127
Biochimica et Biophysiea Acta, 1087 (1990) 127-136 Elsevier BBAEXP 92163
Evidence for D N A binding activity of numatrin (B23), a cell cycle-regulated nuclear matrix protein Nili Feuerstein 1, James J. Mond 2, Paul R. Kinchington 3 Robert Hickey 4 Marja-Liisa Karjalainen Lindsberg 2, Ian Hay 3 and William T. Ruyechan 3 Department of Biochemistry, University of Pennsylvania, Philadelphia, PA, Departments of e Medicine and 3 Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, MD and 4 Department of Pharmacology and Experimental Therapeutics, University of Maryland, School of Medicine, College Park, MD (U.S.A.) (Received 2 February 1990) (Revised manuscript received 30 May 1990)
Key words: DNA binding activity; Numatrin; Nuclear matrix protein
Stimulation of various cell types with growth factors is associated with a rapid induction in the synthesis of a nuclear matrix protein, termed 'numatrin' which was shown to be identical to the nucleolar protein B23. The abundance of numatrin was shown to be correlated with entry and progression through the S-phase. Thus, experiments were undertaken to examine whether numatrin also has DNA binding activity. Using whole nuclear extract, we showed that numatrin binds to both double-stranded (DS) DNA and to single-stranded (SS) DNA cellulose columns. Purified numatrin, which was extracted either under native conditions (in oligomeric form) or under urea conditions (in monomeric form), demonstrated significant binding to either [3H]DS-DNA or |3HIDS-DNA as shown by nitrocellulose filter binding assay. However, numatrin binding to D S - D N A was qualitatively and quantitatively different from its binding to SS-DNA. Thus, the binding of numatrin was several fold higher to D S - D N A as compared to SS-DNA. The binding to D S - D N A was reduced by 77% in the presence of 0.5 M NaCI, while the binding to S S - D N A was not affected under this condition. Furthermore, treatment of the native numatrin under conditions which caused monomerization of the protein resulted in a significant enhancement of numatrin binding to S S - D N A but did not affect the binding to DS-DNA. Following beparin-Sepharose chromatography purification (under native conditions), numatrin at picomole amounts showed significant binding to both D S - D N A and SS-DNA. Finally, numatrin was found to copurify with the complex of DNA polymerase a primase together with other proteins required for SV-40 in vitro replication activity. These results demonstrate that numatrin has DNA binding activity, and imply a possible role for numatrin/B23 in DNA-associated processes.
Introduction
Several nuclear proteins including cyclin/PCNA [1], and proteins encoded by proto-oncogenes such as c-myc, c-fos (for review see Ref. 2), and p53 [3], have been implicated in processes associated with regulation of cellular growth. We have described and characterized another growth regulated nuclear protein (40 k D a / p I 5) which is tightly associated with the nuclear matrix and was termed 'numatrin' [4-6]. We showed that Abbreviations: SS-DNA, single-stranded DNA; DS-DNA, doublestranded DNA; PCNA, proliferating cell nuclear antigen; BSA, bovine serum albumin; FCS, fetal calf serum; PMSF, phenylmethylsufonyl fluoride; DTT, dithiothreitol. Correspondence: N. Feuerstein, The Diabetes Research Center, Room 510, Medical Education Building, University of Pennsylvania, 36 and Hamilton Walk, Philadelphia, PA 19104-6015, U.S.A.
stimulation of B lymphocytes with various mitogens is associated with a rapid stimulation in the synthesis of numatrin [4,5]. This event was shown to be closely correlated with cellular commitment for mitogenesis and not with other parameters of cellular activation in B lymphocytes [5]. Induction of numatrin synthesis was also demonstrated in T lymphocytes stimulated by antiT cell receptor antibody and by concanavalin A as well as in murine Swiss 3T3 fibroblasts stimulated by various growth factors such as insulin and epidermal growth factor [6]. Thus, it was suggested that an increase in the expression of numatrin is a common nuclear event which is closely correlated with cellular induction of mitogenesis in various cell types. Further studies demonstrated elevated synthesis and abundance of numatrin in various malignant cells [4,6], indicating that numatrin might be involved in regulation of cell growth in normal and malignant cells.
0167-4781/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
128 In a recent study we provided evidence to indicate that numatrin is identical to the nucleolar protein B23 [7]. This conclusion was based on: (a) identical migration of numatrin and B23 on two-dimensional gel dectrophoresis; (b) identical tryptic peptide map; and (c) cross-reactivity of antibodies to numatrin and to B23. Protein B23 has been degcribed in 1974 as a nucleolar phosphoprotein [8,9]. Its phosphorylated site has been sequenced and was found to be similar to that of C23 and R-II subunit of cAMP protein kinase [10]. It was found that under native conditions protein B23 exists as an hexamer, composed of 4~ and 2fl monomers [11]. c D N A encoding the human [12] and the rat [13] protein B23 has been recently sequenced and found to have 50% homology with the Xenopus nucleoplasmin [12] and 63% homology to the Xenopus laeuis protein No38 [13]. Protein B23 has been shown to be localized in the granular region of the nucleolus where pre-ribosomal R N A is being assembled [14] and to be associated with pre-ribosomal R N P particles [15]. This evidence led to the hypothesis that protein B23 might be involved in the processing and assembling of pre-ribosomal RNA. However, protein B23 has been shown to translocate from the nucleolus to the nucleoplasm when R N A synthesis is inhibited [15], suggesting that its nucleolar localization is dependent on its association with RNA. Furthermore, only 40% of protein B23 was shown to be associated with R N P particles (80s and 55s) [15], while the nature of the interaction of the other 60% of the protein with other nuclear elements is still not known. In this regard, it is of interest that kinetic studies which examined the accumulation of numatrin during the cell cycle in mitogen-stimulated lymphocytes, demonstrated that the amount of numatrin in the cells reached a peak just prior to the entry of the cells into S-phase, it remained elevated during the S-phase and declined to control levels at the end of the S-phase [6]. This indicates that the abundance of numatrin in the cells is closely correlated with entry and progression through S-phase, namely, with the phase associated with D N A synthesis rather than with R N A synthesis. This, taken collectively with the evidence of numatrin translocation out of the nucleolus when R N A synthesis is inhibited, suggests that numatrin might be involved in different processes during different phases of the cell cycle. To address this possibility, experiments were undertaken to examine whether numatrin can also bind or interact with DNA. Materials and Methods
DNA cellulose chromatography Nuclei were purified from the human promyelocytic leukemic cells HL-60 [18] as previously described [5] and extracted in 2 M NaC1, 40 mM Tris (pH 8.2), 2 m M EDTA and 2 mM /~-mercaptoethanol at 4°C. After 30
min incubation on ice, the samples were centrifuged at 100000 x g for 60 n'fin. The supernatant was removed and dialyzed overnight at 4°C against buffer A (50 ruM NaC1, 20 m M Tris (pH 8.2), 1 mM EDTA, 1 mM /~-mercaptoethanol, 10% glycerol). The dialyzed material was centrifuged at 10000 x g for 30 rain at 4°C.Calf thymus D N A cellulose was prepared by the method of Alberts and Herrick [16]. Prior to application of the proteins, the columns were washed with blocking buffer; buffer A supplemented with 0.05% BSA, and then washed with 4 vol. of buffer A. Equal amounts of nuclear extract ( - 300 /~g) were applied on 5 ml bed volume DNA cellulose columns at flow rate of 1 to 2 ml/h. The unbound proteins were removed by extensive washing of the column with buffer A and then the bound proteins were eluted by stepwise addition to the column of buffer A containing 0.15, 0.5, 1.0 and 2.0 M NaCI. The eluted proteins were precipitated by incubation with 4 vol. of acetone for 2 h at 4°C, followed by centrifugation at 10000 rpm for 15 min. The precipitated proteins were solubilized in SDS sample buffer and analyzed by SDS-PAGE and Western blot.
Western blot analysis Assays were done as previously described [6]. Nuclear proteins were analyzed on SDS-PAGE and electrotransferred onto nitrocellulose membranes at 80 V for 90 min at room temperature. The nitrocellulose membranes were then soaked overnight in Tris-saline buffer (10 mM Tris (pH 7.5), 0.150 mM NaC1) containing 3% BSA and 10% FCS. The membranes were further incubated for 2 h with anti-numatrin antibody 339 (polyclonal antibody, see Ref. 7) (1:150 dilution) in Trissaline buffer containing 5 mM EDTA, 0.25% gelatin, and 0.05% NP-40 for 2 h at room temperature. Detection of immunoreactive bands was done by incubation with 125I-labeled protein A (0.1 /~Ci/ml in the same medium used for antibody incubation). The membranes were washed extensively in buffer containing 50 mM Tris (pH 7.5), 5 mM EDTA, 1 M NaC1, 0.25% gelatin, and 0.8% sarcosyl for 2 h, dried, and exposed to autoradiography. Purification of nucleoli and extraction of nucleolar proteins Nucleoli were isolated using the Nonidet P-40 method previously described [7,11]. Cells were suspended in 20 vol. of RSB buffer (0.01 M Tris-HC1, 0.01 M NaCI, 1.5 mM MgC12, pH 7.2)) for 30 rain and then centrifuged at 630 x g for 8 min. Swollen cells were resuspended in 20 vol. of RSB buffer containing 0.5% Nonidet P-40. The cells were homogenized with a Dounce homogenizer (10 up-and-down strokes), and the crude nuclei were resuspended in 10 vol. of 0.25 M sucrose, 10 mM MgC12 and underlayered with an equal volume of 0.88 M sucrose, 0.05 mM MgC12. After centrifugation at
129 1700 × g for 10 min, the nuclei were resuspended in 10 vol. of 0.34 M sucrose, 0.05 mM MgC12. The suspension was sonicated for 1 min, underlayered with an equal volume of 0.88 M sucrose, 0.05 mM MgC12 and centrifuged at 3000 x g for 18 min. The pellet (nucleoli) was then collected. Nucleoli (0.1 g) were suspended in 3 M urea, 1 mM phenylmethylsulfonyl fluoride, 1 mM leupeptin, 1 mM p-ehloromercuriphenylsulfonic acid and were homogenized with a Dounce homogenizer (20 up-and-down strokes) on ice. The extract was then incubated at 60°C for 30 min and centrifuged at 27 000 x g for 20 min at 5°C. The supernatant contained the partially purified protein (generally > 80% homogenous). Extraction of nucleoli under native conditions was done as previously described [11]. Nucleoli were homogenized at 4°C with Tris extraction buffer containing 10 mM Tris (pH 7.4), 0.5 mM MgC12, 1 mM PMSF, 1 mM leupeptin, and then centrifuged at 27000 x g for 20 min. The supernatant containing nucleolar proteins was collected.
