Biochem. J. (2008) 409, 481–489 (Printed in Great Britain)
481
doi:10.1042/BJ20071358
Amino acids 39–456 of the large subunit and 210–262 of the small subunit
constitute the minimal functionally interacting fragments of the unusual
heterodimeric topoisomerase IB of Leishmania
Somdeb BOSEDASGUPTA*, Benu Brata DAS†, Souvik SENGUPTA*, Agneyo GANGULY*, Amit ROY*, Gayatri TRIPATHI‡ and
Hemanta K. MAJUMDER*1
*Molecular Parasitology Laboratory, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata-700032, India, †Laboratory of Molecular Pharmacology, Centre for Cancer
Research, National Cancer Institute, Bethesda, MD, U.S.A., and ‡Division of Cellular Physiology, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata-700032, India
The unusual, heterodimeric topoisomerase IB of Leishmania
shows functional activity upon reconstitution of the DNA-binding
large subunit (LdTOPIL; or L) and the catalytic small subunit
(LdTOPIS; or S). In the present study, we generated N- and Cterminal-truncated deletion constructs of either subunit and identified proteins LdTOPIL39−456 (lacking amino acids 1–39 and
457–635) and LdTOPIS210−262 (lacking amino acids 1–210) as
the minimal interacting fragments. The interacting region of
LdTOPIL lies between residues 40–99 and 435–456, while for
LdTOPIS it lies between residues 210–215 and 245–262. The
heterodimerization between the two fragments is weak and
therefore co-purified fragments showed reduced DNA binding,
cleavage and relaxation properties compared with the wild-
INTRODUCTION
DNA topoisomerases relieve the torsional strain in DNA that
builds up during vital cellular processes. Type IB enzymes nick
one DNA strand, form a 3 -phosphotyrosyl link and swivel another
strand across the nick by a ‘controlled rotation’ mechanism [1].
The advent of bi-subunit topoisomerase IB in kinetoplastids
is a paradigm shift in the type IB family. The DNA-binding
large subunit with the ‘VAILCNH’ motif associates with the
catalytic small subunit harbouring the consensus ‘SKXXY’ motif
to form an active enzyme within the parasite [2,3]. RNAi (RNA
interference) of one subunit causes concomitant degradation of the
other protein subunit [4]. CPTs (camptothecins) are uncompetitive
inhibitors of topoisomerase IB, which trap the enzyme–DNA
cleavable complex, stall replication and transcription fork and
thereby cause cell death [1].
In monomeric human topoisomerase IB, active complementation of ‘cap’ and ‘catalytic’ domains (linked by a dispensable
‘linker’ domain) and formation of a fully functional enzyme upon
deletion of the N-terminal 214 amino acids have been documented
[5,6]. A 1:1 molar interaction of two subunits in vitro forms the
active LdTOPIL/S (or L/S), which differs from monomeric rat
liver topoisomerase I in having reduced affinity for DNA and
reduced processivity [7]. Catalytic activity is derived chiefly from
five conserved amino acids [8]: Arg314 , Lys352 , Arg410 and His453
from LdTOPIL (or L) and Tyr222 from LdTOPIS (or S).
The bi-subunit Leishmania topoisomerase IB also harbours
stretches of residues in the two subunits, which are dispensable
for activity. Our previous studies show that residues 1–39 of L
modulate the non-covalent interaction with DNA, while residues
type enzyme. The minimal fragments could complement their
respective wild-type subunits inside parasites when the respective
subunits were down-regulated by transfection with conditional
antisense constructs. Site-directed mutagenesis studies identify
Lys455 of LdTOPIL and Asp261 of LdTOPIS as two residues
involved in subunit interaction. Taken together, the present
study provides crucial insights into the mechanistic details
for understanding the unusual structure and inter-subunit cooperativity of this heterodimeric enzyme.
Key words: co-immobilization, DNA topoisomerase, far-Western
analysis, fragment complementation, Leishmania, substrate
competition.
39–99 influence the interaction of the two subunits [9]. In
the present study, we characterize the minimal, functionally
indispensable core domains of this heterodimer by generating
several deletion constructs of each subunit and studying their interaction with each other. Our findings reveal that LdTOPIL39−456
(L) and LdTOPIS210−262 (S) are the minimal interacting
fragments capable of carrying out topoisomerization upon reconstitution and could functionally complement their respective wildtype counterparts in vivo. Two residues have also been identified
that play a crucial role in heterodimerization of the two subunits.
Taken together the present study provides, for the first time, insight
into the mechanistic details of functional subunit interaction.
