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J. Biol. Chem., Vol. 278, Issue 33, 30705-30710, August 15, 2003
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From the Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595
Received for publication, April 2, 2003 , and in revised form, May 29, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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'
subunit of RNA polymerase and the C-terminal domains of E. coli DNA
topoisomerase I, which are homologous to the zinc ribbon domains in a number
of transcription factors. This direct interaction can bring the topoisomerase
I relaxing activity to the site of transcription where its activity is needed.
The zinc ribbon C-terminal domains of other type IA topoisomerases, including
mammalian topoisomerase III, may also help link the enzyme activities to their
physiological functions, potentially including replication, transcription,
recombination, and repair. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In previous studies, Tn5 transposase was found to copurify with E. coli DNA topoisomerase I and inhibit the topoisomerase I activity (10). RNA polymerase was also found to copurify with Tn5 transposase, but the copurification was reduced in extracts from a topA mutant strain, suggesting that the interaction between RNA polymerase and DNA topoisomerase I was responsible for the copurification of RNA polymerase with Tn5 transposase (10). The proposed function of topoisomerase I activity in removal of transcription-driven hypernegative supercoiling (8, 9) would be greatly facilitated by direct protein-protein interaction with RNA polymerase. Experiments described here provide direct evidence for such interaction as well as identifying the domain of topoisomerase I and the subunit in RNA polymerase that are responsible for this protein-protein interaction.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Maltose-binding Protein Affinity Chromatography—A PCR
fragment encoding the E. coli topA gene was generated by Pfu
DNA polymerase (from Stratagene) and cloned into the XmnI site of
pMal-c2X (New England BioLabs) to create a fusion protein with
MBP1 linked to the N
terminus of topoisomerase I. Expression of MBP-topoisomerase I and MBP in
E. coli TB1 cells was induced with
isopropyl-1-thio--D-galactopyranoside. Cells from a 250-ml
culture were lysed by lysozyme treatment combined with freeze-thawing in
phosphate-buffered saline. The soluble extract obtained after centrifugation
was applied to 1 ml of amylose resin (New England BioLabs) equilibrated with
column buffer (20 mM Tris, pH 7.5, 200 mM NaCl, 1
mM EDTA). After extensive washing, the proteins bound to the column
were eluted with column buffer containing 10 mM maltose.
RNA Polymerase Affinity Chromatography—Polyol-responsive
monoclonal antibodies against E. coli RNA polymerase '
subunit (NT73) was purchased from Neoclones and coupled to cyanogen
bromide-activated Sepharose according to published procedures using 0.9 ml of
the antibodies (15). The NT73
affinity matrix was mixed with 1 ml of extracts of E. coli BL21 cells
prepared from 400 ml of culture expressing intact topoisomerase I
(11) or its subdomains
(12–14)
as described for affinity purification of RNA polymerase
(15). After extensive washing,
the bound proteins were eluted with 1 ml of 40% ethylene glycol with 0.75
M NaCl.
Blotting of RNA Polymerase with Digoxigenin (DIG)-labeled Topoisomerase I and Its Subdomains—Purified RNA polymerase (0.8 µg) was electrophoresed in SDS-polyacrylamide gel to separate the subunits. The proteins were transferred onto either supported nitrocellulose membrane for chemiluminescence detection or polyvinylidene difluoride membrane for color detection. Topoisomerase I and its subdomains were labeled with DIG using the labeling kit from Roche Applied Science. Each DIG-labeled protein was incubated with the membrane at 25 °C for 1 h. Anti-DIG antibodies linked to peroxidase were used for detection by the ECL Plus system (Amersham Biosciences). Anti-DIG antibodies linked to alkaline phosphatase were used for color detection with the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate (Roche Applied Sciences). After color detection, the position of the topoisomerase I-binding signal was marked and photographed before staining of the nylon membrane by Coomassie Blue.
In Vitro Transcription—In vitro transcription with E. coli RNA polymerase (3 units) was carried out with procedures similar to those described previously (16). The reaction (30 µl) contained 40 mM Hepes/KOH (pH 7.6), 11 mM magnesium acetate, 100 mM potassium glutamate, 1 mM dithiothreitol, 4 mM ATP, 0.5 mM each of GTP, CTP, and UTP, 0.5 µg of negatively supercoiled plasmid pBR322. Transcription was carried out at 37 °C for 10 min. After deproteinization by phenol extraction, the DNA samples were analyzed by electrophoresis in 1% agarose gel with 0.5x TPE (90 mM Tris, 90 mM phosphate, 2 mM EDTA, pH 8.0) buffer with the indicated chloroquine concentrations for detection of positively supercoiled DNA (17). One-dimensional agarose gels were stained with ethidium bromide and photographed. When analyzed by two-dimensional agarose gel electrophoresis, DNA was visualized by hybridization to 32P-labeled probes as described previously (17).