Heparin-Sepharose chromatography A heparin-Sepharose column CL-6B (Pharmacia, 4 ml bed volume) was equilibrated with T E D G buffer (50 mM Tris (pH 7.5), 5 mM EDTA, 5 mM DTT, 20% glycerol, 1 mM PMSF) containing 50 mM KC1. The column was then rinsed with T E D G buffer containing 100 # g / m l of BSA, and further washed with 5 column volumes of T E D G buffer. Proteins extracted from purified nucleoli under native conditions ( - 2 mg) were applied on the column. The column was then washed with 5 column volumes of T E D G buffer and then proteins were eluted with a linear 0.15 to 2.0 M KC1 gradient. The fractions which were collected (1-1.5 ml) were monitored for the presence of numatrin by ELISA and by SDS-PAGE.
Gel filtration chromatography
cell suspension was added 5 ml of phenol saturated in 10 m M NaC1, 0.5 M Tris-HC1 (pH 8.0), 10 mM EDTA, 0.5% SDS, 5 ml of 95% chloroform, and 5% isoamyl alcohol. Following centrifugation at 5000 rpm for 15 min, the supernatant was collected, and the D N A was precipitated by adding 2.5 vol. of 95% ethanol and 1 / 5 vol. of 3 M N a O H and further incubation at - 2 0 ° C for 16 h. The D N A was pelleted by centrifugation at 3000 rpm for 30 min. The pellet D N A was dried and then resuspended in 10 mM Tris (pH 7.4), and 0.5 mM EDTA by vortexing and incubation overnight at 4°C. The D N A was sheared by passing through a syringe with a 19" gauge needle.
ELISA procedure ELISA was done as previously described [7]. Microtiter plates (Titertek, 96 wells) were coated with proteins for 2 h at room temperature. The plates were then washed and blocked with phosphate-buffered saline containing 3% BSA at 4°C overnight. The solution was aspirated, and the plates were washed three times with phosphate-buffered saline-Tween. Anti-numatrin antibody (339) was added and further incubated for 30 min. The second antibody, alkaline phosphatase-conjugated goat anti-rabbit, was added and incubated at room temperature for 1 h. The plates were then washed, and 150/.tl of substrate (p-nitrophenyl phosphate) was added and incubated for 15 min (until color developed). Results represent average absorbance at 405 nm of triplicate wells (S.E. less than 10%).
Nitrocellulose filter binding assay Nitrocellulose filter binding assay was done as previously described [17]. Numatrin or BSA (used as control) was mixed with 1.65 /~g of [3H]DNA (43000 dpm) at final volume of 100-150 ~1 and incubated for 10 min at room temperature. The samples were then poured on nitrocellulose filters (Grade BA85, Schleicher and Schuel) and washed with 10 mM Tris (pH 7.4), 0.5 mM EDTA three times under vacuum. The filters were dried, and the radioactivity retained on the filters was monitored by liquid scintillation spectrometer.
The fractions containing numatrin from the heparinSepharose columns were concentrated and applied on Sepharose G-100 column (0.8 × 30 cm, 40 ml bed volume). The proteins were eluted with 10 mM Tris (pH 7.4), 0.5 mM MgC12 and proteinase inhibitors. Fractions were collected (2 ml) and monitored for the presence of numatrin by ELISA and by SDS-PAGE.
Two dimensional gel electrophoresis was performed as previously described [18].
Purification of high molecular weight DNA
Results
HeLa cells (5 × 107) were labeled with 1 mCi of [3H]thymidine for 48 h. The ceils were then suspended in 10 mM NaC1, 10 mM Tris-HCl (pH 8.0), 10 m M EDTA, 0.5% SDS in the presence of 50 /Lg/ml proteinase K, and incubated for 2 h at 37°C. Proteinase K (50 t~g/ml) was added again, and the solution was further incubated at 4°C overnight. The D N A was extracted in phenol/chloroform as follows: to 10 ml of
Two-dimensional gel electrophoresis
Binding of numatrin to DNA cellulose columns Nuclear proteins were extracted from nuclei of HL-60 cells as described in Materials and Methods and applied to three columns: SS-DNA cellulose, DS-DNA cellulose and cellulose (not attached to DNA). The D N A binding proteins were eluted with increasing concentrations of NaC1 (in buffer B) at a step gradient of 0.15, 0.5, 1.0
130 SS-DNA NaCI(M)
.15 .5 1.0 2.0
DS.DNA
Cellulose
.15 .5 1.0 2.O(N) .15 .5 1.0 2.0(N)
v,-
×
iF Fig. 1. DNA-cellulose chromatography. Evidence for numatrin binding to SS-DNA and DS-DNA cellulose columns. Equal amounts of nuclear proteins (extracted as described in Materials and Methods) were applied to three columns: DS-DNA cellulose, SS-DNA cellulose and cellulose (not attached to DNA). The bound proteins were eluted by step wise addition of buffer A (see Methods) containing 0.15, 0.5, 1.0 and 2.0 M NaC1. The eluted proteins were precipitated by acetone, solubilized in SDS-sample buffer and analyzed by SDS-PAGE and Western Blot using anti-numatrin antibody. (N) represents numatrin marker.
and 2.0 M, and the proteins in the eluates were further precipitated with acetone. Equal portions of the precipitated proteins were analyzed by SDS-PAGE, electroeluted onto nitrocellulose membranes and immunoblotted with anti-numatrin antibody. As shown in Fig. 1, numatrin was eluted from both SS-DNA column and DS-DNA columns at 0.5 M and 1.0 M NaC1. Small amounts of numatrin were eluted only by 2.0 M NaC1, indicating very strong binding to DNA. Numatrin did not bind the cellulose column, indicating that the bind-
ing of numatrin to the D N A cellulose columns is not due to non-specific interaction to the cellulose matrix. Proteins eluted from DS-DNA columns (at 1.0 M) were further pooled and analyzed by two-dimensional gel electrophoresis (Fig. 2). Silver staining of the gel (panel A) demonstrated the presence of several prominent D N A binding proteins which were eluted from the column. Several of these proteins have a close electrophoretic mobility to that of numatrin. The immunoblot of a parallel gel using anti-numatrin antibody identified the localization of numatrin (both a and /3 chains) among the other nuclear DNA binding proteins which were eluted from the column. D N A binding of numatrin extracted in urea
The results demonstrated in Fig. 2A raised the possibility-that numatrin binding to the D N A columns might be due to its intimate association with other D N A binding proteins. To address this question, further experiments were undertaken to examine the direct binding of purified numatrin to D N A using the nitrocellulose filter assay. In previous studies we have demonstrated that numatrin can be purified to - 9 0 % homogeneity by extracting nucleoli of HL-60 cells with 3 M urea at 60°C [7]. We have used this urea extract to examine numatrin binding to 3H-labeled SS-DNA and DS-DNA using the nitrocellulose filter assay. [3H]DNA was pre-
Silver stain Acid 66--
Immunoblot Base qb
45--
I O
x
31--
=E
21--
14-Fig. 2. Two dimensional gel electrophoresis and immunoblot analysis of proteins eluted from D N A cellulose chromatography. Proteins eluted from DS-DNA column at 1.0 M NaCI were analyzed by two-dimensional gel electrophoresis, and the gel was further stained by silver staining (Bio-Rad kit). A parallel gel was transferred onto nitrocellulose and immunoblotted by anti-numatrin antibody. Arrows indicate the position of numatrin on the gel.
131 400
pared by labeling HeLa cells with [3H]thymidine for 24 hr. After lysing the cells and extensive treatment with proteinase K, the high molecular weight D N A was purified using phenol/chloroform extraction. The specific activity of the D N A was 26 000 dpm//~g. Single stranded heat-denatured D N A was prepared by boiling a part of the purified DS-DNA. Thus, the DS-DNA and the SS-DNA were comparable in every respect. The nitrocellulose D N A binding assay was done as described in Materials and Methods. In short, 1.65 #g of either [3H]SS-DNA or [3H]DS-DNA was mixed with purified numatrin (urea extract) in a total volume of 200/~1 and incubated for 10 min at room temperature. The [3H]DNA-protein complex was then collected and washed on nitrocellulose filters as described in Materials and Methods. These experiments demonstrated (Fig. 3) that numatrin binds to both DS-DNA and SS-DNA in a dose-dependent manner. However, its binding to DS-DNA was 3-15-fold higher than its binding to SS-DNA in various experiments. In the experiment shown in Fig. 3 (bottom) under control conditions (no protein) 120 dpm were retained on the nitrocellulose filters. Addition of 13/~g of numatrin resulted in 3725 dpm associated with SS-DNA (31-fold increase) and 14259 dpm associated with DS-DNA (119-fold increase). Equal amounts of BSA which were used as control did not show any binding to D N A (data not shown).
4000 "
400o] EXP.1
EXP.2
[] BSA []
1ooo
Numalrin
1o0o.
0
~
o OS-DNA
SS-DNA
Sg-DNA
DS-DNA
150o0- EXP.3
10000 ~
~
~ e'=
SS-DNA
•
DS-DNA
5000
0 5
10
15
Numatrin(pg)
Fig. 3. DNA binding of numatrin extracted in urea. Preferential binding to DS-DNA. Binding of purified numatrin ( - 4.0 /~g) extracted in 3 M urea [7] to [3HIDS-DNA and [3H]SS-DNA was examined by using the nitrocellulose filter assay as described in Materials and Methods. In experiment 2, SS-DNA was obtained by boiling DS-DNA just prior to performing the D N A binding study. Experiment 3 (bottom) represents a dose-dependent increase in numatrin binding to D N A . Results represent a typical experiment (average dpm of duplicate samples with less than 10% variability).
300 SS-DNA n
20o
•
[ ] Numatrin 100
o salt
+
salt
6000 -
.00o1 t~
/ 2000"
0
[ ] Numatrin
. . . . .
salt
"
+
salt
Fig. 4. Preferential effect of salt on numatrin binding to DS-DNA as compared to SS-DNA. Binding of purified numatrin ( ~ 4.0 #g) extracted in 3 M urea to [3H]SS-DNA and [3H]DS-DNA was examined in the presence or absence of 0.5 M NaC1, using the nitrocellulose filter assay. Results represent average dpm of duplicate samples with less than 10% variability.
