Plant Molecular Biology 11:3-14 (1988) © KluwerAcademic Publishers, Dordrecht - Printed in the Netherlands
Pea chloroplast topoisomerase I: purification, characterization, and role in replication Brent L. Nielsen and K. K. Tewari Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92717, USA Received 16 November 1987; accepted in revised form 25 February 1988
A DNA-relaxing enzyme was purified 5 000-fold to homogeneity from isolated chloroplasts of Pisum sativum. The enzyme consists of a single polypeptide of 112 kDa. The enzyme was able to relax negatively supercoiled DNA in the absence o f ATP. It is resistant to nalidixic acid and novobiocin, and causes a unit change in the linkage number o f supercoiled DNA. The enzyme shows optimum activity at 37 °C with 50 mM KC1 and 10 mM MgCl 2. From these properties, the enzyme can be classified as a prokaryotic type I topoisomerase. Using a partially purified pea chloroplast DNA polymerase fraction devoid of topoisomerase I activity for in vitro replication on clones containing the pea chloroplast DNA origins o f replication, a 2 - 6-fold stimulation o f replication activity was obtained when the purified topoisomerase I was added to the reaction at 7 0 - 1 0 0 mM KC1. However, when the same reaction was carried out at 125 mM KCI, which does not affect DNA polymerase activity on calf thymus DNA but is completely inhibitory for topoisomerase I activity, a 4-fold drop in activity resulted. Novobiocin, an inhibitor of topoisomerase II, was not found to inhibit the in vitro replication of chloroplast DNA.
The chloroplast genome in higher plants consists of a double-stranded, covalently closed circular DNA molecule of 120-160 kpb . The structural organization o f chloroplast DNA (ctDNA) has been well characterized [2, 3, 10] and genes for proteins, rRNAs and about 30 tRNAs have been identified and mapped in ctDNA [3, 4, 18, 21, 22]. The transcription o f ctDNA has been studied using crude chloroplast extracts and recombinant DNA molecules containing rRNA, tRNA and protein genes . Replication of ctDNA has been known to proceed by the introduction of D-loops and the replication intermediates that have been identified could arise from a Cairns replication model and by the rolling-circle mechanism . The origin of replication in Chlamydomonas ctDNA has been mapped and sequenced [26, 28]. The recombinants
containing the origin of replication of Chlamydomonas ctDNA have been shown to undergo replication in vitro using crude extracts from Chlamydomonas . Similarly, an EcoRI DNA fragment from corn ctDNA, containing sequences present in the Chlamydomonas origin of replication, has been found to be a highly active template in an in vitro DNA synthesis sytem . Recently, Meeker et al.  have mapped the two D-loops on the 12.5 kbp PstI fragment in pea ctDNA, and a clone of this fragment has been found to be an excellent template in an in vitro replication system developed from pea chloroplasts. In spite of the progress made in identifying replicative intermediates of ctDNA and identifying the origins of replication, the proteins that are involved in the replication of ctDNA have not been well studied. Our long-term goal is to identify the proteins that are involved in replication of pea
ctDNA and to analyze the biochemical steps in replication by reconstituting the in vitro replication system using pure proteins. Towards this end, we previously reported a purification procedure which resulted in an essentially homogeneous preparation of DNA polymerase from pea chloroplasts . The chloroplast DNA polymerase has been shown to be involved in replication . The presence o f topoisomerase I and II has been shown in crude fractions of pea chloroplasts , and the topoisomerase I from spinach chloroplasts has been partially purified and characterized, and identified as being prokaryotic-like . We present here the first report of the purification o f a chloroplast topoisomerase I to a homogeneous preparation of a single polypeptide of 112 kDa. The complete purification o f topoisomerase I from DNA polymerase and other proteins involved in replication of ctDNA has enabled us to show that topoisomerase I is required for the optimal level o f replication in vitro.
DNApolymerase assay Ultra-pure ATP, GTP, CTP, UTP, and dATP, dGTP, dCTP, and T T P were from Pharmacia. 3H-TTP (75 Ci/mmol) was from New England Nuclear, and 32p-TTP (3000 Ci/mmol) was from ICN. DNA polymerase activity was assayed as described before .