MATERIALS AND METHODS
Construction of recombinant plasmids, overexpression
and purification
LdTOPIL and its deletion constructs were cloned in the
BamHI/HindIII site of pET28c, while LdTOPIS and its deletion
constructs were cloned in the BamHI/EcoRI site of pGEX5X2. All constructs were transformed into BL21(DE3)pLysS. For
reconstituted protein purification, co-transformation was carried
out in combinations as stated in Supplementary Table 1 (http://
www.BiochemJ.org/bj/409/bj4090481add.htm). Proteins were
purified through Ni-NTA (Ni2+ -nitrilotriacetate)–agarose [9] or
GST (glutathione transferase)–Sepharose 4B column followed
by phosphocellulose column (P11 cellulose; Whatman). See
Supplementary information I at http://www.BiochemJ.org/bj/409/
bj4090481add.htm for more details.
Abbreviations used: AP, alkaline phosphatase; CPT, camptothecin; DTT, dithiothreitol; GST, glutathione transferase; LdTOPIL (or L), large subunit of
topoisomerase IB of Leishmania ; LdTOPIS (or S), small subunit of topoisomerase IB of Leishmania ; L, LdTOPIL39−456 ; S, LdTOPIS210−262 ; Ni-NTA,
2+
Ni -nitrilotriacetate; NLS, nuclear localization signal; SV40, simian virus 40.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2008 Biochemical Society
482
Figure 1
S. BoseDasgupta and others
Schematic diagram of recombinant constructs and purification of proteins
Schematic representation of wild-type and deletion constructs of LdTOPIL (A, left panel) and LdTOPIS (B, left panel). The right panels show Coomassie Brilliant Blue-stained SDS/12 % PAGE of
recombinant proteins. Proteins were loaded as shown in the left panel.
Far-Western analysis
Purified L and its deletion constructs (Figure 1A) were
electrophoresed in SDS/12 % PAGE gel and transferred on to
a PVDF membrane (two sets). The control set was blotted using
polyclonal anti-L antibody. The other membrane was rinsed in
1×PBS, washed four times (1 h each) with renaturation buffer
[50 mM Tris/HCl, pH 7.5, 50 mM KCl, 5 mM MgCl2 , 0.5 mM
EDTA, 1 mM DTT (dithiothreitol), 5 % (v/v) glycerol and 0.05 %
Tween 20] containing decreasing concentrations of guanidinium
chloride (6, 4, 2 and 1 M) at 4 ◦C and blocked with 5 % (w/v)
BSA (2 h) in renaturation buffer [10]. Next, the membrane was
incubated overnight at 4 ◦C with bait protein, S, in renaturation
buffer and thereafter blotted using anti-S antibody. Similar
experiments were carried out with S and its deletion constructs.
One blot was incubated with L as bait and immunoblotted with
anti-L antibody. The control set was blotted with anti-S. For all
blots, AP (alkaline phosphatase)-conjugated anti-rabbit secondary
antibody was used.
Co-immobilization assay
6 × His-tagged L and LdTOPIL
(L) were co-expressed
separately with GST-tagged S and LdTOPIS210−262 (S) and
purified through Ni-NTA–agarose or GST–Sepharose 4B. Elution
fractions were run in SDS/12 % PAGE (three sets). One set
was stained with Coomassie Brilliant Blue, while the other two
sets were transferred on to nitrocellulose and blotted separately
with anti-L and anti-S antibodies respectively, followed by APconjugated anti-rabbit secondary antibody [9].
39−456
Plasmid relaxation and equilibrium cleavage assay
Co-expressed empty vector-induced lysates and reconstituted
enzymes L/S, L/S, L/S and L/S were purified
through Ni-NTA–agarose followed by phosphocellulose column
chromatography (final protein concentration was 0.8 mg/ml).
Purified proteins and pHOT1 plasmid substrate were mixed in
1:2 molar ratios in a relaxation buffer (25 mM Tris/HCl, pH 7.5,
c The Authors Journal compilation c 2008 Biochemical Society
5 % glycerol, 0.5 mM DTT, 50 mM KCl, 10 mM MgCl2 , 2.5 mM
EDTA and 125 µg/ml BSA) at 37 ◦C for the indicated time
periods. Samples were run in 1 % agarose gel and later stained
with EtBr (ethidium bromide) [7].