When a similar experiment was performed in the presence of NaC1 (at a final concentration of 0.5 M) it was found that the binding of numatrin to D S - D N A was reduced by 77% while the binding of numatrin to SS-DNA was not at all affected (Fig. 4). These results indicate that the binding of numatrin to SS-DNA when compared to its binding to DS-DNA is different quantitatively as well as qualitatively. D N A binding of numatrin extracted under native conditions The nucleolar protein B23 was reported to be an hexamer under native conditions, and to dissociate into monomers of - 4 0 kDa in the presence of urea [11]. Thus, the D N A binding of the purified numatrin in urea extract (Fig. 3) indicates that numatrin can bind D N A directly under monomerie conditions. Further experiments were undertaken to examine whether numatrin can bind D N A also as an oligomer when extracted under native conditions. To this end, nucleoli were purified from HL-60 cells and extracted as previously described [11] in 10 mM Tris (pH 7.4), 0.5 mM MgC12 and proteinase inhibitors. As shown in Fig. 5, this extraction condition results in variable yields of numatrin. The extraction in experiment 2 (in Fig. 5) demonstrates - 9 0 % purified numatrin. This material was used for study of numatrin binding to DNA, using the nitrocellulose filter assay. As shown in Table IA, numatrin extracted under native conditions (oligomer)
132
2
34
A. ELISA
56
2.0
.70
I
o v
x
1.0
.35
==
Fig. 5. Extraction of numatrin under native conditions. Nucleoli were purified from HL-60 cells and extracted by homogenization in 10 mM Tris (pH 7.4), 0.5 mM MgCl2 and proteinase inhibitors. The suspension was further centrifuged at 11000 rpm for 20 min. The supernatant was collected and analyzed by SDS-PAGE. The results represent Coomassie blue staining of six different experiments. 0
15
30
45
Fraction Number
b o u n d both SS-DNA and D S - D N A . However, similarly to numatrin extracted in urea (Fig. 3) the binding to D S - D N A was several-fold higher as c o m p a r e d to the binding to SS-DNA. T o c o m p a r e the binding of numatrin as an oligomer to its binding as a m o n o m e r , the Tris extract was heated in the presence of 3 M urea (conditions which were previously shown to dissociate B 2 3 / n u m a t r i n into monomers). As shown in Table I, the dissociation of numatrin to m o n o m e r s resulted in 3.2-fold enhancement of binding to SS-DNA, but had only a small effect on its binding to D S - D N A . This is in agreement with the previous results (Fig. 4) which indicated that the nature of numatrin binding to D S - D N A is qualitatively different from its binding to SS-DNA.
TABLE I
DNA binding of numatrin extracted under native conditions. Preferential binding to DS-DNA N u m a t r i n extracted u n d e r native conditions (see e x p e r i m e n t 2 in Fig. 5) was used to e x a m i n e D N A b i n d i n g in the nitrocellulose filter assay. Results (of a typical experiment) represent average d p m of duplicates with less t h a n 10% variability. To c o m p a r e b i n d i n g of n u m a t r i n ( - 2.0 t~g) as an oligomer to its b i n d i n g as a m o n o m e r (A) the native extract was heated to 6 0 ° C in the presence of 3 M urea a n d D N A b i n d i n g was further examined. In (B), the n u m a t r i n extract was treated with 5 ~ t g / m l of R N A s e A (Sigma) for 60 rain at r o o m t e m p e r a t u r e a n d the b i n d i n g to D N A was further e x a m i n e d u s i n g the nitrocellulose filter assay. (A)
Protein
Oligomer
SS-DNA
+
47 393
DS-DNA
+
34 4 921
B. Coomassie blue stain Fraction Number Lead 11 12 13 14 15 16 17
18
30
i
92- i 66--! I
4~-
X
~E~
: :
31ii
ZI--
Fig. 6. Purification of n u m a t r i n by heparin-Sepharose column. Nucleolar proteins extracted u n d e r native conditions (see Fig. 5) were loaded on a h e p a r i n - S e p h a r o s e c o l u m n CL-6B (4 ml bed volume). T h e u n b o u n d proteins were w a s h e d with T E D G b u f f e r (see Materials and M e t h o d s ) a n d then the b o u n d proteins were eluted with linear gradient of KC1 r a n g i n g from 0.05 to 2.0 M. Fractions (1-1.5 ml) were collected a n d m o n i t o r e d for the presence of n u m a t r i n by ELISA (A) a n d by S D S - P A G E (B). T h e peak elution of n u m a t r i n was obtained at 0.7 M KCI.
F u r t h e r experiments were undertaken to examine the D N A binding of numatrin after treatment of the extract with R N A s e A. This treatment had only a small effect on increasing the binding of numatrin to SS-DNA. Thus, under this condition numatrin still b o u n d significantly better to D S - D N A indicating that the lower level of n u m a t r i n binding to S S - D N A as c o m p a r e d to DSD N A is not due to competition of the SS-DNA with R N A which m a y be present in the Tris extract.
Monomer/ Oligomer
Chromatography purification of numatrin by heparin-Sepharose column
82 1280
3.2
15 6 067
1.2
F u r t h e r experiments were undertaken to purify numatrin to homogeneity by c h r o m a t o g r a p h y techniques. Nucleoli were purified from 2 g of HL-60 cell nuclei and further extracted in 10 m M Tris (pH 7.4), 0.05 m M MgC12 (Tris extract, see Fig. 4). 2 ml of extracted material which contained about 2 mg of nucleolar proteins were applied on heparin-Sepharose CL-6B column and extracted with T E G P buffer at a continuous gradient of KC1 ranging from 50 to 2000 mM. The fractions were analyzed for the presence of
Monomer
(B) SS-DNA
Protein +
- RNAse 79 472
+ RNAse 49 612
DS-DNA
+
15 3210
18 3395
133
A. ELISA
were applied on a gel filtration Sephadex G-100 column and the proteins were eluted with Tris extraction buffer. The fractions were screened for the presence of numatrin by ELISA and by silver staining of SDS-PAGE. As shown in Fig. 7, the major peak of numatrin was eluted at fraction 6, corresponding to the elution of catalase which was used as a molecular mass marker (232 kDa) to indicate the void volume of the column. This result indicates that the natively purified numatrin is indeed not found as a monomer of 40 kDa, but rather is found in oligomeric form as previously reported [11]. The minor peak which was detected by ELISA may be the monomeric form of numatrin. However, it could not be detected by silver staining of the gel (data not shown).
Catalyse (232 kDa)
t
.5@
g
c u~ o qr
.25
DNA binding of numatrin following heparin.Sepharose
chromatography Further studies examined the D N A binding of numatrin -purified by heparin-Sepharose using the nitrocellulose filter assay. The results in Fig. 8 show that numatrin binds both SS-DNA and D S - D N A in a dose-dependent manner, thus confirming our previous .00
I 5
0
, I 26
50 SS DNA
Fraction Number 300
A
B. Sliver Stain
DS D N A
EXP 1
EXP 1
B
H SS D N A e--~ DS D N A BSA
Fraction Number 4
5
6
7
8
9
10
11
12
13
14
15 200(
j
n c3 I
100(
X
d 00"
I.J 16 32
I 64
I 128
I
I
I
J
• 16
32
64
128
PROTEIN (picornoles) EXP 2
Fig. 7. Gel filtration chromatography of purified numatrin. Fractions from the heparin-Sepharose column which contained numatrin were loaded on gel filtration column Sephadex G-100 and eluted with Tris extraction buffer. The fractions were monitored for the presence of numatrin by ELISA (A) and by SDS-PAGE (B). Gel (in B) was stained by the silver staining method.
numatrin by ELISA and by SDS-PAGE (Fig. 6). Numatrin was eluted at - 0 . 7 M KC1 as shown by ELISA and by SDS-PAGE. The recovery of numatrin after the chromatography purification was about 40%. (Silver staining of fraction 16 indicated only one band, data not shown). To examine whether numatrin which was eluted from the heparin-Sepharose column is an oligomer or is found as a monomer, the fractions which contained numatrin
2000
C
EXP 2
/
D
/
a. 1000 D
I
I
0~ 16 32
I
6i4
128
--I
I
16 32 PROTEIN 3icomoles)
I
J
64
128
Fig. 8. Binding of numatrin purified by heparin-Sepharose chromatography to D S - D N A and SS-DNA. Numatrin purified from heparinSepharose column (or comparable amounts of BSA which were used as control) were mixed with [3H]SS-DNA or [3H]DS-DNA, and the binding to D N A was examined using the nitrocellulose filter assay. Experiments 1 and 2 were identical. Results represent average dpm of duplicate samples with less than 10% variability.
134 Purified numatrin 4000
Numatrin +a 110 kDa protein
A
B SS DNA DS-DNA o--,o
D
2000
0~'
64
128
I
I
64
128
PROTEIN 3icomoles) C
T FI# 1
~" 97 6 8 ~ x 45
2
3
4
5
6
7
8
9
"0
11 12 13 14
15
~:
numatrin in two purification steps [19,20]; (a) PEGtreated NE/S-3 supernatant which contains the low salt nuclear extraet-postmicrosomal supernatant; and (b) the final purified fraction of Q-Sepharose chromatography which contained the purified DNA polymerase c~ primase activity [20]. Equal amounts of proteins of these two fractions (kindly provided by Dr. Earl F. Baril, Worcester Foundation, MA) were applied on SDS-PAGE and transferred onto nitrocellulose membrane, Using immunoblotting technique with anti-numatrin antibody, we show that purification of DNA polymerase a primase activity is associated with copurification of numatrin. indicating that numatrin is found in complex with the DNA polymerase c~ primase. Discussion
Fig. 9. A protein at a 110 kDa which copurifies with numatrin on heparin-Sepharose column conferred a preferential enhancement of binding to SS-DNA. (C) Coomassie blue staining of SDS-PAGE of fractions eluted from heparin-Sepharose column loaded with nucleolar proteins (as described in Fig. 6). Binding of purified numatrin (fractions 9 and 10) to SS-DNA and DS-DNA is shown in (A). DNA binding of numatrin copurified with the 110 kDa protein (fractions ll and 12) is shown in (B).
results in this work. Significant DNA binding could be detected at 64 pmol of numatrin and was dramatically increased at 128 pmol. Notably, following chromatography purification, numatrin did not demonstrate a preferential binding to DS-DNA as found in the previous experiments. This might be due to the presence of high salt concentration in the purified fraction (0.7 M KC1). In certain preparations of heparin-Sepharose column etution of purified numatrin was followed by copurification of a 110 kDa protein together with numatrin (see fractions 11, 12, 13 in Fig. 9). The fractions which contained purified numatrin and the fractions which contained mixture or numatrin and the 110 kDa protein were pooled separately and assayed for DNA binding. As shown in Fig. 9 (panels A and B), the-presence of the 110 kDa protein conferred a preferential enhancement of binding to SS-DNA, but it did not affect at all the binding to DS-DNA.