Column chromatography DEAE cellulose (DE 52) was obtained from Whatman and used without further treatment. Cellulose phosphate Pll (Whatman) was precycled by first washing with 0.5 N HCI in 50°70 ethanol, followed by repeated washing with water and finally with 0.5 N NaOH. After the alkali treatment, the phosphocellulose was washed with water until neutral. Heparin-Sepharose and Superose-12 gel filtration columns were from Pharmacia.
Materials and methods
DNA Isolation of chloroplasts Purified chloroplasts from discontinuous sucrose gradients were suspended in 2 0 - 4 0 ml buffer A (50 mM Tris-HCl pH 8.0, 50 mM 2mercaptoethanol, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 25 °70 glycerol) containing 0.025 M NaCI, and stored frozen at - 7 0 ° C until used for protein isolation.
Plasmid pBR322 DNA, pCP12-7 (containing the 12.5 kbp Pst I ctDNA fragment) and pCBI-12 (containing the 10 kbp Bam HI ctDNA fragment, see Fig. 7) were purified through two rounds of cesium chloride/ethidium bromide centrifugation as described before .
SDS-gel electrophoresis Topoisomerase assay The topoisomerase activity was assayed using 0.6/zg of supercoiled pBR322 DNA in a 20 #l volume containing 50 mM Tris-HC1 pH 8.0, 10 mM MgC12, I mM dithiothreitol. A typical reaction contained 5 #l o f enzyme. After incubation at 37 °C, the reaction was stopped by adding 4/zl of 2007o Ficoll containing 0.5°70 SDS and 0.5°7o bromophenol blue. The samples were heated to 65 °C for 2 min and then analyzed in a 0.8°7o agarose gel using Tris-acetate buffer .
Samples from the various stages of purification were analyzed in 10°70SDS polyacrylamide gels according to the method o f Laemmli , and stained with either Coomassie Blue or the BioRad silver stain.
Protein assay Protein concentrations of the various fractions were determined using the BioRad protein assay system.
In vitro replication Replication assays were carried out in a 100/~1 reaction mixture containing 50 mM Tris-HC1 (pH 7.0), 12 mM MgCI 2, 70-125 mM KCI, 100/xM each o f dATP, dCTP, dGTP and 1/~Ci of [3H]-TTP, 10 ttM each of ATP, GTP, U T P and C T P along with 1 /~g of supercoiled DNA templates, and 10/~g o f the topoisomerase I-deficient replication system and, when present, 20 units (0.25/zg) topoisomerase I. For the analysis of products o f DNA synthesis, assays were carried out as described above except that 25/~Ci of [ot-a2p]-TTP was used instead o f [3H]TTP, the concentration of dATP, dGTP, d C T P was decreased to 20/~M and 2/~M o f cold T T P was included. After 30 min at 37°C, the reaction was stopped by adding 10/~1 of 3 M sodium acetate, 20 mM EDTA and 5/~1 o f 10070 SDS. The mixture was extracted with phenol-chloroform, precipitated by ethanol and the sample analyzed in a 0.9070 agarose gel in Tris-acetate-EDTA buffer. After electrophoresis for 18 h at 30 V, the gel was washed with double-distilled water for 10 rain, the DNA bands fixed with 5070 TCA for 30 min, and washed again with water for 10 min. The gel was then dried between many layers of filter paper and exposed for 18 hours against Kodak X-OMat film using an intensifying screen.
Other materials PMSF, novobiocin, nalidixic acid, agarose and heparin were purchased from Sigma. Calf thymus topoisomerase I was purchased from BRL. Purified E. coli topoisomerase I was the generous gift o f Jim Hejna, Department o f Molecular Biology and Biochemistry, University of California, Irvine.