↓
The 25-mer oligonucleotide ML25 (5 -GAAAAAAGACTTAGAAAAATTTTTA-3 ; where the residues in bold indicate the
topoisomerase 1B cleavage site and the arrow indicates the position of the cleavage) was 5 -end-labelled with [γ -32 P]ATP
and annealed to reverse 25-mer MC25 (5 -TAAAAATTTTTCTAAGTCTTTTTTC-3 ). Excess of annealed oligonucleotides in the absence and presence of 100 µM CPT was mixed
with L/S (50 nM) or L/S (50–1000 nM) in a cleavage reaction
buffer (25 mM Tris/HCl, pH 7.5, 0.5 mM DTT, 10 mM MgCl2 ,
50 mM KCl, 1 mM EDTA and 150 µg/ml BSA) and assayed at
37 ◦C. It was electrophoresed in a denaturing 20 % PAGE gel
containing 7 M urea [9].
Substrate competition and fluorescence polarization assay
The same radiolabelled 25-mer duplex (7.5 nM) was either mixed
with L/S or L/S (25 and 50 nM) or together with L/S (50 nM)
and increasing amounts of L/S (50–600 nM) in a cleavage
buffer (25 mM Tris/HCl, pH 7.5, 0.5 mM DTT, 10 mM MgCl2 ,
50 mM KCl, 1 mM EDTA and 150 µg/ml BSA) for 30 min at
37 ◦C to induce substrate competition. Samples were boiled with
1 % SDS and run in SDS/15 % PAGE [11].
Freshly purified S and S were incubated with FITC and dimethylformamide in 100 mM phosphate buffer (pH 8) [12] and incubated at 16 ◦C for 30 min (Pierce Biotechnology). Samples were
desalted and free FITC was removed by passing through PD10
columns (Amersham Biosciences). Labelled S and S (50 or
100 nM) were mixed with increasing amounts of L (50–500 nM)
and L (100–1000 nM) separately. Using an F-3010, Hitachi/
Japan Polarization system at λex = 495 nm and λem = 520 nm, the
changes in fluorescence polarization were calculated. Thereafter,
the fraction of bound proteins (S and S) was calculated as stated
in Supplementary information II (http://www.BiochemJ.org/bj/
Minimal fragments of bi-subunit topoisomerase IB of Leishmania
483
409/bj4090481add.htm) and plotted against respective protein
concentration of L and L.
Conditional antisense knockouts, immunoblotting,
immunofluorescence and functional complementation
Antisense constructs of L (antiL) and S (antiS) were prepared
(detailed in Supplementary information III at http://www.
BiochemJ.org/bj/409/bj4090481add.htm) and transfected into
Leishmania tarentolae T7.TR strain (Jena Bioscience). DsRed
and SV40 (simian virus 40) T-antigen NLS (nuclear localization
signal)-tagged L (RLn) and GFP (green fluorescent protein)tagged S (GS) were transfected in parasites harbouring antiL
and antiS respectively. The transfectant promastigotes
antiL, antiL + Rn, antiL + RLn, antiS, antiS + G and antiS +
GS were used in functional complementation [13,14].
Site-directed mutagenesis
Single mutations were introduced into the Leishmania heterodimeric topoisomerase I at positions Lys436 , Asn441 and Lys455
of the large subunit and Lys249 , Asn256 and Asp261 of the small
subunit. Mutagenesis was performed using the Stratagene (La
Jolla, CA, U.S.A.) QuikChange® XL kit following the manufacturer’s protocol [15]. To carry out the desired mutations,
pET28c/LdTOPIL and pGEX-5X2/LdTOPIS were used as
templates. The following sense primers (along with antisense
counterparts) with their substitution sites in boldface were used:
LdTOPILK436A , 5 -GGTCCACCGCGGACGCGCTGGCCTACTTCAAC-3 ; LdTOPILN441A , 5 -GCTGGCCTACTTCGCCAAGGCGAACACC-3 ; LdTOPILK455A , 5 -CTGTGCAACCATCAAGCGTCCGTCTCGAAG-3 ; LdTOPISK249A , 5 -CCGCAACCATCCAGGCGAAGTTTCCGTGGGCC-3 ; LdTOPISN256A ,
LdTO5 -CCGTGGGCCATGGCCGCCGAGAACTTCG-3 ;
PISD261A , 5 -CGAGAACTTCGCTTTTTGAGGATCCC-3 . Mutations were confirmed through DNA sequencing in a PerkinElmer
(Norwalk, CT, U.S.A.) ABI PrismTM DNA sequencer.