Evidence that numatrin copurifies with the complex of DNA polymerase a primase It has been shown that the cell cycle regulated nuclear protein PCNA/cyclin [1] coeluted with the purified complex of DNA polymerase a primase [20] together with other enzymes which are required for in vitro DNA replication of SV-40, such as DNA-dependent ATPase, DNA ligase, RNAse H and topoisomerase I [19-21]. To examine whether numatrin is also found in this purified complex, we examined the presence of
The results of this study demonstrate that numatrin is a DNA binding protein. It binds both DS-DNA and SS-DNA as shown by using DNA cellulose chromatography and by using the nitrocellulose filter assay. Evidence is presented to suggest that there are certain differences in numatrin interaction with DS-DNA as compared to its interaction with SS-DNA: (a) Studies using numatrin extracted under native conditions or under urea conditions showed a several-fold higher binding affinity of numatrin to DS-DNA as compared to SS-DNA. This difference could not be overcome by pretreatment of numatrin with RNAse, indicating that the lower affinity to SS-DNA is not due to competition with RNA, which may be present in the preparation. (b) The binding to DS-DNA demonstrated a high sensitivity to the ionic strength as it was reduced by 77% in the presence of 0.5 M NaC1, while the binding to SS-DNA was not at all affected under this ionic condition. This indicates that the binding to SS-DNA may involve less ionic interaction as compared to the binding to DSDNA. (c) Treatment of the native numatrin under conditions which cause monomerization of the protein resulted in a significant enhancement of numatrin binding to SS-DNA but did not affect the binding of numatrin to DS-DNA, implying that the affinity of numatrin to SS-DNA may be higher as a monomeric form as compared to the oligomeric form, while this factor is not pertinent in numatrin binding to DS-DNA. Taken collectively, these data indicate that numatrin binding to DS-DNA is qualitatively and quantitatively different from its binding to SS-DNA, However, the higher affinity to DS-DNA could not be demonstrated following heparin-Sepharose chromatography. Since numatrin was eluted from the column at 0.7 M KCI, it is possible that the presence of the salt in the preparation of the purified protein is responsible for the decrease in theaffinity to DS-DNA (as was demonstrated the binding to DS-DNA in contrast to
135 the binding to SS-DNA was significantly reduced with the increase in the ionic strength of the buffer). Since we could not remove the salt without losing the protein, the discrepancy of the results regarding the preferential affinity to DS-DNA could not be resolved at this point. However, the results of this paper dearly demonstrate that numatrin can bind significantly to DNA, both DS-DNA and SS-DNA. The T antigen of SV-40 has been shown to bind DS-DNA as well as SS-DNA in two different mechanisms: its binding to DS-DNA is sequence specific while its binding to SS-DNA is nonspecific [22]. It has been suggested that its binding to DS-DNA may be involved in initiation of replication while its binding to SS-DNA may be involved in further unwinding and helix destabilization [22]. Extensively characterized SSDNA binding proteins such as UPI, adenovirus DBP, T4 gene and 32 protein were often implied in DNA unwinding, helix destabilization and stimulation of the activity of their cognate DNA polymerase (for review see Ref. 23). Studies on nuclear onco-proteins have demonstrated that certain of these proteins such as myc [24] and fos [25] bind to DNA nonspecifically, while others such as jun [26] and myb [27] are sequencespecific DNA binding proteins. Both )Cosand jun were implicated as transcription factors [26,28]. Interestingly, it has been shown that interaction between los and jun (via the leucine zipper domain) resulted in enhancement of the binding of jun to its specific DNA binding domain [29,30]. This demonstrates a novel mechanism by which a nonspecific DNA binding protein may exert a crucial physiological effect via its binding to a sequence-specific DNA binding protein. In this regard the finding that a protein at 110 kDa which copurified with numatrin in certain column fractions, conferred an enhancement of the binding to SS-DNA but not to DS-DNA is of interest. Thus, it is possible that other proteins (such as this 110 kDa protein) associate with numatrin and by this association regulate its binding to DNA. Conversely, numatrin binding to other proteins may regulate their binding to DNA. These potential p r o t e i n / D N A interactions are particularly pertinent to the finding that numatrin copurifies with DNA polymerase a primase (Fig. 10). This observation indicates that numatrin is found in association with a complex of enzymes which are required for in vitro DNA replication [19-21] and thus may have a physiological significance in the enzymatic activity of this complex. Studies are currently in progress to examine this possibility. The physiological significance of numatrin binding to DNA and its complexing with DNA polymerase a primase may be of particular importance in view of the finding that B23 (numatrin) has been shown to translocate from the nucleolus to the nucleoplasm when RNA synthesis is inhibited [15]. Taken collectively with
a 9?. " ,-. ~
b (o) N E / S - 5
~
6 6 .....,~ ~
~ (b)Purified
~ x45- ~i~!~'
supernatant
DNApolymerase
a primase fraction from Q - Sepharose chromatography
:E Fig. 10. Numatrin copurifies with the complex of DNA polymerase a primase. Equal amounts of proteins of two purification steps in the process of purification of DNA polymerase a primase [19-21] were applied on SDS-PAGE, transferred onto nitrocellulose membrane and immunoblotted with anti-numatrin antibody. (a) NE/S-3 supernatant (containing the low salt nuclear extract postmicrosomal supernatant); and (b) the final purified fraction of Q-Sepharose chromatography which contains the purified complex of DNA polymerase a primase activity [20].
the evidence that numatrin accumulation in the cells is correlated with entry and progression of the cells through S-phase [6], namely a phase which is associated with a decrease in RNA synthesis and an increase in DNA synthesis, it may be postulated that the ability of numatrin to bind DNA and its association with DNA polymerase a primase may indicate that numatrin may have different functions during different phases of the cell cycle, and it may be involved in DNA-associated processes. Acknowledgments The authors would like to thank Dr. Earl Baril (Worcester Foundation, MA) for providing the chromatography purification fractions of DNA polymerase a primase, and Ms. Mary Wise for excellent secretarial assistance. This work was supported by National Institutes of Health Grants R29 CA48667-01, RO-AI24273 and AI18449, and Uniformed Services University of the Health Sciences Protocol RO83BQ. References 1 Celis, J. and Cells, A. (1985) Proc. Natl. Acad. Sci. USA 82, 3262-3266. 2 Bishop, J.M. (1985) Cell 42, 23-38. 3 Reich, N.C. and Levine, A.J. (1984) Nature 308, 199-201. 4 Feuerstein, N. and Mond, J.J. (1987) J. Immunol. 139, 1818-1822. 5 Feuerstein, N. artd Mond, J.J. (1987) J. Biol. Chem. 262, 1138911397. 6 Feuerstein, N., Speigel, S. and Mond, J.J. (1988) J. Cell Biol. 107, 1629-1642. 7 Feuerstein, N., Chan, P.K. and Mond, J.J. (1988) J. Biol. Chem. 263, 10608-10612. 8 Kang, Y.J., Olson, M.O.J. and Busch, H. (1974) J. Biol. Chem. 249, 5580-5585. 9 Kang, Y.J., Olson, M.O.J. and Busch, H. (1975) Cancer Res. 35, 1470-1475. 10 Chan, P.K., Aldrich, M.B., Cook, R.G. and Busch, H. (1986) J. Biol. Chem. 261, 1868-1872.
136 11 Yung, B.Y-M. and Chart, P.K. (1987) Bioehim. Biophys. Acta 925, 74 82. 12 Chart, W-Y., Liu, Q-R., Borjigin, J., Busch, H., Rennert, O.H., Tease, L.A. and Chan, P.K. (1989) Biochemistry 28, 1033-1039. 13 Chang, J-H., Dunbar, T.S. and Olson, M.O.J. (1988) J. Biol. Chem. 263, 12824-12827. 14 Spector, D.L., Och, R.I. and Busch, H. (1984) Chromosoma 90, 139-148. 15 Yung, B.Y.M., Busch, H. and Chan, P.K. (1985) Biochim. Biophys. Acta 826, 167-173. 16 Alberts, B. and Herrick, G. (1962) Methods Enzymol. 210, 198217. 17 Ruyechan, W.T. (1983) J. Virol. 46, 661-666. 18 Feuerstein, N. and Cooper, H.L. (1983) J. Biol. Chem. 258, 1078610793. 19 Baril, E.F., Malkas, L.M., Hickey, R.H., El, C.-J., Coughlin, S. and Vishwanatha, J.K. (1988) in Cancer Cells 6: Eukaryotic DNA Replication (Kelly, T.J. and Stillman, B., eds.), pp. 373-384, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 20 Hickey, R.J., Malkas, L.H., Pedersen, N., Li, C. and Baril, E.F. (1988) in DNA Replication and Mutagenesis (Moses. R. and
21 22 23 24 25 26 27 28 29 30
Summers, W., eds.), pp. 41-54, American Society of Microbiology Publications, Washington, DC. Malkas, L.M. and Baril, E.F. (1989) Proc. Natl. Acad. Sci. USA 86, 70-74. Aubom, K.J., Markowitz, R.B., Wang, E., Yu, Y.T., and Prives, C. (1988) J. Virol. 62, 2204-2208. Chase, J.W. and Williams, K.R. (1986) Annu. Rev. Biochem. 55, 103-136. Watt, R.A., Shatzman, A.R. and Rosenberg, M. (1985) Mol. Ceil. Biol. 5,448-456. Sambucetti, L.C. and Curran, T. (1986) Science 234, 1417-t419. Angel, P., Allegretto~ E.A., Okino, S,T., Hattori, K,, Boyle, W.J., Hunter, T. and Karin, M. (1988) Nature 332, 166-171. Biedenkapp, H., Borgmeyer, U., Sippel, A.E. and Klempnauer, K.H. (1988) Nature 335, 835-837. Setoyma, C.R., Frunzio, F., Lian, G., Mudryj, M. and (_rombrugghe, B.D. (1986) Proc. Natl. Acad. Sci. USA 83, 3213-3217. Kouzarides, T. and Ziff, E. (1988) Nature 336,646 651. Sassone-Corsi, P., Ransone, L.J., Lamph, W.W. and Verma, I.M. (1988) Nature 336, 692-695.