Purification o f topoisomerase The purified chloroplasts were disrupted with Triton X-100 as described before  and the chloroplast supernatant was loaded on a 20 ml DEAE-cellulose
column equilibrated with buffer A containing 0.025 M NaC1. After loading, the column was washed with 100 ml of the same buffer. The bound proteins were eluted with a 100 ml gradient from 0.025 M NaC1 to 0.425 M NaC1 in buffer A. The enzyme fractions were assayed for DNA polymerase and topoisomerase activities as described in Materials and methods. The peak fractions o f topoisomerase activity eluted just before the peak DNA polymerase activity from the DEAE-cellulose column at about 0.15 to 0.28 M NaC1 (Fig. 1). The pooled topoisomerase activity containing about 10-15°70 of the DNA polymerase activity was dialyzed against buffer B (buffer A + 0.1070Triton X-100 and 0.1 mM EDTA) containing 0.1 M NaC1. The dialyzed enzyme was loaded over a 10 ml heparin-Sepharose column equilibrated with the dialysis buffer. The column was washed with 15 ml buffer B + 0.1 M NaC1, followed by 40 ml buffer B + 0.3 M NaCI. No topoisomerase activity was eluted with either of these washes. The enzyme was eluted with 0.6 M NaCI in buffer B and dialyzed against buffer A + 0.1 M NaC1. The enzyme fractions from the heparin-Sepharose column were loaded onto a 1.5 ml phosphocellulose column equilibrated in buffer A + 0.1 M NaCI. The column was washed with buffer A + 0.1 M NaC1 followed by buffer A + 0.35 M NaCI, which removedthe remaining DNA polymerase activity, and the topoisomerase was eluted with buffer A + 0.70 M NaC1 (Fig. 2). The progress o f purification of the enzyme through various columns is shown in Table 1. It may be pointed out that the enzyme fractions from the heparin-Sepharose column could only be assayed after dialysis because some of the heparin always bled from the column and strongly inhibited the topoisomerase reaction at very low concentration (see Table 2). In order to determine the level of purification of the enzyme, one unit of topoisomerase was taken as the amount of enzyme required to catalyze relaxation of 50°7o of the supercoiled DNA molecules from 1.0/~g of pBR322 DNA under our experimental conditions. About 61070 o f the original topoisomerase activity was obtained in the heparinSepharose fraction with 240-fold purification. At this stage of the purification, the enzyme fraction
Fig. 1. Elution profile of the DEAE cellulose column. The column was loaded and washed as described in the text, and then eluted with a 100 ml gradient of buffer A containing 0.025 M NaCl to 0.425 M NaC1. The molar salt concentration is indicated by the dashed line. Topoisomerase activity ( • ) is indicated as the number of units (defined in Table 1) in 5/~1 of sample. DNA polymerase activity ( • ) was assayed as described, and is expressed as CPM × 10-3. The inset photograph illustrates agarose gel analysis of topoisomerase activity, with the lower band being supercoiled plasmid DNA, and the upper band relaxed circular DNA. Lane C is DNA without enzyme added. Numbers indicate the elution volume of the column.
c o n t a i n e d n u m e r o u s b a n d s . W h e n t h e e n z y m e was f r a c t i o n a t e d o n t h e p h o s p h o c e l l u l o s e c o l u m n , the recovery o f the e n z y m e was r e d u c e d to 21.5%. T h e e n z y m e h a d been p u r i f i e d a b o u t 2 2 0 0 - f o l d at this step. W h e n the p h o s p h o c e l l u l o s e f r a c t i o n was p a s s e d t h r o u g h a F P L C Superose-12 sizing c o l u m n , t h e e n z y m e was p u r i f i e d a b o u t 5 000-fold with 7.7°7o recovery o f the enzyme.
Molecular size of the topoisomerase and polypeptide composition A t every step o f t h e p u r i f i c a t i o n , the p o l y p e p t i d e c o m p o s i t i o n o f the fractions was a n a l y z e d by S D S gel electrophoresis. T h e p h o s p h o c e l l u l o s e f r a c t i o n c o n t a i n e d a n u m b e r o f p o l y p e p t i d e s a l o n g with a
p o l y p e p t i d e o f a b o u t 110 k D a (Fig. 3). I n o r d e r to f i n d o u t the m o l e c u l a r size o f the native t o p o i s o m e r ase a n d to remove o t h e r c o n t a m i n a t i n g p o l y p e p tides, a n a l i q u o t o f p e a k e n z y m e activity f r o m the p h o s p h o c e l l u l o s e c o l u m n was f r a c t i o n a t e d o n a n F P L C Superose-12 gel f i l t r a t i o n c o l u m n . T h e c o l u m n was e q u i l i b r a t e d a n d eluted with b u f f e r A + 0.1 M NaC1 at a c o n s t a n t rate o f 0.20 m l / m i n a n d the e l u t i o n p r o f i l e was m o n i t o r e d at 280 nm. F r a c tions o f 0.25 ml were collected a n d assayed for activity. T h e m o l e c u l a r weight o f t h e e n z y m e f r o m the F P L C gel f i l t r a t i o n c o l u m n was d e t e r m i n e d to be 112 k D a by c o m p a r i s o n with a m i x t u r e o f p r o t e i n s t a n d a r d s c o n t a i n i n g ferritin, catalase, b o v i n e s e r u m a l b u m i n a n d c y t o c h r o m e c (Fig. 4). W h e n the p e a k f r a c t i o n f r o m t h e Superose-12 c o l u m n was a n a l y z e d by S D S gel electrophoresis a n d silver-stained, o n l y
Fig. 2. Elution profile of the phosphocellulose column. The column was loaded and washed as described in the text. Remaining DNA polymerase activity ( • ) was eluted with buffer A + 0.35 M NaC1. Topoisomerase activity ( • ) was eluted with buffer A + 0.7 M NaCI. Activity units are defined as in Fig. 1. The inset Photograph shows the topoisomerase activity of the 0.35 M elution and 0.7 M NaC1 elution.