RESULTS AND DISCUSSION
Recombinant protein constructs and their purification
Bi-subunit topoisomerase IB (L/S) may be the phylogenetic
predecessor of the latter reconstituted ‘cap’ and ‘catalytic’
domains of its monomeric counterparts, but it originates as
distinct subunits from different genes. The crystal structure of the
truncated bi-subunit topoisomerase IB of Leishmania indicates
that the two subunits contain separate interacting regions around a
pocket region, which encompasses the DNA [8]. Knowledge of
a functional inter-subunit co-operativity is obtained by identifying
the minimal, functionally indispensable interacting fragments of
this heterodimer. We had previously reported two N-terminal
deletion constructs of L, i.e. LdTOPIL39−635 and LdTOPIL99−635
where residues 1–39 influenced the non-covalent interaction with
DNA and residues 39–99 had some role in mediating subunit
interaction [9].
In the present study, we generated several N- and C-terminal
deletion constructs of L and S. The construct LdTOPIL1−456 was
generated keeping the conserved ‘VAILCNH’ motif, while the
construct LdTOPIL1−435 deletes this motif. Since LdTOPIL39−635
has been shown to be active [9], the smallest fragment
LdTOPIL39−456 was generated that deletes both the unconserved Nand C-termini of L. The smaller subunit has an unconserved
N-terminal extension of 200 amino acids, which bears an unusual
stretch of serine residues. The construct LdTOPIS80−262 starts
Figure 2
Far-Western analysis
Constructs of L and S as indicated in Figure 1 were subjected to SDS/12 % PAGE (two sets
each) and transferred on to a PVDF membrane and renatured. (A) Immunoblot using anti-L.
(B) Incubated with bait protein S and immunoblotted with anti-S. (C) Immunoblot using anti-S.
(D) Incubated with bait protein L and immunoblotted with anti-L.
with this serine stretch at the N-terminus. Next, the construct
LdTOPIS200−262 truncates the unconserved N-terminus, which
harbours the serine stretch. Two further N-terminal truncations
were carried out to generate the constructs LdTOPIS210−262 and
LdTOPIS215−262 . The C-terminal deletion constructs LdTOPIS1−255
and LdTOPIS1−245 were generated keeping the conserved
‘SKXXY’ motif harbouring the active-site tyrosine residue intact.
Another deletion construct LdTOPIS210−255 was generated deleting
the unconserved N-terminal end as well as part of the small Cterminus (Figures 1A and 1B, left panels). The deletion constructs
of S were prepared in the pGEX-5X2 vector so that the 26 kDa,
GST tag could stabilize the small protein fragments of S.
All recombinant proteins were transformed into
BL21(DE3)pLysS cells and purified using Ni-NTA–agarose
(constructs of L) or GST–Sepharose 4B (constructs of S)
affinity chromatography followed by phosphocellulose column
purification. SDS/PAGE analysis (Figures 1A and 1B, right
panels) shows that the proteins were purified to near homogeneity.
LdTOPIL39−456 (L) and LdTOPIS210−262 (S) constitute the
minimal interacting fragments
Far-Western analysis was carried out to study the in vitro
interaction between the proteins as well as to find the interacting
regions of two subunits. Using this method, we have identified
the minimal fragment of one subunit that interacts with the
other intact subunit [11]. The large subunit and its deletion
constructs were electrophoresed in two separate polyacrylamide
gels and transferred on to a nitrocellulose membrane. One set
was immunoblotted with anti-L antibody (Figure 2A). It shows
the exact position of the constructs and therefore served as the
control. The other membrane was incubated overnight with S and
then probed with anti-S antibody (Figure 2B). The proteins
LdTOPIL100−635 and LdTOPIL1−435 (lanes 3 and 5) were absent
from the blot, indicating that these two proteins failed to interact with intact S. The presence of bands corresponding to
LdTOPIL39−635 , LdTOPIL1−456 and LdTOPIL39−456 (lanes 2, 4 and
c The Authors Journal compilation c 2008 Biochemical Society
484
S. BoseDasgupta and others
6) indicates that these proteins interact with S. Deletion further
downstream of N-terminal residue 39 and upstream of C-terminal
residue 456 results in loss of interaction. Hence LdTOPIL39−456
(L) is the minimal fragment of L that interacts with S and the
interacting regions for L could be lying between residues 39–99
and residues 435–456.
Similarly, the small subunit and its deletion constructs
were electrophoresed in two separate polyacrylamide gels and
transferred on to nitrocellulose membrane. One set was immunoblotted with anti-LdTOPIS antibody as shown in Figure 2(C).