Biochimica et Biophysiea Acta, 1087 (1990) 127-136 Elsevier BBAEXP 92163
Evidence for D N A binding activity of numatrin (B23), a cell cycle-regulated nuclear matrix protein Nili Feuerstein 1, James J. Mond 2, Paul R. Kinchington 3 Robert Hickey 4 Marja-Liisa Karjalainen Lindsberg 2, Ian Hay 3 and William T. Ruyechan 3 Department of Biochemistry, University of Pennsylvania, Philadelphia, PA, Departments of e Medicine and 3 Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, MD and 4 Department of Pharmacology and Experimental Therapeutics, University of Maryland, School of Medicine, College Park, MD (U.S.A.) (Received 2 February 1990) (Revised manuscript received 30 May 1990)
Key words: DNA binding activity; Numatrin; Nuclear matrix protein
Stimulation of various cell types with growth factors is associated with a rapid induction in the synthesis of a nuclear matrix protein, termed 'numatrin' which was shown to be identical to the nucleolar protein B23. The abundance of numatrin was shown to be correlated with entry and progression through the S-phase. Thus, experiments were undertaken to examine whether numatrin also has DNA binding activity. Using whole nuclear extract, we showed that numatrin binds to both double-stranded (DS) DNA and to single-stranded (SS) DNA cellulose columns. Purified numatrin, which was extracted either under native conditions (in oligomeric form) or under urea conditions (in monomeric form), demonstrated significant binding to either [3H]DS-DNA or |3HIDS-DNA as shown by nitrocellulose filter binding assay. However, numatrin binding to D S - D N A was qualitatively and quantitatively different from its binding to SS-DNA. Thus, the binding of numatrin was several fold higher to D S - D N A as compared to SS-DNA. The binding to D S - D N A was reduced by 77% in the presence of 0.5 M NaCI, while the binding to S S - D N A was not affected under this condition. Furthermore, treatment of the native numatrin under conditions which caused monomerization of the protein resulted in a significant enhancement of numatrin binding to S S - D N A but did not affect the binding to DS-DNA. Following beparin-Sepharose chromatography purification (under native conditions), numatrin at picomole amounts showed significant binding to both D S - D N A and SS-DNA. Finally, numatrin was found to copurify with the complex of DNA polymerase a primase together with other proteins required for SV-40 in vitro replication activity. These results demonstrate that numatrin has DNA binding activity, and imply a possible role for numatrin/B23 in DNA-associated processes.
Introduction
Several nuclear proteins including cyclin/PCNA [1], and proteins encoded by proto-oncogenes such as c-myc, c-fos (for review see Ref. 2), and p53 [3], have been implicated in processes associated with regulation of cellular growth. We have described and characterized another growth regulated nuclear protein (40 k D a / p I 5) which is tightly associated with the nuclear matrix and was termed 'numatrin' [4-6]. We showed that Abbreviations: SS-DNA, single-stranded DNA; DS-DNA, doublestranded DNA; PCNA, proliferating cell nuclear antigen; BSA, bovine serum albumin; FCS, fetal calf serum; PMSF, phenylmethylsufonyl fluoride; DTT, dithiothreitol. Correspondence: N. Feuerstein, The Diabetes Research Center, Room 510, Medical Education Building, University of Pennsylvania, 36 and Hamilton Walk, Philadelphia, PA 19104-6015, U.S.A.
stimulation of B lymphocytes with various mitogens is associated with a rapid stimulation in the synthesis of numatrin [4,5]. This event was shown to be closely correlated with cellular commitment for mitogenesis and not with other parameters of cellular activation in B lymphocytes [5]. Induction of numatrin synthesis was also demonstrated in T lymphocytes stimulated by antiT cell receptor antibody and by concanavalin A as well as in murine Swiss 3T3 fibroblasts stimulated by various growth factors such as insulin and epidermal growth factor [6]. Thus, it was suggested that an increase in the expression of numatrin is a common nuclear event which is closely correlated with cellular induction of mitogenesis in various cell types. Further studies demonstrated elevated synthesis and abundance of numatrin in various malignant cells [4,6], indicating that numatrin might be involved in regulation of cell growth in normal and malignant cells.
0167-4781/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
128 In a recent study we provided evidence to indicate that numatrin is identical to the nucleolar protein B23 [7]. This conclusion was based on: (a) identical migration of numatrin and B23 on two-dimensional gel dectrophoresis; (b) identical tryptic peptide map; and (c) cross-reactivity of antibodies to numatrin and to B23. Protein B23 has been degcribed in 1974 as a nucleolar phosphoprotein [8,9]. Its phosphorylated site has been sequenced and was found to be similar to that of C23 and R-II subunit of cAMP protein kinase [10]. It was found that under native conditions protein B23 exists as an hexamer, composed of 4~ and 2fl monomers [11]. c D N A encoding the human [12] and the rat [13] protein B23 has been recently sequenced and found to have 50% homology with the Xenopus nucleoplasmin [12] and 63% homology to the Xenopus laeuis protein No38 [13]. Protein B23 has been shown to be localized in the granular region of the nucleolus where pre-ribosomal R N A is being assembled [14] and to be associated with pre-ribosomal R N P particles [15]. This evidence led to the hypothesis that protein B23 might be involved in the processing and assembling of pre-ribosomal RNA. However, protein B23 has been shown to translocate from the nucleolus to the nucleoplasm when R N A synthesis is inhibited [15], suggesting that its nucleolar localization is dependent on its association with RNA. Furthermore, only 40% of protein B23 was shown to be associated with R N P particles (80s and 55s) [15], while the nature of the interaction of the other 60% of the protein with other nuclear elements is still not known. In this regard, it is of interest that kinetic studies which examined the accumulation of numatrin during the cell cycle in mitogen-stimulated lymphocytes, demonstrated that the amount of numatrin in the cells reached a peak just prior to the entry of the cells into S-phase, it remained elevated during the S-phase and declined to control levels at the end of the S-phase [6]. This indicates that the abundance of numatrin in the cells is closely correlated with entry and progression through S-phase, namely, with the phase associated with D N A synthesis rather than with R N A synthesis. This, taken collectively with the evidence of numatrin translocation out of the nucleolus when R N A synthesis is inhibited, suggests that numatrin might be involved in different processes during different phases of the cell cycle. To address this possibility, experiments were undertaken to examine whether numatrin can also bind or interact with DNA. Materials and Methods
DNA cellulose chromatography Nuclei were purified from the human promyelocytic leukemic cells HL-60 [18] as previously described [5] and extracted in 2 M NaC1, 40 mM Tris (pH 8.2), 2 m M EDTA and 2 mM /~-mercaptoethanol at 4°C. After 30
min incubation on ice, the samples were centrifuged at 100000 x g for 60 n'fin. The supernatant was removed and dialyzed overnight at 4°C against buffer A (50 ruM NaC1, 20 m M Tris (pH 8.2), 1 mM EDTA, 1 mM /~-mercaptoethanol, 10% glycerol). The dialyzed material was centrifuged at 10000 x g for 30 rain at 4°C.Calf thymus D N A cellulose was prepared by the method of Alberts and Herrick [16]. Prior to application of the proteins, the columns were washed with blocking buffer; buffer A supplemented with 0.05% BSA, and then washed with 4 vol. of buffer A. Equal amounts of nuclear extract ( - 300 /~g) were applied on 5 ml bed volume DNA cellulose columns at flow rate of 1 to 2 ml/h. The unbound proteins were removed by extensive washing of the column with buffer A and then the bound proteins were eluted by stepwise addition to the column of buffer A containing 0.15, 0.5, 1.0 and 2.0 M NaCI. The eluted proteins were precipitated by incubation with 4 vol. of acetone for 2 h at 4°C, followed by centrifugation at 10000 rpm for 15 min. The precipitated proteins were solubilized in SDS sample buffer and analyzed by SDS-PAGE and Western blot.
Western blot analysis Assays were done as previously described [6]. Nuclear proteins were analyzed on SDS-PAGE and electrotransferred onto nitrocellulose membranes at 80 V for 90 min at room temperature. The nitrocellulose membranes were then soaked overnight in Tris-saline buffer (10 mM Tris (pH 7.5), 0.150 mM NaC1) containing 3% BSA and 10% FCS. The membranes were further incubated for 2 h with anti-numatrin antibody 339 (polyclonal antibody, see Ref. 7) (1:150 dilution) in Trissaline buffer containing 5 mM EDTA, 0.25% gelatin, and 0.05% NP-40 for 2 h at room temperature. Detection of immunoreactive bands was done by incubation with 125I-labeled protein A (0.1 /~Ci/ml in the same medium used for antibody incubation). The membranes were washed extensively in buffer containing 50 mM Tris (pH 7.5), 5 mM EDTA, 1 M NaC1, 0.25% gelatin, and 0.8% sarcosyl for 2 h, dried, and exposed to autoradiography. Purification of nucleoli and extraction of nucleolar proteins Nucleoli were isolated using the Nonidet P-40 method previously described [7,11]. Cells were suspended in 20 vol. of RSB buffer (0.01 M Tris-HC1, 0.01 M NaCI, 1.5 mM MgC12, pH 7.2)) for 30 rain and then centrifuged at 630 x g for 8 min. Swollen cells were resuspended in 20 vol. of RSB buffer containing 0.5% Nonidet P-40. The cells were homogenized with a Dounce homogenizer (10 up-and-down strokes), and the crude nuclei were resuspended in 10 vol. of 0.25 M sucrose, 10 mM MgC12 and underlayered with an equal volume of 0.88 M sucrose, 0.05 mM MgC12. After centrifugation at
129 1700 × g for 10 min, the nuclei were resuspended in 10 vol. of 0.34 M sucrose, 0.05 mM MgC12. The suspension was sonicated for 1 min, underlayered with an equal volume of 0.88 M sucrose, 0.05 mM MgC12 and centrifuged at 3000 x g for 18 min. The pellet (nucleoli) was then collected. Nucleoli (0.1 g) were suspended in 3 M urea, 1 mM phenylmethylsulfonyl fluoride, 1 mM leupeptin, 1 mM p-ehloromercuriphenylsulfonic acid and were homogenized with a Dounce homogenizer (20 up-and-down strokes) on ice. The extract was then incubated at 60°C for 30 min and centrifuged at 27 000 x g for 20 min at 5°C. The supernatant contained the partially purified protein (generally > 80% homogenous). Extraction of nucleoli under native conditions was done as previously described [11]. Nucleoli were homogenized at 4°C with Tris extraction buffer containing 10 mM Tris (pH 7.4), 0.5 mM MgC12, 1 mM PMSF, 1 mM leupeptin, and then centrifuged at 27000 x g for 20 min. The supernatant containing nucleolar proteins was collected.