Table L Purification of pea chloroplast topoisomerase. Fraction
Lysed chloroplasts DEAE-cellulose Heparin-Sepharose Phosphocellulose FPLC Superose-12
520 5.1 1.32 0.05 0.008
26000 b 25000 16 000 5600 2000
50 4900 12120 112000 250000
98 242 2240 5000
96 61.5 21.5 7.7
a One unit of topoisomerase is defined as the amount of enzyme required to catalyze relaxation of 50% of the supercoiled DNA molecules from 1.0/~g pBR322 DNA in 30 min at 37°C. b This value is an estimate due to the presence of nucleases at this step which mask the topoisomerase activity.
o n e p o l y p e p t i d e o f 112 k D a w a s f o u n d t o b e p r e s e n t
Fig. 5. T h e a c t i v i t y o f t h e p h o s p h o c e l l u l o s e - p u r i f i e d
( F i g . 3, p a n e l B).
a linear time
120 m i n . T h e e n z y m e r e m a i n e d a c t i v e o v e r a b r o a d r a n g e o f t e m p e r a t u r e ( 1 2 - 37 ° C ) , w i t h p e a k a c t i v i t y a t 37 ° C, b u t t h e e n z y m e w a s u n s t a b l e a t h i g h e r t e m -
Properties o f topoisomerase
peratures Typical assays of the topoisomerase
are shown in
Topoisomerase was found to be sensitive to high salt
8 Table 2. Effect of inhibitors on pea chloroplast topoisomerase I a. Inhibitor
Activity retained (°70)
1 #M 2.5 #M 5 #M
0 0 0
0.5 mM 1 mM 2 mM
80 20 0
10 #g/ml 2.5 #g/ml
a Assays were carried out as described in Materials and methods. The enzyme was preincubated with the inhibitor for 3 - 4 min on ice, and then 1 #g pBR322 was added and the reaction incubated at 37 °C. The degree of inhibition of activity was estimated by comparison with dilutions of enzyme without inhibitor in the same gel.
concentrations (Fig. 5). The enzyme showed peak activity when the reaction contained 2 5 - 5 0 mM KCI or NaC1, but very little activity was detected at 100 mM salt. The enzyme exhibited a broad optimum for Mg 2+ between 5 and 15 mM; the activity
was, however, significantly inhibited by 20 mM M g 2+ . A b o u t 2 0 % o f o p t i m u m
a c t i v i t y c o u l d be
o b t a i n e d in t h e a b s e n c e o f a d d e d M g 2÷ , i f t h e react i o n c o n t a i n e d 50 m M KCI. M n 2÷ c o u l d b e subs t i t u t e d f o r M g 2+ , t h o u g h r e s u l t i n g in a d e c r e a s e o f u p to a p p r o x i m a t e l y o n e h a l f o f o p t i m u m activity. T h i s d i f f e r s f r o m t h e r e p o r t o f S e i d l e c k i et al. , w h o f o u n d t h a t M g 2+ was a b s o l u t e l y r e q u i r e d f o r activity of the spinach chloroplast topoisomerase. Because the interaction between topoisomerase I and t h e D N A t e m p l a t e is e s s e n t i a l l y electrostatic, t h e salt c o n c e n t r a t i o n is t h e b i g g e s t d e t e r m i n i n g f a c t o r o f
Fig. 3. SDS-polyacrylamide gel of pea chloroplast topoisomerase purification. Samples from various stages of purification were analyzed in a 10070polyacrylamide denaturing gel electrophoresed at 120 V. Panel A, Coomassie blue-stained gel. Lane 1, protein molecular weight markers, from top to bottom, myosin (200kDa), B-galactosidase (116.25 kDa), phosphorylase b (92.5 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa). Lane 2, DEAE cellulose gradient fraction. Lane 3, heparin-Sepharose fraction. Lane 4, phosphocellulose. Panel B, silver-stained gel. Lane 1, molecular weight markers, same as in A. Lane 2, FPLC Superose-12 fraction 13. The arrow shows the topoisomerase band, at approximately 112 kDa.