Induction of Synthesis of the 14-kDa C-terminal Fragment in Vivo— DNA coding for the 14-kDa C-terminal fragment of E. coli DNA topoisomerase I was generated by PCR using the Pfu DNA polymerase and inserted into the pBADThio-TOPO expression vector (from Invitrogen). The resulting plasmid pBAD14K has the 14-kDa C-terminal fragment fused to the carboxyl end of thioredoxin and under the control of pBAD promoter. E. coli strain TOP10 with wild-type topoisomerase genotypes (from Invitrogen) was transformed with either pBAD14K or the control plasmid pBAD/Thio expressing thioredoxin. Cells were grown at 37 °C in LB containing 100 µg/ml ampicillin until A600 reached 0.5, when transcription from the PBAD promoter was induced by addition of 0.005% arabinose. Cell growth was continued for 4 h at 37 °C before harvest of the cell pellets for plasmid preparation using the Qiagen kit. Plasmid DNA supercoiling was analyzed by two-dimensional gel electrophoresis using chloroquine concentrations specific for identification of hypernegatively supercoiled DNA (16, 17) and was visualized by hybridization to a 32P-labeled probe and by autoradiography.
Survival Rates after N-Ethylmaleimide (NEM) Treatment—RFM445
(gyrB22(couR)gyrB203(Ts)), RFM475
(gyrB22(couR)gyrB203(Ts), and
(topAcysB)) and its transformants were cultured in LB broth at
37 °C to stationary phase (>18 h). Plasmid pJW312
(18) was used for expression
of full-length topoisomerase I, whereas its linker insertion derivative
pJW2277ter (18) was used for
expressing Top85 lacking the 14-kDa C-terminal fragment
(19). Plasmid for expression
of Top67 lacking the 30-kDa terminal fragment was constructed by introduction
of a termination codon in pJW312 via site-directed mutagenesis
(14). The cells were washed
and resuspended in M9 medium as described
(20) before treatment for 1 h
with 8 mM NEM at 37 °C. The rate of survival was determined by
serial dilutions and plating of treated cells as well as control cells not
treated with NEM. Colonies on LB plates were counted after 36 h of incubation
at 37 °C.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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and ' subunits were detected in the eluate
fractions from cells expressing MBP-topoisomerase I but not in the eluate
fractions prepared from cells expressing MBP
(Fig. 1). This demonstrates
that the linkage of DNA topoisomerase I to MBP is required for binding of RNA
polymerase to the amylose resin.
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The Zinc Ribbon Domains of Topoisomerase I Are Responsible for the
Interaction with RNA Polymerase—An RNA polymerase affinity column
was prepared using polyol-responsive monoclonal antibodies against the
' subunit of E. coli RNA polymerase
(15). Soluble extracts of
E. coli BL21 cells expressing DNA topoisomerase I
(11) or its subdomains
(12–14)
were applied individually to the affinity column in separate experiments
carried out with identical procedures (Fig.
2). Topoisomerase I could be detected in the bound proteins eluted
from this affinity column with buffer containing 40% ethylene glycol and 0.75
M NaCl. In similar experiments carried out separately with lysates
expressing an individual topoisomerase I subdomain, it was determined that the
67-kDa N-terminal transesterification domain (Top67) did not bind to the
affinity column but the 20-kDa (ZD2) and 14-kDa C-terminal fragments could
bind to the RNA polymerase affinity column. These two fragments contain three
and two copies of zinc ribbon domains, respectively, and are homologous to the
zinc ribbon domains in transcription regulators including RPB9, TFIIS, and
TFIIB (21). However, only the
three zinc ribbon domains on ZD2 have Zn(II) bound to tetracysteine motifs
(13). Binding of the RNA
polymerase subunits to this affinity column was confirmed by both Coomassie
Blue staining of the eluted proteins and Western blot analysis using
antibodies against the , , and ' subunits
(Fig. 2c).