Heparin-Sepharose chromatography A heparin-Sepharose column CL-6B (Pharmacia, 4 ml bed volume) was equilibrated with T E D G buffer (50 mM Tris (pH 7.5), 5 mM EDTA, 5 mM DTT, 20% glycerol, 1 mM PMSF) containing 50 mM KC1. The column was then rinsed with T E D G buffer containing 100 # g / m l of BSA, and further washed with 5 column volumes of T E D G buffer. Proteins extracted from purified nucleoli under native conditions ( - 2 mg) were applied on the column. The column was then washed with 5 column volumes of T E D G buffer and then proteins were eluted with a linear 0.15 to 2.0 M KC1 gradient. The fractions which were collected (1-1.5 ml) were monitored for the presence of numatrin by ELISA and by SDS-PAGE.
Gel filtration chromatography
cell suspension was added 5 ml of phenol saturated in 10 m M NaC1, 0.5 M Tris-HC1 (pH 8.0), 10 mM EDTA, 0.5% SDS, 5 ml of 95% chloroform, and 5% isoamyl alcohol. Following centrifugation at 5000 rpm for 15 min, the supernatant was collected, and the D N A was precipitated by adding 2.5 vol. of 95% ethanol and 1 / 5 vol. of 3 M N a O H and further incubation at - 2 0 ° C for 16 h. The D N A was pelleted by centrifugation at 3000 rpm for 30 min. The pellet D N A was dried and then resuspended in 10 mM Tris (pH 7.4), and 0.5 mM EDTA by vortexing and incubation overnight at 4°C. The D N A was sheared by passing through a syringe with a 19" gauge needle.
ELISA procedure ELISA was done as previously described [7]. Microtiter plates (Titertek, 96 wells) were coated with proteins for 2 h at room temperature. The plates were then washed and blocked with phosphate-buffered saline containing 3% BSA at 4°C overnight. The solution was aspirated, and the plates were washed three times with phosphate-buffered saline-Tween. Anti-numatrin antibody (339) was added and further incubated for 30 min. The second antibody, alkaline phosphatase-conjugated goat anti-rabbit, was added and incubated at room temperature for 1 h. The plates were then washed, and 150/.tl of substrate (p-nitrophenyl phosphate) was added and incubated for 15 min (until color developed). Results represent average absorbance at 405 nm of triplicate wells (S.E. less than 10%).
Nitrocellulose filter binding assay Nitrocellulose filter binding assay was done as previously described [17]. Numatrin or BSA (used as control) was mixed with 1.65 /~g of [3H]DNA (43000 dpm) at final volume of 100-150 ~1 and incubated for 10 min at room temperature. The samples were then poured on nitrocellulose filters (Grade BA85, Schleicher and Schuel) and washed with 10 mM Tris (pH 7.4), 0.5 mM EDTA three times under vacuum. The filters were dried, and the radioactivity retained on the filters was monitored by liquid scintillation spectrometer.
The fractions containing numatrin from the heparinSepharose columns were concentrated and applied on Sepharose G-100 column (0.8 × 30 cm, 40 ml bed volume). The proteins were eluted with 10 mM Tris (pH 7.4), 0.5 mM MgC12 and proteinase inhibitors. Fractions were collected (2 ml) and monitored for the presence of numatrin by ELISA and by SDS-PAGE.
Two dimensional gel electrophoresis was performed as previously described [18].
Purification of high molecular weight DNA
Results
HeLa cells (5 × 107) were labeled with 1 mCi of [3H]thymidine for 48 h. The ceils were then suspended in 10 mM NaC1, 10 mM Tris-HCl (pH 8.0), 10 m M EDTA, 0.5% SDS in the presence of 50 /Lg/ml proteinase K, and incubated for 2 h at 37°C. Proteinase K (50 t~g/ml) was added again, and the solution was further incubated at 4°C overnight. The D N A was extracted in phenol/chloroform as follows: to 10 ml of
Two-dimensional gel electrophoresis
Binding of numatrin to DNA cellulose columns Nuclear proteins were extracted from nuclei of HL-60 cells as described in Materials and Methods and applied to three columns: SS-DNA cellulose, DS-DNA cellulose and cellulose (not attached to DNA). The D N A binding proteins were eluted with increasing concentrations of NaC1 (in buffer B) at a step gradient of 0.15, 0.5, 1.0
130 SS-DNA NaCI(M)
.15 .5 1.0 2.0
DS.DNA
Cellulose
.15 .5 1.0 2.O(N) .15 .5 1.0 2.0(N)
v,-
×
iF Fig. 1. DNA-cellulose chromatography. Evidence for numatrin binding to SS-DNA and DS-DNA cellulose columns. Equal amounts of nuclear proteins (extracted as described in Materials and Methods) were applied to three columns: DS-DNA cellulose, SS-DNA cellulose and cellulose (not attached to DNA). The bound proteins were eluted by step wise addition of buffer A (see Methods) containing 0.15, 0.5, 1.0 and 2.0 M NaC1. The eluted proteins were precipitated by acetone, solubilized in SDS-sample buffer and analyzed by SDS-PAGE and Western Blot using anti-numatrin antibody. (N) represents numatrin marker.
and 2.0 M, and the proteins in the eluates were further precipitated with acetone. Equal portions of the precipitated proteins were analyzed by SDS-PAGE, electroeluted onto nitrocellulose membranes and immunoblotted with anti-numatrin antibody. As shown in Fig. 1, numatrin was eluted from both SS-DNA column and DS-DNA columns at 0.5 M and 1.0 M NaC1. Small amounts of numatrin were eluted only by 2.0 M NaC1, indicating very strong binding to DNA. Numatrin did not bind the cellulose column, indicating that the bind-
ing of numatrin to the D N A cellulose columns is not due to non-specific interaction to the cellulose matrix. Proteins eluted from DS-DNA columns (at 1.0 M) were further pooled and analyzed by two-dimensional gel electrophoresis (Fig. 2). Silver staining of the gel (panel A) demonstrated the presence of several prominent D N A binding proteins which were eluted from the column. Several of these proteins have a close electrophoretic mobility to that of numatrin. The immunoblot of a parallel gel using anti-numatrin antibody identified the localization of numatrin (both a and /3 chains) among the other nuclear DNA binding proteins which were eluted from the column. D N A binding of numatrin extracted in urea
The results demonstrated in Fig. 2A raised the possibility-that numatrin binding to the D N A columns might be due to its intimate association with other D N A binding proteins. To address this question, further experiments were undertaken to examine the direct binding of purified numatrin to D N A using the nitrocellulose filter assay. In previous studies we have demonstrated that numatrin can be purified to - 9 0 % homogeneity by extracting nucleoli of HL-60 cells with 3 M urea at 60°C [7]. We have used this urea extract to examine numatrin binding to 3H-labeled SS-DNA and DS-DNA using the nitrocellulose filter assay. [3H]DNA was pre-
Silver stain Acid 66--
Immunoblot Base qb
45--
I O
x
31--
=E
21--
14-Fig. 2. Two dimensional gel electrophoresis and immunoblot analysis of proteins eluted from D N A cellulose chromatography. Proteins eluted from DS-DNA column at 1.0 M NaCI were analyzed by two-dimensional gel electrophoresis, and the gel was further stained by silver staining (Bio-Rad kit). A parallel gel was transferred onto nitrocellulose and immunoblotted by anti-numatrin antibody. Arrows indicate the position of numatrin on the gel.
131 400
pared by labeling HeLa cells with [3H]thymidine for 24 hr. After lysing the cells and extensive treatment with proteinase K, the high molecular weight D N A was purified using phenol/chloroform extraction. The specific activity of the D N A was 26 000 dpm//~g. Single stranded heat-denatured D N A was prepared by boiling a part of the purified DS-DNA. Thus, the DS-DNA and the SS-DNA were comparable in every respect. The nitrocellulose D N A binding assay was done as described in Materials and Methods. In short, 1.65 #g of either [3H]SS-DNA or [3H]DS-DNA was mixed with purified numatrin (urea extract) in a total volume of 200/~1 and incubated for 10 min at room temperature. The [3H]DNA-protein complex was then collected and washed on nitrocellulose filters as described in Materials and Methods. These experiments demonstrated (Fig. 3) that numatrin binds to both DS-DNA and SS-DNA in a dose-dependent manner. However, its binding to DS-DNA was 3-15-fold higher than its binding to SS-DNA in various experiments. In the experiment shown in Fig. 3 (bottom) under control conditions (no protein) 120 dpm were retained on the nitrocellulose filters. Addition of 13/~g of numatrin resulted in 3725 dpm associated with SS-DNA (31-fold increase) and 14259 dpm associated with DS-DNA (119-fold increase). Equal amounts of BSA which were used as control did not show any binding to D N A (data not shown).
4000 "
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10000 ~
~
~ e'=
SS-DNA
•
DS-DNA
5000
0 5
10
15
Numatrin(pg)
Fig. 3. DNA binding of numatrin extracted in urea. Preferential binding to DS-DNA. Binding of purified numatrin ( - 4.0 /~g) extracted in 3 M urea [7] to [3HIDS-DNA and [3H]SS-DNA was examined by using the nitrocellulose filter assay as described in Materials and Methods. In experiment 2, SS-DNA was obtained by boiling DS-DNA just prior to performing the D N A binding study. Experiment 3 (bottom) represents a dose-dependent increase in numatrin binding to D N A . Results represent a typical experiment (average dpm of duplicate samples with less than 10% variability).
300 SS-DNA n
20o
•
[ ] Numatrin 100
o salt
+
salt
6000 -
.00o1 t~
/ 2000"
0
[ ] Numatrin
. . . . .
salt
"
+
salt
Fig. 4. Preferential effect of salt on numatrin binding to DS-DNA as compared to SS-DNA. Binding of purified numatrin ( ~ 4.0 #g) extracted in 3 M urea to [3H]SS-DNA and [3H]DS-DNA was examined in the presence or absence of 0.5 M NaC1, using the nitrocellulose filter assay. Results represent average dpm of duplicate samples with less than 10% variability.