Fig. 5. Salt requirements of pea chloroplast topoisomerase.
Standard reactions were carried out as described in the text, at 37 °C for 30 min. Lanes 1- 5, each with 5 mM MgC!2 and 0, 50, 100, 150, and 200 mM KC1, respectively. Lanes 6-10, each with 50 mM KC1 and 0, 5, 10, 15, and 20 mM MgC12, respectively.
Fig. 4. Analysis of topoisomerase activity by gel filtration chro-
matography. A. Topoisomerase activity eluted from FPLC Superose-12 column. Lane 1, supercoiled plasmid DNA. Lane 2, phosphocellulose topoisomerase fraction. Lanes 3-10, assays of 0.5 ml FPLC fractions 12.5-16 ml. L indicates linear form of the plasmid. B. Calculation of molecular weight of pea chloroplast topoisomerase eluted from FPLC Superose-12 column. The molecular weight of the topoisomerase activity in A was plotted with molecular weight markers eluted through the same column under the same conditions as described in the text. • Ferritin, 440 kDa, o catalase, 220 kDa; • pea chloroplast topoisomerase I, about 112 kDa; • bovine serum albumin, 66 kDa; and o cytochrome c, 13 kDa.
t o p o i s o m e r a s e I activity . T h e size a n d p r o p e r t i e s o f the e n z y m e are similar to t h o s e r e p o r t e d for t h e s p i n a c h c h l o r o p l a s t t o p o i s o m e r a s e  a n d w h e a t g e r m m i t o c h o n d r i a l a n d n u c l e a r t o p o i s o m e r a s e [5, 6]. T h e p e a c h l o r o p l a s t t o p o i s o m e r a s e was f o u n d to cause a unit c h a n g e in the linkage n u m b e r b y c o m p a r i s o n with p u r i f i e d c a l f t h y m u s t o p o i s o m e r a s e I activity a n a l y z e d in t h e s a m e gel ( d a t a n o t shown), as p r e v i o u s l y d e t e r m i n e d for t h e s p i n a c h e n z y m e . T h e presence o f ATP, even at high c o n c e n t r a tions o f 100/~M, d i d n o t affect the t o p o i s o m e r a s e . T h e e n z y m e was s t r o n g l y i n h i b i t e d by Ne t h y l m a l e i m i d e , which indicates t h a t s u l f h y d r y l g r o u p s are required for e n z y m e activity. T h e presence o f e t h i d i u m b r o m i d e , even at a c o n c e n t r a t i o n o f 1.0/~g/ml, c o m p l e t e l y i n h i b i t e d t h e p e a c h l o r o p l a s t t o p o i s o m e r a s e (Table 2). This shows t h a t p e a c h l o r o p l a s t t o p o i s o m e r a s e is o n l y able to relax negatively s u p e r c o i l e d D N A a n d n o t the positive supercoils i n d u c e d by e t h i d i u m b r o m i d e - i n t e r c a l a t i o n into the D N A d o u b l e helix. T h e e n z y m e is n o t inh i b i t e d by n o v o b i o c i n o r nalidixic acid, w h i c h are inh i b i t o r s o f t y p e II t o p o i s o m e r a s e . F r o m these properties, t h e p e a c h l o r o p l a s t e n z y m e is o f t h e p r o k a r y o t i c t y p e I t o p o i s o m e r a s e , w h i c h is a D N A relaxing enzyme.
