estimated from absorbancies at 280 rnr and 260 rnp (II). Ribonucleic acid and DNA were det,ermined by orcinol reaction (12) and Burton’s method (13), respectively. Calf thymus DNA was prepared according to Thomas et al. (14). Heat-denaturation of DNA was achieved by heating DNA in boiling water for 10 minutes and rapidly cooling it in ice-water. RESULTS
Composition of the nonhistone chromatin proteins. The total nonhist,one chromatin prot’eins have on the average 94.2 % protein, 3.2% DNA, and 2.6% RNA. Table I shows the diskibution of protein and nucleic acids among t’he fractions. The largest fractions are the pH 6 and the ammonium sulfate fractions, which account for about threefourths of the total nonhistone chromatin proteins. It’ is also seen that all four fractions contain some RKA and DNA. Amino acid compositions of the nonhistone proteins are shown in Tables II and III. Except for the pellet fract’ion, which has only a slightly higher acidic than basic amino acid residues, all other three fractions are TABLE
I
DISTRIBUTION AND I)NA-POLYMERSSE OF THE NONHIST~NE CHROMATIN PROTEIN FRXTIONS
ACTIVITY
Fractions
-____ Pellet fraction PH 6 PH 5 A.8. fractions S mpmoles 30 minutes.
1.2
13.0
30.7
37.1
0.74
4.0 1.3 2.7
43.4 14.5 29.1
28.7 23.1 17.9
24.3 22.8 15.8
0.55 0.17 0.07
3H-dTTP
incorporated/mg
enzyme/
631
PROTEINS TABLE
II
AMINO ARID COMPOSITION OF CHHOM~TIN ACIDK PROTEINSOFC.~LFTHYMUS(MOLESAMINOACIDS PER 100 MOLES AMINO ACIDS I~ECOVERED) Amino
acid
I RRNP
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine !$-Cystine Valine Methionine Isoleucine Lerlcine Tyrosine Phenylaline (Asp + Glu) (Lys + His + Arg)
6
PH 5
AS. raction
7.75 2.52 8.07 9.25 5.47 6.12 10.7 3.86 7.13 8.35 0.48 6.09 2.29 4.80 9.57 3.82 4.11
7.30 2.34 6.24 9.02 4.81 5.40 13.2 4.88 G.60 7.30 0.88 6.29 4.00 4.93 9.91 3.14 3.80
6.89 2.00 5.66 9.78 4.60 5.36 15.7 4.48 6.60 9.19 0.47 5.95 3.42 4.36 9.31 2.65 3.59
6.94 1.74 4.25 9.78 5.76 5.79 14.4 6.38 7.65 7.65 0.54 6.39 3.74 4.70 8.23 2.20 3.66
1.09
1.40
1.75
1.87
TABLE AMIXO
PH
III
ACID COMPOSITION OF AMMONIUM SULFATE SUBFR.~WIONS FROM THE CHROM~TIN ACIDIC PROTEINS Amino
acid
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine >$-Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine (Asp + Glu) (Lys + IIis + Arg)
-
AS-30
AS-40
AS-50
i1%60-80
4.64 1.67 4.06 8.92 5.71 5.70 14.0 8.84 8.06 8.36 0.44 6.38 4.30 4.60 8.60 2.15 3.51
6.63 2.01 4.71 10.1 5.20 5.54 14.3 6.50 7.69 7.55 0.67 6.63 3.67 4.82 8.19 2.23 3.49
8.11 1.76 3.78 10.4 5.42 6.06 14.7 5.11 7.69 7.15 0.50 6.30 3.95 4.84 7.98 2.34 3.96
10.1 1.47 5.38 9.71 8.13 5.48 14.7 3.81 6.58 7.74 0.72 G.33 1.87 4.42 8.19 1.87 3.44
2.21
1.83
1.85
1.44
-
632
WANG TABLE
PURIFICATION CHROM~TIN
OF
IV
DNA-POLYMERASE ACIDIC
CALF
PHOTEINS
OF
THYMUS Total protein
Fraction
FROM
b%)
Total activitya
Specific
activity
(mf.anoles/ w)
1. Total chromatin acidic proteins
800
102.0
0.13
2. pH 6 3. DEAE
314 40
172.7 68.6
0.55 1.51
4. Ammonium
sulfate
8.8
25.9
2.94
a mrmoles of 3H-dTTP incorporated/30 minutes lmder conditions as described in Mat,erials and Methods.