When a similar experiment was performed in the presence of NaC1 (at a final concentration of 0.5 M) it was found that the binding of numatrin to D S - D N A was reduced by 77% while the binding of numatrin to SS-DNA was not at all affected (Fig. 4). These results indicate that the binding of numatrin to SS-DNA when compared to its binding to DS-DNA is different quantitatively as well as qualitatively. D N A binding of numatrin extracted under native conditions The nucleolar protein B23 was reported to be an hexamer under native conditions, and to dissociate into monomers of - 4 0 kDa in the presence of urea [11]. Thus, the D N A binding of the purified numatrin in urea extract (Fig. 3) indicates that numatrin can bind D N A directly under monomerie conditions. Further experiments were undertaken to examine whether numatrin can bind D N A also as an oligomer when extracted under native conditions. To this end, nucleoli were purified from HL-60 cells and extracted as previously described [11] in 10 mM Tris (pH 7.4), 0.5 mM MgC12 and proteinase inhibitors. As shown in Fig. 5, this extraction condition results in variable yields of numatrin. The extraction in experiment 2 (in Fig. 5) demonstrates - 9 0 % purified numatrin. This material was used for study of numatrin binding to DNA, using the nitrocellulose filter assay. As shown in Table IA, numatrin extracted under native conditions (oligomer)
132
2
34
A. ELISA
56
2.0
.70
I
o v
x
1.0
.35
==
Fig. 5. Extraction of numatrin under native conditions. Nucleoli were purified from HL-60 cells and extracted by homogenization in 10 mM Tris (pH 7.4), 0.5 mM MgCl2 and proteinase inhibitors. The suspension was further centrifuged at 11000 rpm for 20 min. The supernatant was collected and analyzed by SDS-PAGE. The results represent Coomassie blue staining of six different experiments. 0
15
30
45
Fraction Number
b o u n d both SS-DNA and D S - D N A . However, similarly to numatrin extracted in urea (Fig. 3) the binding to D S - D N A was several-fold higher as c o m p a r e d to the binding to SS-DNA. T o c o m p a r e the binding of numatrin as an oligomer to its binding as a m o n o m e r , the Tris extract was heated in the presence of 3 M urea (conditions which were previously shown to dissociate B 2 3 / n u m a t r i n into monomers). As shown in Table I, the dissociation of numatrin to m o n o m e r s resulted in 3.2-fold enhancement of binding to SS-DNA, but had only a small effect on its binding to D S - D N A . This is in agreement with the previous results (Fig. 4) which indicated that the nature of numatrin binding to D S - D N A is qualitatively different from its binding to SS-DNA.
TABLE I
DNA binding of numatrin extracted under native conditions. Preferential binding to DS-DNA N u m a t r i n extracted u n d e r native conditions (see e x p e r i m e n t 2 in Fig. 5) was used to e x a m i n e D N A b i n d i n g in the nitrocellulose filter assay. Results (of a typical experiment) represent average d p m of duplicates with less t h a n 10% variability. To c o m p a r e b i n d i n g of n u m a t r i n ( - 2.0 t~g) as an oligomer to its b i n d i n g as a m o n o m e r (A) the native extract was heated to 6 0 ° C in the presence of 3 M urea a n d D N A b i n d i n g was further examined. In (B), the n u m a t r i n extract was treated with 5 ~ t g / m l of R N A s e A (Sigma) for 60 rain at r o o m t e m p e r a t u r e a n d the b i n d i n g to D N A was further e x a m i n e d u s i n g the nitrocellulose filter assay. (A)
Protein
Oligomer
SS-DNA
+
47 393
DS-DNA
+
34 4 921
B. Coomassie blue stain Fraction Number Lead 11 12 13 14 15 16 17
18
30
i
92- i 66--! I
4~-
X
~E~
: :
31ii
ZI--
Fig. 6. Purification of n u m a t r i n by heparin-Sepharose column. Nucleolar proteins extracted u n d e r native conditions (see Fig. 5) were loaded on a h e p a r i n - S e p h a r o s e c o l u m n CL-6B (4 ml bed volume). T h e u n b o u n d proteins were w a s h e d with T E D G b u f f e r (see Materials and M e t h o d s ) a n d then the b o u n d proteins were eluted with linear gradient of KC1 r a n g i n g from 0.05 to 2.0 M. Fractions (1-1.5 ml) were collected a n d m o n i t o r e d for the presence of n u m a t r i n by ELISA (A) a n d by S D S - P A G E (B). T h e peak elution of n u m a t r i n was obtained at 0.7 M KCI.
F u r t h e r experiments were undertaken to examine the D N A binding of numatrin after treatment of the extract with R N A s e A. This treatment had only a small effect on increasing the binding of numatrin to SS-DNA. Thus, under this condition numatrin still b o u n d significantly better to D S - D N A indicating that the lower level of n u m a t r i n binding to S S - D N A as c o m p a r e d to DSD N A is not due to competition of the SS-DNA with R N A which m a y be present in the Tris extract.
Monomer/ Oligomer
Chromatography purification of numatrin by heparin-Sepharose column
82 1280
3.2
15 6 067
1.2
F u r t h e r experiments were undertaken to purify numatrin to homogeneity by c h r o m a t o g r a p h y techniques. Nucleoli were purified from 2 g of HL-60 cell nuclei and further extracted in 10 m M Tris (pH 7.4), 0.05 m M MgC12 (Tris extract, see Fig. 4). 2 ml of extracted material which contained about 2 mg of nucleolar proteins were applied on heparin-Sepharose CL-6B column and extracted with T E G P buffer at a continuous gradient of KC1 ranging from 50 to 2000 mM. The fractions were analyzed for the presence of
Monomer
(B) SS-DNA
Protein +
- RNAse 79 472
+ RNAse 49 612
DS-DNA
+
15 3210
18 3395
133
A. ELISA
were applied on a gel filtration Sephadex G-100 column and the proteins were eluted with Tris extraction buffer. The fractions were screened for the presence of numatrin by ELISA and by silver staining of SDS-PAGE. As shown in Fig. 7, the major peak of numatrin was eluted at fraction 6, corresponding to the elution of catalase which was used as a molecular mass marker (232 kDa) to indicate the void volume of the column. This result indicates that the natively purified numatrin is indeed not found as a monomer of 40 kDa, but rather is found in oligomeric form as previously reported [11]. The minor peak which was detected by ELISA may be the monomeric form of numatrin. However, it could not be detected by silver staining of the gel (data not shown).
Catalyse (232 kDa)
t
.5@
g
c u~ o qr
.25
DNA binding of numatrin following heparin.Sepharose
chromatography Further studies examined the D N A binding of numatrin -purified by heparin-Sepharose using the nitrocellulose filter assay. The results in Fig. 8 show that numatrin binds both SS-DNA and D S - D N A in a dose-dependent manner, thus confirming our previous .00
I 5
0
, I 26
50 SS DNA
Fraction Number 300
A
B. Sliver Stain
DS D N A
EXP 1
EXP 1
B
H SS D N A e--~ DS D N A BSA
Fraction Number 4
5
6
7
8
9
10
11
12
13
14
15 200(
j
n c3 I
100(
X
d 00"
I.J 16 32
I 64
I 128
I
I
I
J
• 16
32
64
128
PROTEIN (picornoles) EXP 2
Fig. 7. Gel filtration chromatography of purified numatrin. Fractions from the heparin-Sepharose column which contained numatrin were loaded on gel filtration column Sephadex G-100 and eluted with Tris extraction buffer. The fractions were monitored for the presence of numatrin by ELISA (A) and by SDS-PAGE (B). Gel (in B) was stained by the silver staining method.
numatrin by ELISA and by SDS-PAGE (Fig. 6). Numatrin was eluted at - 0 . 7 M KC1 as shown by ELISA and by SDS-PAGE. The recovery of numatrin after the chromatography purification was about 40%. (Silver staining of fraction 16 indicated only one band, data not shown). To examine whether numatrin which was eluted from the heparin-Sepharose column is an oligomer or is found as a monomer, the fractions which contained numatrin
2000
C
EXP 2
/
D
/
a. 1000 D
I
I
0~ 16 32
I
6i4
128
--I
I
16 32 PROTEIN 3icomoles)
I
J
64
128
Fig. 8. Binding of numatrin purified by heparin-Sepharose chromatography to D S - D N A and SS-DNA. Numatrin purified from heparinSepharose column (or comparable amounts of BSA which were used as control) were mixed with [3H]SS-DNA or [3H]DS-DNA, and the binding to D N A was examined using the nitrocellulose filter assay. Experiments 1 and 2 were identical. Results represent average dpm of duplicate samples with less than 10% variability.
134 Purified numatrin 4000
Numatrin +a 110 kDa protein
A
B SS DNA DS-DNA o--,o
D
2000
0~'
64
128
I
I
64
128
PROTEIN 3icomoles) C
T FI# 1
~" 97 6 8 ~ x 45
2
3
4
5
6
7
8
9
"0
11 12 13 14
15
~:
numatrin in two purification steps [19,20]; (a) PEGtreated NE/S-3 supernatant which contains the low salt nuclear extraet-postmicrosomal supernatant; and (b) the final purified fraction of Q-Sepharose chromatography which contained the purified DNA polymerase c~ primase activity [20]. Equal amounts of proteins of these two fractions (kindly provided by Dr. Earl F. Baril, Worcester Foundation, MA) were applied on SDS-PAGE and transferred onto nitrocellulose membrane, Using immunoblotting technique with anti-numatrin antibody, we show that purification of DNA polymerase a primase activity is associated with copurification of numatrin. indicating that numatrin is found in complex with the DNA polymerase c~ primase. Discussion
Fig. 9. A protein at a 110 kDa which copurifies with numatrin on heparin-Sepharose column conferred a preferential enhancement of binding to SS-DNA. (C) Coomassie blue staining of SDS-PAGE of fractions eluted from heparin-Sepharose column loaded with nucleolar proteins (as described in Fig. 6). Binding of purified numatrin (fractions 9 and 10) to SS-DNA and DS-DNA is shown in (A). DNA binding of numatrin copurified with the 110 kDa protein (fractions ll and 12) is shown in (B).
results in this work. Significant DNA binding could be detected at 64 pmol of numatrin and was dramatically increased at 128 pmol. Notably, following chromatography purification, numatrin did not demonstrate a preferential binding to DS-DNA as found in the previous experiments. This might be due to the presence of high salt concentration in the purified fraction (0.7 M KC1). In certain preparations of heparin-Sepharose column etution of purified numatrin was followed by copurification of a 110 kDa protein together with numatrin (see fractions 11, 12, 13 in Fig. 9). The fractions which contained purified numatrin and the fractions which contained mixture or numatrin and the 110 kDa protein were pooled separately and assayed for DNA binding. As shown in Fig. 9 (panels A and B), the-presence of the 110 kDa protein conferred a preferential enhancement of binding to SS-DNA, but it did not affect at all the binding to DS-DNA.