This served as the control, indicating the positions of S and its
deletion constructs. Another membrane was incubated overnight
with L and thereafter probed with anti-L antibody as shown in
Figure 2(D). The proteins LdTOPIS215−262 , LdTOPIS210−255 and
LdTOPIS1−245 (lanes 5, 6 and 7) failed to interact with L; hence
their respective bands were absent from the blot. The presence
of bands corresponding to LdTOPIS80−262 , LdTOPIS210−262 and
LdTOPIS1−255 (lanes 2, 4 and 8) indicates their interaction with
L. A peptide flanking residues 215–235, harbouring the catalytic
tyrosine residue (Tyr222 ), also failed to interact with L (results
not shown). Since deletion further downstream of N-terminal
residue 210 and C-terminal residue 255 of S results in loss of
interaction, LdTOPIS210−262 (S) is the minimal fragment of S
that interacts with L. The interacting regions of S therefore lie
between residues 210–215 and 245–262. From the above results,
it is evident that both L and S harbour two stretches of residues on
either side of their respective conserved motifs (‘VAILCNH’ for
L and ‘SKXXY’ for S), which together are involved in subunit
interaction at a time.
Co-immobilization assay was used to find out whether L and
S formed the minimal interacting fragments [10]. The 6 × Histagged constructs L and L and the GST-tagged constructs S
and S were separately co-transformed and co-expressed. Purification of His-tagged L and L using Ni-NTA–agarose
separately co-eluted GST-tagged S and S (Figure 3A, lanes 1–4),
while purification of GST-tagged S and S using GST–Sepharose
4B separately co-eluted His-tagged L and L (Figure 3A, lanes
5–8). Two similar polyacrylamide gels were electrophoresed and
the proteins were transferred on to a nitrocellulose membrane and
blotted using specific antibodies in order to confirm the identity
of the co-eluted proteins. Immunoblotting with anti-L antibody
lights up bands of 6 × His–L (73 kDa) in lanes 1, 2, 5 and 6
and bands of 6 × His–L (48.7 kDa) in lanes 3, 4, 7 and 8 in
Figure 3(B). The appearance of the bands 6 × His–L in lanes 5 and
6 and 6 × His–L in lanes 7 and 8 respectively indicate that both
GST–S and GST–S can separately co-elute the wild-type as well
as the minimal fragment of L through a GST–Sepharose column.
When anti-S antibody was used, bands corresponding to GST–
S (56 kDa) appear in lanes 1, 3, 5 and 7 and bands of GST–S
(34 kDa) appear in lanes 2, 4, 6 and 8 (Figure 3C). The appearance
of bands corresponding to GST–S in lanes 1 and 3 and GST–S
in lanes 2 and 4 respectively indicates that both 6 × His–L and
6 × His–L can separately co-elute the wild-type S as well as its
minimal fragment through an Ni-NTA column. Therefore L and
S were identified as the minimal interacting fragments of L
and S.
LdTOPIL39−456 /LdTOPIS210−262 (L/S) is active but 20-fold less
sensitive to CPT
Supplementary Table 1 shows all the co-expressed constructs that
were tested for plasmid relaxation activity, where L/S formed
the minimal functional heterodimer. The two empty vectors
pET28c and pGEX-5X2, transformed into BL21(DE3)pLysS,
were induced and the cell extracts were purified as stated in
c The Authors Journal compilation c 2008 Biochemical Society
Figure 3
Co-immobilization assay
6 × His-tagged L and L co-expressed with GST-tagged constructs of S and S, purified
and electrophoresed in SDS/12 % PAGE gel (three sets). (A) SDS/PAGE of Ni-NTA–agarose
(lanes 1–4) and GST–Sepharose purified proteins (lanes 5–8). Lanes 1 and 2, L co-elutes S
and S. Lanes 3 and 4, L co-elutes S and S. Lanes 5 and 6, S and S co-elutes L. Lanes
7 and 8, S and S co-elutes L. (B, C) Similar gels were transferred on to nitrocellulose and
immunoblotted with anti-L and anti-S antibodies.
the Methods section. The eluted fraction served as the negative
control. The purified subunits were found to be inactive in the
relaxation assay, indicating the absence of contaminating bacterial
topoisomerases. Time kinetics was carried out using co-expressed
empty vectors and reconstituted enzymes (shown in boldface in
Supplementary Table 1) mixed in 1:2 molar ratio with pHOT1
DNA substrate. Figure 4(A) shows the assay with the eluted
fraction of the empty vector control. Figures 4(B)–4(D) show
the relaxation activity of the reconstituted wild-type and deletion
constructs of L and S. The presence of a large number of
intermediate supercoils and a lesser amount of fully relaxed band
formation indicate the distributive nature of these enzymes. L/S
completely relaxes the substrate in 1 min (Figure 4B, lane 3).