Replication system without topoisomerase I The DEAE fractions containing DNA polymerase, RNA polymerase and very little topoisomerase I were pooled together and loaded on a 10 ml heparinSepharose column equilibrated with buffer B (buffer A + 0.1% Triton X-100 + 0.1 mM EDTA) + 0.1 M (NH4)2SO 4. The column was washed with buffer B + 0.3 M (NH4)2SO4 and the bound proteins were eluted from the column with 0.5 M (NH4)2SO4 in buffer B. DNA polymerase and RNA polymerase activities were found to be present in the same fractions. The peak enzyme fractions were pooled, dialyzed against buffer A + 0.1 M (NH4)2SO4 and loaded on a phosphocellulose column equilibrated with the same buffer. The DNA polymerase and RNA polymerase activities were eluted from the column with 0.2 M (NH4)2SO 4 in buffer A. This phosphocellulose fraction contained no topoisomerase activity.
Role of topoisomerase I in replication The role of topoisomerase I in the replication o f ctDNA was studied by using the replication system lacking topoisomerase I and the pure topoisomerase I. The recombinants pCP12-7 and pCBI-12, both o f which contain the two D-loops, were used as templates in the polymerase reaction (Fig. 6). These templates have been previously established to undergo replication in vitro when an enzyme fraction containing DNA polymerase, RNA polymerase and
topoisomerase I was used . The in vitro replication o f the recombinants was analyzed in the presence and absence o f topoisomerase I (Table 3). When pCP12-7 was used as a template for the DNA synthesis at 80 mM KC1, addition of 20 units (0.25/~g) o f purified topoisomerase I resulted in 3to 6-fold increase in DNA synthesis. Similarly, DNA synthesis showed a 1.8-fold increase in the presence of topoisomerase I using pCBI-12 as template. Similar levels o f stimulation were obtained when purified E. coli topoisomerase I was added to the reactions. However, calf thymus topoisomerase I did not cause any significant stimulation of replication activity on pCP12-7 (Table 3). If the DNA synthesis was carried out in the presence of 125 mM KCI, there was about a four-fold decrease in the DNA synthesis in the presence or absence of pea chloroplast topoisomerase I. This salt concentration has been found to completely inhibit chloroplast topoisomerase I (see Fig. 5). When activated calf thymus DNA, a nonspecific control, was used as a template there was a somewhat higher level o f DNA synthesis at high salt concentration, but no effect was seen by adding purified topoisomerase I (Table 3). To analyze whether the incorporation of [3H]T T P with the D-loop containing recombinants truly reflects DNA synthesis, in vitro synthesized DNA labelled with [32p]-TTP was analyzed by gel electrophoresis (Fig. 7). A 4 - 5-fold increase in product was obtained when 20 units (0.25/~g) topoisomerase I was added to the reaction using the pCP12-7 template (lanes 2 and 3) at 70 mM KC1. When pCBI-12 was used as template (lanes 5 and 6) at 70 mM KCI,
I kbp Fig. 6. Restriction map of pCPI2-7 and pCBI-12, and location of D-loop replication origins. Plasmid pCP12-7 contains the 12.5 kbp Pst I fragment from pea chloroplast DNA in pACYC-177. Plasmid pCBI-12 contains the 10.0 kpb Bam HI fragment from pea chloroplast
DNA in pBR322. The two clones overlap as shown. The thick lines represent the vector DNA, and the filled boxes represent the two D-loops. The restriction sites for Eco RI (circles), Pst I (triangles over the line), and Barn HI (triangles under the line) are shown. A - K are the restriction fragments generated by digestion with Pst I and Eco RI, and have the following sizes: A, 3.40 kbp; B, 3.7 kbp; C, 3.5 kbp; D, 2.2 kbp; E, 2.1 kbp; F, 1.9 kbp; G, 1.6 kbp; H, 1.2 kbp; I, 1.1 kbp; J, 0.94 kbp; and K, two small fragments of 0.27 kbp and 0.06 kbp.