characterized by high glutamic acid content. In general, amino acid analyses show differences among the four chromatin acidic protein fractions mainly by their relative acidic and basic amino acid contents. Amino acid analyses of the ammonium sulfate subfractions show that, they are also distinctly different in acidic to basic amino acids ratios as well as in other amino acids. Notable differences are manifested by their lysine and arginine contents. This indicates that the differences in acidic character is due to variances in basic amino acids. Other differences in amino acid residues are observed in their proline, glycine, and methionine contents. Properties of the partially purijied DNApolymeruse. Of the four nonhistone acidic protein fractions, only the pellet and the pH 6 fractions show substantial DNA-polymerase activity (Table I). While the pellet fraction activity,
has a higher its total activity
specific is only
enzymic
25% that of the pH 6 fraction. When the pH 6 fraction is fractionated 011 DEAE-cellulose, most of the DNA-polymerase activity is eluted between 0.01 and 0.14 M NaCI. The DEAE-purification step gives a threefold increase in enzymic activity. Precipitation of the DEAE-fraction with 40-60% ammonium sulfate results in an overall 22-fold purification of the enzyme from the whole nonhistone proteins. The partial purification steps of the DNA-polymerase are tabulated in Table IV. Table V summarizes some of the charac-
teristics of the partially purified DNApolymerase. The calf thymus preparation is dependent on the presence of all four deoxyribonucleoside triphosphates for optimal activity. It preferentially uses heatdenatured DNA as the template. The incorporation of 3H-dTTP into DNA in the presence of all four deoxynucleoside triphosphates is inhibited by actinomycin D and pyrophosphate. The presence of a thiol appears to be essential, for in the absenceof 2mercaptoethano1, incorporation of 3HdTTP is reduced to 50%. DISCUSSION
The amino acid composition of the calf thymus chromatin acidic protein fractions in general showsa close similarity to that of the corresponding rat liver fractions (2) with the exception of the pellet fraction. The calf thymus pellet fraction has a higher arginine content (8%) and lower glutamic acid content than the other three fractions and the rat liver fractions. The pellet fraction also shows relatively lower proline and methionine content. Since the pellet fraction was obtained by 105,000 g centrifugation, any histone contamination would be likely in this fraction. The difference in relative amounts of arginine and glutamic acid in the calf thymus pellet fract’ion and its comparatively high DiYA content suggest that, if histone is the contaminant, it perhaps exists as the arginine-rich histone in association with DNA. TABLE
V
OF 3H-dTTP INTO DNA BY CALF CHEOM~TIN DNA-POLYMERASE’
INCORPORATION THYMUS
m~moles 3H-dTTP
System
1. Complete
incorporated
system
0.30 0.010 0.094 0.16 0.27 0.070 0.022 0.38 0.14
-DNA -dATP, -dCTP, -dGTP -2-mercaptoethanol 2. Complete system +50 pg actinomycin D
+15 rmoles Na-pyrophosphate 3. Complete -DNA,
+
system native
a In all three enzyme (Fraction tions was used.
DNA
experiments, 4) from three
about different
100 pg of prepara-
NONHISTONE
CFIROMATIN
Among the four nlajor chromat#in acidic protein fractions, the ammonium sulfate fraction has the highest proportion of acidic amino acids relative t’o basic amino acids, indicating strong acidity. Differential salting-out of this ammonium sulfate fraction into subfractions shows that the nlain difference among these subfractions resides in t*heir variance in lysine content, ranging from 4.64 to 10.1 moles per cent, and to a lesser extent in proline, glycine, and methionine. The amount of glutamic acid among these subfractions remains practically COINstant. In spite of the present preliminary fractionation procedure, the differences in amino acid compositions among t,he many fractions demonstrate their distinction as well as t,heir heterogeneity. An early sedimentation st’udy of t,he calf thymus chromatin a,cidic prot,eins (15) has indeed shown a polydisperse pattern similar to that of the dilute saline nuclear ext’ract. These observations t,hus indicate the complex nature and the high heterogeneity of the chromatin acidic proteins. Calf