L/S and L/S show that reduced activity and complete
relaxation occur in 10 min (Figure 4C, lane 5) and in 15 min
(Figure 4D, lane 6) respectively. L/S shows a gradual appearance of intermediate topoisomers; therefore the reconstituted
enzyme is capable of DNA relaxation (Figure 4E). Since it
did not follow steady-state conditions in the estimated time, its
fold reduction in relaxation activity was not determined. The
reconstituted construct L/LdTOPIS1−255 was found to be active
although it lacks few residues at the C-terminal interacting region
of S. The presence of the entire wild-type structure of L along with
255 residues of S probably provides the required structural balance
to attain an active conformation. Support for our hypothesis
is provided by the fact that truncated constructs LdTOPIL1−456
and LdTOPIL39−456 fail to interact with LdTOPIS1−255 (results not
shown). But LdTOPIS1−255 was not the minimal fragment of S that
interacts with L and hence it was not used in further studies. Davies
et al. [8] have recently reported a 2.27 Å (1 Å = 0.1 nm) crystal
structure of an active truncated L/S comprising residues 27–456
of L and residues 200–262 of S [8]. Therefore the functional
activity exhibited by the minimal fragments L/S indicates
their structural conformation close to the reported structure.
The sensitivity of an inhibitor of DNA topoisomerase I is
generally assessed based on relaxation kinetics in the absence and
presence of the drug. Since L/S did not follow steady-state
kinetics, its CPT-sensitivity was elucidated using the equilibrium
Minimal fragments of bi-subunit topoisomerase IB of Leishmania
Figure 4
L/S
485
Relaxation of pHOT1 DNA by using (A) eluted fraction of empty vector-induced bacterial lysate and enzymes (B) L/S, (C) L/S, (D) L/S and (E)
Lane 1, 150 fmol of supercoiled pHOT1 DNA; lanes 2–9, same as lane 1 but incubated with 75 fmol of reconstituted enzymes for indicated time periods.
cleavage assay [9]. CPT is known to trap the cleavable complex,
and in this assay it releases the cleaved product under denaturing
conditions. An equimolar concentration of L/S, compared
with L/S, did not form any cleaved product even in the presence
of CPT. A 20-fold molar excess of L/S produces 80 % of
the cleavable complex formed by L/S in the presence of CPT
(Figure 5). This indicated that L/S was 20-fold less sensitive
to CPT compared with L/S. Since L/S showed reduced CPTsensitivity, its relative affinity for DNA and interaction between
the fragments were studied.
LdTOPIL39−456 /LdTOPIS210−262 (L/S) has reduced affinity for
DNA and decreased association between them
A critical amount of a radiolabelled substrate induces competition
between L/S and L/S to form the cleavable complex. For
the wild-type L/S, phosphotyrosyl linkage is formed between
radiolabelled oligonucleotide and Tyr222 of S, while for L/S
it is formed by the same oligonucleotide and Tyr222 of S. In
an SDS/15 % PAGE, the two intact subunits or their fragments
are separated but the covalently linked oligonucleotide migrates
with S or S and is detected in an autoradiograph indicative
of the extent of cleavage. When the concentration of L/S is
Figure 5 Equilibrium cleavage assay using 5 -end-labelled 25-mer duplex
as shown
Lane 1, labelled 25-mer duplex DNA. Lanes 2 and 3, labelled DNA incubated with
50 nM L/S, in the absence (−) and presence (+) of CPT. Lanes 4–11, same as lane
1, but incubated with indicated amounts of L/S in the absence and presence of CPT
alternately.
c The Authors Journal compilation c 2008 Biochemical Society
486
S. BoseDasgupta and others
polarization. The fraction of bound protein (f B ) was obtained
from the equation stated in Supplementary information II and
was plotted against the protein concentrations (F) for S and S
separately with L (Figure 7A) and L (Figure 7B). The K D value
for the interaction between L and S is 6.1 × 10−8 M, that between
L and S is 1.53 × 10−7 M, that between L and S is 2.2 × 10−7 M
and that between L and S is 3.9 × 10−7 M. Therefore the extent
of interaction in descending order is L/S>L/S>L/S>L/S
where L/S manifests a 6.5-fold reduced association between
themselves. Therefore it can be stated that the reduced association
between the fragments forms a stringent conformation, which
is reflected by its decreased affinity for the DNA substrate and
reduced relaxation activity.