11 Table 3. Role of topoisomerase I in replication o f chloroplast DNA. Template
m M KCI
_+ topoisomerase I a
+_ novobiocin b
64000 103 000
+ + 20 units calf t h y m u s topoismerase I + 20 units E. coli topoisomerase I + +
6 700 47 700 9570
+ + -
21000 3 8 000 9000
Activated calf thymus DNA
80 80 80
80 80 80 125 125 80 80 125
4 740 c 32 360
2020 36 360 3 570 2090
a W h e n added, 10 #1 of the purified topoisomerase I (ca. 20 units) was added to 10 #1 o f the crude D N A polymerase fraction, except for the reactions carried out with calf t h y m u s or E. coli topoisomerase I, as shown. b W h e n present, novobiocin was added to 250 #g/ml, which is inhibitory for topoisomerase II activity. c Results of assays done at a later time.
an approximately product
2-fold increase in the amount
ded. In each case the entire template as each of the expected fragments
Pst I + Eco RI restriction
( s e e F i g . 7) w e r e f o u n d t o b e l a b e l l e d . I n -
hibition of replication periments
I was ad-
b y 125 m M K C 1 is s e e n i n e x -
where pCBI-12 was used as template
7), a n d o n l y w i t h a l o n g e x p o s u r e
could the bands
Fig. 7. The role oftopoisomerase I in in vitro replication. In vitro replication reactions were carried out as described in Materials a n d methods, using pCP12-7 (lanes 2 a n d 3) or pCBI-12 (lanes 5 - 7 ) as template. The D N A was then digested with Eco RI and P s t I and the products analyzed in a 1% agarose gel. The autoradiogram showed that fragments o f the expected sizes were labelled (fragments identified at the right, see also Fig. 6). Lanes 1 and 4, phage lambda D N A digested with H i n d III and 5 ' end-labelled with 32p, with sizes as shown at the left. Lanes 2 and 5, reactions at 70 m M KCI with the partially purified D N A polymerase fraction only. Lanes 3 and 6, reactions at 70 m M KCl with 10 t~l of purified topoisomerase added to the partially purified D N A polymerase fraction. Lane 7, the reaction carried out with topoisomerase addition but at 125 m M KC1, which is inhibitory for topoisomerase I but permissive for D N A polymerase on a nonspecific template (see Table 3).
12 Role of topoisomerase H in the replication of chloroplast DNA? The phosphocellulose fraction used for replication studies was analyzed for the presence o f topoisomerase II. There was a low but detectable amount of topoisomerase II in this fraction as assayed by following the ATP-dependent supercoiling of relaxed circular molecules . This activity was, however, inhibited by novobiocin. When this enzyme fraction was used for the in vitro replication o f recombinants pCP 12-7 and pCB 1-12 in the presence of novobiocin, the replication activity was not inhibited (Table 3).
In order to establish the role of topoisomerase I in the replication o f ctDNA, we had to develop a method that will produce a pure enzyme. Even though the topoisomerase I has been reported in pea  and corn  and partially purified (ll0-fold) from spinach , the complete purification of this enzyme was found to be even more difficult than the purification o f chloroplast DNA polymerase. Both chloroplast DNA polymerase and topoisomerase I in pea chloroplasts have been found to be present in about the same relative protein concentrations. Topoisomerase I, however, was found to be more labile than DNA polymerase. Purified pea chloroplast enzyme has properties similar to those reported from other sources. In the present study, the topoisomerase I from pea chloroplasts has been purified 5 000-fold to a single polypeptide having a molecular weight o f 112 kDa. Pea chloroplast topoisomerase I required 5 - 1 5 mM Mg 2+ for optimum activity, but ATP caused no change in activity. The enzyme was not able to relax positively supercoiled DNA. The enzyme was found to cause a change of _+ 1 in the linkage number o f supercoiled DNA. The purified topoisomerase I from pea chloroplasts is totally resistant to 250/~g/ml novobiocin, contrary to what was reported for a crude fraction from pea chloroplasts . Topoisomerase I from pea chloroplasts has also been found not to be inhibited by nalidixic acid. These results are similar to those reported for spinach chlo-