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The ' Subunit of RNA Polymerase Interacts with
Topoisomerase I—E. coli RNA polymerase was expected to bind to the
affinity matrix as a multisubunit complex. To determine which subunit was
responsible for the interaction with DNA topoisomerase I, the RNA polymerase
subunits were electrophoresed in a 7% SDS-polyacrylamide gel and transferred
onto membrane. Overlay blotting was carried out using DIG-labeled
topoisomerase I, DIG-labeled Top67, DIG-labeled ZD2, and DIG-labeled 14-kDa
fragment. All of these DIG-labeled proteins, except DIG-labeled Top67, gave a
positive chemiluminescence signal with peroxidase-linked anti-DIG antibody at
a position where RNA polymerase and ' subunits would
migrate in the 7% SDS-polyacrylamide gel
(Fig. 3a). This result
was in agreement with the data in Fig.
2b and showed that DNA that might have been present in
the E. coli protein extract during binding to the affinity columns
was not required for interaction between DNA topoisomerase I and RNA
polymerase. There was also no signal when the RNA polymerase subunits on the
membrane were blotted with DIG-labeled bovine serum albumen.
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To determine whether the or ' subunit was interacting
with topoisomerase I, a 5% polyacrylamide gel was used for SDS-gel
electrophoresis of the RNA polymerase subunits to better separate these two
high molecular weight subunits. After transfer, the membrane was incubated
with DIG-labeled ZD2 followed by alkaline-phosphatase linked anti-DIG
antibodies. The signal of the DIG-topoisomerase I fragment bound to the nylon
membrane was developed with the nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate color reagent. The position
of the signal was marked on the membrane and photographed alongside a ruler.
The nylon membrane was then stained with Coomassie Blue to visualize both the
and ' subunits in the gel lane analyzed previously with the
alkaline-phosphatase linked anti-DIG antibodies. A comparison with the
previously marked and photographed signal from alkaline phosphatase indicated
that the topoisomerase I subdomain was interacting with the '
subunit (Fig. 3b). The
experiment was repeated using DIG-topoisomerase I or DIG 14-kDa fragment, and
both of these also interacted with the ' subunit
(Fig. 3b and data not
shown).
Direct Interaction between E. coli RNA Polymerase and Topoisomerase I Is Important for Removal of Transcription-driven Negative Supercoils—It has been proposed that topoisomerase I plays an important physiological role in the removal of negative supercoils formed during transcription because of the movement of the RNA polymerase complex (6–9). In an in vitro transcription reaction in the presence of DNA topoisomerase I, positive supercoiling of the DNA template results from the removal of transcription-driven negative supercoils by topoisomerase I (7, 16). To demonstrate that the direct interaction between E. coli RNA polymerase and topoisomerase I is important for the removal of transcription-driven negative supercoils, a recombinant 14-kDa C-terminal fragment (purified from an overexpression system (12) at >99% purity as determined by Coomassie Blue staining of SDS gel (data not shown)) was also added to the in vitro transcription reaction. The 14-kDa C-terminal fragment could compete with topoisomerase I for interaction with E. coli RNA polymerase, and the formation of positively supercoiled DNA template was found to be inhibited (Fig. 4). In a relaxation reaction in the absence of RNA polymerase, the 14-kDa fragment has no significant effect on the relaxation of negatively supercoiled DNA by DNA topoisomerase I; thus so the 14-kDa fragment does not inhibit the catalytic activity of DNA topoisomerase I (Fig. 4c).
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The significance of the RNA polymerase-topoisomerase I interaction for removal of transcription driven supercoils was also demonstrated in vivo. The 14-kDa C-terminal domain was expressed in E. coli as a thioredoxin fusion protein via the tightly regulated PBAD promoter (22). Accumulation of hypernegative supercoils in the plasmid DNA was observed when synthesis of the 14-kDa C-terminal fragment by pBAD14K was induced by addition of 0.005% arabinose to the culture (Fig. 5a). The induced 14-kDa C-terminal domain was expected to compete with topoisomerase I for interaction with RNA polymerase, impeding the removal of transcription-driven negative supercoils by DNA topoisomerase I. The level of 14-kDa C-terminal domain synthesis and resulting accumulation of hypernegatively supercoiled DNA did not vary significantly when the arabinose concentration was varied between 0.001 and 0.1% (data not shown). Induction of expression of thioredoxin in the control pBAD/THIO plasmid under the same experimental conditions did not result in accumulation of hypernegatively supercoiled DNA (Fig. 5b).