Figure 6 Substrate competition assay using 5 -end-labelled 25-mer duplex
(as shown)
Lanes 1 and 2, incubated with 25 and 50 nM each of L/S; lanes 3 and 4, incubated with 25 and
50 nM each of L/S. Lanes 5–11, same substrate incubated with 50 nM of L/S mixed with
increasing (50–600 nM) amounts of L/S.
increased, the amount at which it reduces the cleavable complex
of L/S by 50 % is the equivalence competitive concentration. Fold
excess of L/S at the equivalence concentration required over
wild-type L/S gives the K D (equilibrium dissociation constant)
value, since it shares a linear relationship [11]. The amount of
cleavable complex formed was obtained by densitometry of the
autoradiograph in Figure 6. An 8-fold molar excess of L/S
was required to reduce substrate binding of L/S by 50 %. The K D
value of L/S for the oligonucleotide substrate is 3.1 × 10−7 M [7];
hence the relative K D of L/S for the oligonucleotide substrate
is 2.5 × 10−6 M. The K D values of L/S and L/S obtained by
similar experiments were 1.03 × 10−6 and 1.64 × 10−6 M respectively. Reconstitution of ‘cap’ and ‘topo31’ of human topoisomerase IB occurs at 2:1 or greater molar ratio. It binds DNA with
reduced affinity but retains the processive nature. Therefore it
differs considerably from reconstituted L/S fragments [6].
Using fluorescence polarization, the association of L and L
separately with FITC-labelled S and S was measured at a λex
of 495 nm and a λem of 520 nm [12]. When free the smaller
FITC-labelled S and S exhibit rapid molecular rotation, thereby
depolarizing the polarized beam of light. When these proteins
are bound by L or L, their molecular rotation ceases with the
extent of binding, thereby causing an increase in fluorescence
Figure 7
LdTOPIL39−456 (L) and LdTOPIS210−262 (S) separately
complement their wild-type subunits in Leishmania
For complementation studies, wild-type genes were downregulated using a conditional antisense approach [14]. Figure 8(A)
shows the constructs antiL, Rn and RLn, which were transfected
into the L. tarentolae T7.TR strain to generate transfectant
parasites antiL, antiL + Rn and antiL + RLn as stated in
Supplementary information III. Figure 8(B) shows the nuclear
localization of Rn in antiL + Rn parasites and the kinetoplast
and nuclear localization of L in antiL + RLn parasites. The
NLS of SV40 T-antigen (n) facilitates its nuclear localization.
The construct antiL was generated from the nucleotide sequence
1725–1908 of wild-type L that was absent from L. Wild-type
L was down-regulated in antiL, antiL + Rn and antiL + RLn
parasites after 24 h of tetracycline induction of antiL construct
in these parasites (results not shown). Since one subunit is
unstable in the absence of the other subunit [4], a concomitant
decrease in wild-type S occurs within 72 h in antiL and antiL + Rn
and these parasites die. In antiL + RLn, tetracycline induction
reduces wild-type L but simultaneously overexpressed RLn
interacts with wild-type S and stabilizes it and RLn/S, similar
to the active L/S shown in Figure 4(D), is formed. Hence L
functionally complements wild-type L (Figure 8C). Figure 8(D)
shows the constructs antiS, G and GS, which were transfected
into L. tarentolae T7.TR strain to generate transfectant parasites
antiS, antiS + G and antiS + GS as stated in Supplementary
information III. G is localized in the cytoplasm of antiS + G,
while GS is localized in the nucleus and kinetoplast of
antiS + GS (Figure 8E). The construct antiS was generated from
the nucleotide sequence 240–420 of wild-type S that was absent
Fluorescence polarization assay
(A) The change in polarization value was measured at 518 nm and the fraction bound for 50 nM of S and S mixed with increasing amounts of L (50–500 nM) was plotted. Results shown represent
means +
− S.D. (n = 3). (B) Same as (A) but using 100 nM of S and S mixed with increasing amounts (100–1000 nM) of L.
c The Authors Journal compilation c 2008 Biochemical Society
Minimal fragments of bi-subunit topoisomerase IB of Leishmania
Figure 8
487
Fragment complementation experiments
Schematic representation of (A) transfected constructs of L and (B) localization of Rn and RLn. (C) Functional complementation by RLn. Promastigotes (1 × 105 ) were cultured with (+) or without
(−) tetracycline (T). Live promastigotes were calculated at 12 h intervals and plotted against time. (D) Transfected constructs of S and (E) localization of G and GS. (F) Functional complementation
by GS as stated in (C).