roplast topoisomerase I, and support the identification of the pea chloroplast enzyme as a prokaryotic type I topoisomerase. Wheat germ mitochondrial topoisomerase I, on the other hand, has been found to relax positive as well as negative supercoils , thus resembling eukaryotic topoisomerase I. The phosphocellulose-purified pea chloroplast topoisomerase I activity was stable for several months when stored at - 2 0 °C in 50°70 glycerol. Fully purified topoisomerase, however, only retained activity for a few weeks under these conditions. Analysis on a SDS-polyacrylamide gel showed that the 112 kDa band diminished in correlation with the loss in activity. However, no specific smaller bands could be identified. In this paper, we provide the first report of the role of topoisomerase I in replication of ctDNA. We have been able to establish the role of topoisomerase I in replication because of our success in obtaining a pure homogeneous preparation o f topoisomerase I o f a single polypeptide and the development of an in vitro system that can replicate D-loop-containing recombinants in vitro. The data presented in Table 3 and Fig. 7 show that the addition of purified pea chloroplast topoisomerase I to a replication system containing DNA polymerase, DNA-binding proteins, and other proteins involved in replication, resulted in a significant ( 2 - 6 - f o l d ) stimulation of replication activity on the two templates containing the pea ctDNA origins of replication. The analysis of the in vitro synthesized DNA in agarose gel electrophoresis confirmed the observed stimulation of replication by topoisomerase I. Similarly, in vitro replication activity with the pCBI-12 template, which contains the two D-loop origins of replication o f pea chloroplast DNA, is inhibited 4-fold when the reaction is carried out at 125 mM KCI. This salt concentration completely inhibits topoisomerase I activity but does not affect DNA polymerase activity. Very little product could be seen when the reaction products o f the 125 mM KCI reaction were analyzed by autoradiography o f the gel, as compared to the reaction without topoisomerase I addition. These two experiments clearly show that the topoisomerase I is involved in in vitro replication of pea chloroplast DNA. Our data implicating topoisomerase I in the repli-
13 cation o f c t D N A are supported by observations in other systems. For example, in an SV40 in vitro replication system using purified proteins, the removal o f topoisomerase I reduced by a factor o f 2 - 3 the inc o r p o r a t i o n o f radioactive nucleotides . This agrees with the result we obtained with pea chloroplast topoisomerase I (Table 3 and Fig. 7). Similarly, in E. coli a replication system containing primase, D N A polymerase, topoisomerase II, and other enzymes k n o w n to be involved in replication, but lacking topoisomerase I, supports only a low level o f replication on a specific template in vitro . The addition o f purified E. coli topoisomerase I caused a 6 - 8-fold stimulation o f replication activity in vitro. The topoisomerase I was identified as a specificity factor required to suppress replication initiation at locations other t h a n the correct initiation site . In these experiments eukaryotic topoisomerase I was unable to substitute for the E. coli topoisomerase I, suggesting specificities to the topoisomerases, which have different abilities to relax positively supercoiled D N A , cleave at different sequences, and nick at opposite sides o f the phosphate groups in the D N A . This is similar to the results we obtained, where E. coli, but n o t calf thymus, topoisomerase I was able to cause the same level o f stimulation o f replication. Topoisomerase II, or D N A gyrase, has been f o u n d to be essential for initiation o f replication in bacterial systems [7, 9, 15, 271. Topoisomerase II activity has been reported in whole Chlamydomonas cell extracts  and in pea chloroplasts . This ATPdependent supercoiling enzyme was f o u n d to be inhibited by low concentrations o f novobiocin or nalidixic acid, a characteristic o f type II topoisomerase. W h e n Chlamydomonas cells were grown in the presence o f low concentrations o f these inhibitors, the a c c u m u l a t i o n o f various chloroplast transcripts was significantly affected. Some transcripts increased in a b u n d a n c e while others decreased [251. Similar results were obtained when crude pea chloroplast extracts were used to transcribe various chloroplast genes . O u r replication system contained barely detectable a m o u n t s o f topoisomerase II. W h e n novobiocin was used in the replication system, there was no reduction in the synthesis o f c t D N A . These results m a y be interpreted to m e a n that topoisomer-
ase II is not required, but a more direct demonstration o f the role o f topoisomerase II in replication will have to await its purification. In this connection, it is interesting to m e n t i o n that in yeast, a temperature-sensitive (ts) m u t a t i o n in the gene for topoisomerase II caused a defect in replication termination, but not initiation, while a m u t a t i o n in topoisomerase I caused no growth defects. However, a double m u t a t i o n in b o t h genes caused a rapid halt in growth at the nonpermissive temperature . These results show that in yeast cells b o t h topoisomerases play a role in replication.
Acknowledgements This work was supported by a grant f r o m the N.I.H. ( G M 33725). B. L. N. was the recipient o f a postdoctoral fellowship f r o m the C a m p u s Biotechnology Training Program.
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