t hynlus DSA-polymerase has generally been prepared from soluble extract of tissue (16) or nuclei (17). A preliminary study on the intranuclear fractions of calf thpmus, hwever, also suggests a chromosomal origin for this enzyme (4). Mazia and Hinegardner (18) have correlated DNApolymerase activity of sea urchin embryos with t,he number of chromosomal sets. Since t,he acidic proteins can only be obtained by dissociation of the DNA-protein complex, the partial purification of a D?JApolymerase from the chromatin acidic proteins thus demonstrates the chromosomal location of this enzyme. The association of DSA-polymerase with chromatin is logical, for D1\JA is almost exclusively present in the chromat,in, a nat,ural sit’e for DNA svnthesis. - The chromnt,in DNA-polymerase of calf t,hymus described here does not exhibit an absolute dependence on the complete supplement of all four deoxyribonucleoside triphosphates. In the absence of dATP, dCTP, and dGTP, incorporation of 3HdTTP int)o DSA is about 30 5%of that in the presence of the other three unlabeled
633
PROTEINS
deoxyribonucleosidc triphosphates. Addition of actinomycin D inhibits 3H-dTTP incorporation to about t)he same level. These results suggest that, most of the enzymic act(ivit,y of the calf t8hymus chromatin DKA-polymerase is of the replicative type, which uses DNA as template. DXAnucleot~idyltransferase catalyzing the t,erminal addition of single deoxyribonucleoside triphosphate has been isolated from calf t’hymus by Krakow et al. (19), Yoneda and Bollum (20), and Keir and Snmh (21). The lack of absolute dependence on a complete supplement’ of four deoxynucleoside triphosphates of t’he chromatin enzyme perhaps indicat’es the presence in the chromatin preparat~ion of two such enzymes possessing different, specificities. Like the calf t,hymus soluble DNApolymerase (22), t,he chromat,in enzyme of calf t,hymus uses heat-denatured DNA as t,hc template. Similar template preference has also been shown with DNA-polymerase prepared from Landshutz ascites-tumor (23). However, DKA-polymerases purified from rat liver soluble prot,eins (7) and chromatin acidic: proteins (24) exhibit a preferential use of na.tive DXA as the template. Thus, t,he specificity of template requirement’ in DNA synthesis appears to be related t,o specific: t#issue or cells rat,her than to the intracellular fract,ions. ACKNOWLEDGMENT The author wishes to thank Mrs. I<. Bogdan for her technical assistance in this work. REFERENCES A. I&, .iNI) POLIJSTER, A. W., J. Gem Physiol. 30, 117 (1946). 2. WANG, T. Y., J. Biol. Chem., 242, 1220 (1967). 3. WaNG, T. Y. in “The Cell Nllclells: Metabolism and I~adioserlsitivit.4,” p. 243. Taylor and Francis, Ltd., l,ondon (1966). 4. Waxc, T. Y., .txu P.\‘L’EI,, (+., I,ije Sri. 4, 1893 (lYG5). 5. ALLFREY, V. G., MIILHKY, A. E., .AND Oxawa, S., J. Gen. Ph.!ysioZ. 40, 451 (1957). 6. UOUXCE, A. L., WITTER, lL F.,MoNTY,K. J., PhT'E, s., AND COTTOXE, M. A., J. Biophys. Bioch,em. Cytol. 1, 139 (1955). 7. MANTUVINOS. R.., 1. Viol. Chem. 239, 3431 (1964). 8. BUY, A. A., Anal. Hiochem. 1, 279 (1960). 1. MIRSKY,
634
WANG
9. MOORE, s., SPACKMAN, D. H., AND STEIN, W. H., Anal. Chem. 30, 1185 (1958). 10. LOIVRY, 0. IS., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, 13. J., J. Biol. Chem. 193, 265 (1951). 11. LAYNE, E., Methods Enzymol. 3, 457 (1957). 12. LUSEN.~, C. V., Can. J. Biochem. Physiol. 29, 107 (1951). 13. BURTON, K., Biochem. J. 62, 315 (1956). 14. THOMAS, C. A., JR., BERNS, K. I., AND KELLY, T. J., JR., in “Procedures in Nucleic Acid Research” (G. L. Cantoni and It. Davies, eds.), p. 535. Harper, New York (1966). 15. WANG, T. Y., Biochim. Biophys. Acta 49, 239 (1961). 16. BOLLUM, F. J., J. Biol. Chem. 236, 2399 (1960).
17. SMITH, M. J., AND KIER, H. M., Biochim. Biophys. dcta 68, 578 (1903). 18. MAZIA, D., AND HINEGARDNER, R. T., Proc. ‘l;d. ;Icad. sci. u. s. 60, 148 (1963). 19. KRIKOJV, J., COUTSOGEORGOPOULOS, C., AND CANELLAKIS, E. H., Biochim. Biophys. dcta 66, G39 (1962). 20. YOXEDA, M., AND BOLLUM, F. J., J. Biol. Chem. 240, 3385 (1965). 21. HEIR, H. M., AND SMITH, M. J., Biochim. Biophys. Acta 68, 589 (1963). 22. BOLLUM, F. J., J. Biol. Chem. 234, 2733 (1959). 23. KEIR, H. M., BINNIE, B., AND SMELLIE, R. M. S., Biochem. J. 82, 493 (1962). 24. P.&TEL, C., HOWK, It., AND WANG, T. Y., Nature, in press.