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Zinc Ribbons Are Important for in Vivo Function of Topoisomerase I during Stress Response—An 85-kDa truncated topoisomerase I lacking the 14-kDa C-terminal fragment (Top85) has been shown previously to be active in relaxation of supercoiled DNA (18, 19). The in vitro relaxing activity of Top85 was >75% of that of the full-length enzyme, albeit with lower processivity in high salt (19). In vivo, Top85 could fully complement the viability of the E. coli strain AS17 that has a temperature-sensitive topoisomerase I (18, 23). Top85 has three of the five zinc ribbon domains present in topoisomerase I. We hypothesize that during stress response, when rapid transcription of induced genes takes place, direct interaction between topoisomerase I and RNA polymerase via the zinc ribbon domains may be important for survival. We have previously shown that the E. coli strain RFM475 with the topA gene deleted is much more sensitive to killing by the toxic electrophile NEM than the isogenic topA+ strain RFM445 (20). The increased sensitivity of RFM475 to NEM can be reversed by the presence of a plasmid expressing DNA topoisomerase I (20). Strain RFM475 was transformed with plasmids expressing full-length topoisomerase I or its truncated forms lacking the C-terminal fragments. Comparison of the survival rates of the RFM475 transformants after NEM treatment (Fig. 6) showed that the survival rate of the transformant expressing Top85 was higher than the transformant expressing Top67 but still about 100-fold lower than that of the transformant expressing the full-length topoisomerase I. Although not required for viability of E. coli under optimal laboratory growth conditions, the absence of the 14-kDa fragment in topoisomerase I thus affected the function of topoisomerase I in stress response significantly.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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' subunit of RNA polymerase, so that negative DNA supercoiling
formed during transcription can be removed immediately. The rapid removal of
transcription-driven negative supercoils would prevent the formation of
R-loops (8,
9). This suppression of R-loops
during transcription has been proposed to be an essential function for E.
coli DNA topoisomerase I
(24).
Sequence analysis shows that the number of zinc ribbon domains in type IA
DNA topoisomerases found in different organisms varies from zero to five
(20). If present, these zinc
ribbon domains can potentially interact with RNA polymerase in these organisms
during transcription. Other DNA topoisomerases have also been shown previously
to be involved directly in transcription. Human topoisomerase I, a type IB
topoisomerase, has been shown to be a cofactor of RNA polymerase II
transcription (25,
26), whereas topoisomerase
II is required for RNA polymerase II transcription on chromatin
templates (27).
We have observed previously that a recombinant plasmid expressing the 14-kDa C-terminal domain of E. coli DNA topoisomerase I under the T7 promoter was unstable in E. coli BL21DE3 (12). Attempts to construct a recombinant plasmid expressing the 14-kDa C-terminal domain as a MBP-fusion protein in pMal-c2X were unsuccessful. The results showing the interference of E. coli DNA topoisomerase I function during transcription by the 14-kDa C-terminal domain would account for the instability of these recombinant plasmids. It may be possible for topoisomerase I function or DNA supercoiling to be modulated via targeting of the protein-protein interactions between topoisomerase I and its partners in E. coli.
Besides Tn5 transposase and RNA polymerase in E. coli, cellular
proteins in other organisms may also interact with type IA topoisomerases.
This is particularly intriguing for mammalian type IA topoisomerases. There
are two type IA topoisomerases (TOP3 and TOP3) present in both
human and mouse
(28–32)
with four zinc ribbon domains present in each of these enzymes
(21). Human TOP3 has
been shown to interact with the Bloom Syndrome helicase, BLM
(33,
34), whereas both TOP3
and TOP3 interact with human RecQ5 helicase
(35). There is evidence that
interaction between Bloom Syndrome helicase and human TOP3 is important
for genomic stability (36).
The domains in TOP3 and TOP3 responsible for the interactions
with the RecQ family of helicases have not been identified experimentally.
Multiple transcripts from alternative splicing with tissue specific expression
pattern give rise to variant forms of TOP3 that have different numbers
of zinc ribbon domains (31).
This can affect the interaction of TOP3 with other cellular proteins.
The potential involvement of these zinc ribbon domains in protein-protein
interactions may play the important role of directing different forms of
mammalian type IA topoisomerases to complexes involved in replication,
transcription, recombination, or DNA repair.
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FOOTNOTES |
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Present address: Dept. of Physiology, 194 Xuefu Rd., Harbin Medical
University, Harbin 150086, China.
Present address: The Inst. for Genomic Research, 9712 Medical Center Dr.,
Rockville, MD 20850.
¶ Supported by National Institutes of Health Grant GM54226. To whom correspondence should be addressed. Tel.: 914-594-4061; Fax: 914-594-4058; E-mail: yuk-ching_tse-dinh{at}nymc.edu.
1 The abbreviations used are: MBP, maltose-binding protein; DIG, digoxigenin;
NEM, N-ethylmaleimide.
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ACKNOWLEDGMENTS |
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' subunit
identification, and Marc Drolet for helpful discussions. |
REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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