Evidence that numatrin copurifies with the complex of DNA polymerase a primase It has been shown that the cell cycle regulated nuclear protein PCNA/cyclin [1] coeluted with the purified complex of DNA polymerase a primase [20] together with other enzymes which are required for in vitro DNA replication of SV-40, such as DNA-dependent ATPase, DNA ligase, RNAse H and topoisomerase I [19-21]. To examine whether numatrin is also found in this purified complex, we examined the presence of
The results of this study demonstrate that numatrin is a DNA binding protein. It binds both DS-DNA and SS-DNA as shown by using DNA cellulose chromatography and by using the nitrocellulose filter assay. Evidence is presented to suggest that there are certain differences in numatrin interaction with DS-DNA as compared to its interaction with SS-DNA: (a) Studies using numatrin extracted under native conditions or under urea conditions showed a several-fold higher binding affinity of numatrin to DS-DNA as compared to SS-DNA. This difference could not be overcome by pretreatment of numatrin with RNAse, indicating that the lower affinity to SS-DNA is not due to competition with RNA, which may be present in the preparation. (b) The binding to DS-DNA demonstrated a high sensitivity to the ionic strength as it was reduced by 77% in the presence of 0.5 M NaC1, while the binding to SS-DNA was not at all affected under this ionic condition. This indicates that the binding to SS-DNA may involve less ionic interaction as compared to the binding to DSDNA. (c) Treatment of the native numatrin under conditions which cause monomerization of the protein resulted in a significant enhancement of numatrin binding to SS-DNA but did not affect the binding of numatrin to DS-DNA, implying that the affinity of numatrin to SS-DNA may be higher as a monomeric form as compared to the oligomeric form, while this factor is not pertinent in numatrin binding to DS-DNA. Taken collectively, these data indicate that numatrin binding to DS-DNA is qualitatively and quantitatively different from its binding to SS-DNA, However, the higher affinity to DS-DNA could not be demonstrated following heparin-Sepharose chromatography. Since numatrin was eluted from the column at 0.7 M KCI, it is possible that the presence of the salt in the preparation of the purified protein is responsible for the decrease in theaffinity to DS-DNA (as was demonstrated the binding to DS-DNA in contrast to
135 the binding to SS-DNA was significantly reduced with the increase in the ionic strength of the buffer). Since we could not remove the salt without losing the protein, the discrepancy of the results regarding the preferential affinity to DS-DNA could not be resolved at this point. However, the results of this paper dearly demonstrate that numatrin can bind significantly to DNA, both DS-DNA and SS-DNA. The T antigen of SV-40 has been shown to bind DS-DNA as well as SS-DNA in two different mechanisms: its binding to DS-DNA is sequence specific while its binding to SS-DNA is nonspecific [22]. It has been suggested that its binding to DS-DNA may be involved in initiation of replication while its binding to SS-DNA may be involved in further unwinding and helix destabilization [22]. Extensively characterized SSDNA binding proteins such as UPI, adenovirus DBP, T4 gene and 32 protein were often implied in DNA unwinding, helix destabilization and stimulation of the activity of their cognate DNA polymerase (for review see Ref. 23). Studies on nuclear onco-proteins have demonstrated that certain of these proteins such as myc [24] and fos [25] bind to DNA nonspecifically, while others such as jun [26] and myb [27] are sequencespecific DNA binding proteins. Both )Cosand jun were implicated as transcription factors [26,28]. Interestingly, it has been shown that interaction between los and jun (via the leucine zipper domain) resulted in enhancement of the binding of jun to its specific DNA binding domain [29,30]. This demonstrates a novel mechanism by which a nonspecific DNA binding protein may exert a crucial physiological effect via its binding to a sequence-specific DNA binding protein. In this regard the finding that a protein at 110 kDa which copurified with numatrin in certain column fractions, conferred an enhancement of the binding to SS-DNA but not to DS-DNA is of interest. Thus, it is possible that other proteins (such as this 110 kDa protein) associate with numatrin and by this association regulate its binding to DNA. Conversely, numatrin binding to other proteins may regulate their binding to DNA. These potential p r o t e i n / D N A interactions are particularly pertinent to the finding that numatrin copurifies with DNA polymerase a primase (Fig. 10). This observation indicates that numatrin is found in association with a complex of enzymes which are required for in vitro DNA replication [19-21] and thus may have a physiological significance in the enzymatic activity of this complex. Studies are currently in progress to examine this possibility. The physiological significance of numatrin binding to DNA and its complexing with DNA polymerase a primase may be of particular importance in view of the finding that B23 (numatrin) has been shown to translocate from the nucleolus to the nucleoplasm when RNA synthesis is inhibited [15]. Taken collectively with
a 9?. " ,-. ~
b (o) N E / S - 5
~
6 6 .....,~ ~
~ (b)Purified
~ x45- ~i~!~'
supernatant
DNApolymerase
a primase fraction from Q - Sepharose chromatography
:E Fig. 10. Numatrin copurifies with the complex of DNA polymerase a primase. Equal amounts of proteins of two purification steps in the process of purification of DNA polymerase a primase [19-21] were applied on SDS-PAGE, transferred onto nitrocellulose membrane and immunoblotted with anti-numatrin antibody. (a) NE/S-3 supernatant (containing the low salt nuclear extract postmicrosomal supernatant); and (b) the final purified fraction of Q-Sepharose chromatography which contains the purified complex of DNA polymerase a primase activity [20].
the evidence that numatrin accumulation in the cells is correlated with entry and progression of the cells through S-phase [6], namely a phase which is associated with a decrease in RNA synthesis and an increase in DNA synthesis, it may be postulated that the ability of numatrin to bind DNA and its association with DNA polymerase a primase may indicate that numatrin may have different functions during different phases of the cell cycle, and it may be involved in DNA-associated processes. Acknowledgments The authors would like to thank Dr. Earl Baril (Worcester Foundation, MA) for providing the chromatography purification fractions of DNA polymerase a primase, and Ms. Mary Wise for excellent secretarial assistance. This work was supported by National Institutes of Health Grants R29 CA48667-01, RO-AI24273 and AI18449, and Uniformed Services University of the Health Sciences Protocol RO83BQ. References 1 Celis, J. and Cells, A. (1985) Proc. Natl. Acad. Sci. USA 82, 3262-3266. 2 Bishop, J.M. (1985) Cell 42, 23-38. 3 Reich, N.C. and Levine, A.J. (1984) Nature 308, 199-201. 4 Feuerstein, N. and Mond, J.J. (1987) J. Immunol. 139, 1818-1822. 5 Feuerstein, N. artd Mond, J.J. (1987) J. Biol. Chem. 262, 1138911397. 6 Feuerstein, N., Speigel, S. and Mond, J.J. (1988) J. Cell Biol. 107, 1629-1642. 7 Feuerstein, N., Chan, P.K. and Mond, J.J. (1988) J. Biol. Chem. 263, 10608-10612. 8 Kang, Y.J., Olson, M.O.J. and Busch, H. (1974) J. Biol. Chem. 249, 5580-5585. 9 Kang, Y.J., Olson, M.O.J. and Busch, H. (1975) Cancer Res. 35, 1470-1475. 10 Chan, P.K., Aldrich, M.B., Cook, R.G. and Busch, H. (1986) J. Biol. Chem. 261, 1868-1872.
136 11 Yung, B.Y-M. and Chart, P.K. (1987) Bioehim. Biophys. Acta 925, 74 82. 12 Chart, W-Y., Liu, Q-R., Borjigin, J., Busch, H., Rennert, O.H., Tease, L.A. and Chan, P.K. (1989) Biochemistry 28, 1033-1039. 13 Chang, J-H., Dunbar, T.S. and Olson, M.O.J. (1988) J. Biol. Chem. 263, 12824-12827. 14 Spector, D.L., Och, R.I. and Busch, H. (1984) Chromosoma 90, 139-148. 15 Yung, B.Y.M., Busch, H. and Chan, P.K. (1985) Biochim. Biophys. Acta 826, 167-173. 16 Alberts, B. and Herrick, G. (1962) Methods Enzymol. 210, 198217. 17 Ruyechan, W.T. (1983) J. Virol. 46, 661-666. 18 Feuerstein, N. and Cooper, H.L. (1983) J. Biol. Chem. 258, 1078610793. 19 Baril, E.F., Malkas, L.M., Hickey, R.H., El, C.-J., Coughlin, S. and Vishwanatha, J.K. (1988) in Cancer Cells 6: Eukaryotic DNA Replication (Kelly, T.J. and Stillman, B., eds.), pp. 373-384, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 20 Hickey, R.J., Malkas, L.H., Pedersen, N., Li, C. and Baril, E.F. (1988) in DNA Replication and Mutagenesis (Moses. R. and
21 22 23 24 25 26 27 28 29 30
Summers, W., eds.), pp. 41-54, American Society of Microbiology Publications, Washington, DC. Malkas, L.M. and Baril, E.F. (1989) Proc. Natl. Acad. Sci. USA 86, 70-74. Aubom, K.J., Markowitz, R.B., Wang, E., Yu, Y.T., and Prives, C. (1988) J. Virol. 62, 2204-2208. Chase, J.W. and Williams, K.R. (1986) Annu. Rev. Biochem. 55, 103-136. Watt, R.A., Shatzman, A.R. and Rosenberg, M. (1985) Mol. Ceil. Biol. 5,448-456. Sambucetti, L.C. and Curran, T. (1986) Science 234, 1417-t419. Angel, P., Allegretto~ E.A., Okino, S,T., Hattori, K,, Boyle, W.J., Hunter, T. and Karin, M. (1988) Nature 332, 166-171. Biedenkapp, H., Borgmeyer, U., Sippel, A.E. and Klempnauer, K.H. (1988) Nature 335, 835-837. Setoyma, C.R., Frunzio, F., Lian, G., Mudryj, M. and (_rombrugghe, B.D. (1986) Proc. Natl. Acad. Sci. USA 83, 3213-3217. Kouzarides, T. and Ziff, E. (1988) Nature 336,646 651. Sassone-Corsi, P., Ransone, L.J., Lamph, W.W. and Verma, I.M. (1988) Nature 336, 692-695.