from S. Wild-type S was down-regulated in antiS, antiS + G
and antiS + GS parasites after 24 h of tetracycline induction
of antiS construct in these parasites (results not shown). Since
one subunit is unstable in the absence of the other subunit [4],
a concomitant decrease in wild-type L occurs within 72 h in
antiS and antiS + G and these parasites die. In antiS + GS,
tetracycline induction reduces wild-type S, but simultaneously
overexpressed GS interacts with wild-type L and stabilizes it,
and L/GS, similar to active L/S shown in Figure 4(C), is
formed. Hence S functionally complements the wild-type S
(Figure 8F).
Residues Lys455 of LdTOPIL and Asp261 of LdTOPIS are involved in
heterodimerization
Alanine substitutions of three charged residues were carried out
in the C-terminal interacting region of both subunits. For L, the
residues were Lys436 , Asn441 and Lys455 , while for S they were
Lys249 , Asn256 and Asp261 respectively. The reconstituted mutant
enzymes LdTOPILK436A /S, LdTOPILN441A /S, L/LdTOPISK249A and
L/LdTOPISN256A did not show any appreciable change in the
relaxation pattern compared with the wild-type enzyme L/S.
Besides, the reconstitution of each of the two mutants of L and
S with each other also showed negligible changes in the plasmid
relaxation pattern compared with L/S (results not shown). Time
kinetics of relaxation was carried out using wild-type enzyme
L/S (Figure 9A) and two mutant constructs. Figure 9(B) shows
that reconstitution of L with LdTOPISD261A (SD261A ) causes a
15-fold decrease in the relaxation activity compared with L/S.
This is evident from the fact that complete relaxation by the
mutant enzyme L/SD261A occurs in 15 min compared with 1 min
for L/S. Similarly, reconstitution of LdTOPILK455A (LK455A ) with
S causes a 25-fold decrease in the relaxation activity compared
c The Authors Journal compilation c 2008 Biochemical Society
488
Figure 9
S. BoseDasgupta and others
Relaxation of pHOT1 DNA using reconstituted wild-type and mutant enzymes
(A) L/S (B) L/SD261A , (C) LK455A /S and (D) LK455A /SD261A . Lane 1, 150 fmol of supercoiled pHOT1 DNA; lanes 2–9, same as lane 1 but incubated with 75 fmol of reconstituted enzymes for the indicated
time periods.
Table 1
Various properties of the reconstituted wild-type and minimal fragments
Enzyme
LdTOPIL/S
(L/S)
LdTOPIL/LdTOPIS210−262
(L/S)
LdTOPIL39−456 /LdTOPIS
(L/S)
LdTOPIL39−456 /LdTOPIS210−262
(L/S)
Relaxation activity (fold
reduced compared with L/S)
DNA-binding affinity
(K D )DNA
CPT sensitivity (fold
reduced compared with L/S)
Inter-subunit affinity
(K D )protein
100-fold
3.1 × 10−7 M
82-fold
1.03 × 10−6 M
7-fold
1.53 × 10−7 M
65-fold
1.64 × 10−6 M
9-fold
2.2 × 10−7 M
21-fold
2.5 × 10−6 M
20-fold
3.9 × 10−7 M
with L/S, since complete relaxation by the mutant enzyme LK455A /S
occurs in 25 min compared with 1 min for L/S (Figure 9C). The
reconstitution of LK455A and SD261A did not show any relaxation
activity (Figure 9D). The reconstituted enzymes formed by
one mutant and the other wild-type subunit induce a structural
stringency at one end of the subunit harbouring the mutation.
But the intact subunit balances the overall structure by forming a
partially interacted conformer having reduced relaxation activity.
The inhibitory effect on relaxation by mutation in LK455A of the
reconstituted enzyme LK455A /S is greater than that of the mutation
in SD261A of the reconstituted enzyme L/SD261A . Reconstitution
of two mutant subunits, LK455A and SD261A , fails to assume an
interacted conformation and hence it does not show any relaxation
activity.
CONCLUSION
Overall, the present study identifies the minimal functionally
active fragments of the heterodimeric topoisomerase IB of
Leishmania (Table 1). The search for important residues
governing subunit interaction is narrowed to the active L/S.
Therefore, taking this into account, we have identified residues
Lys455 of L and Asp261 of S as two key amino acids modulating
c The Authors Journal compilation c 2008 Biochemical Society
0
6.1 × 10−8 M
In vivo complementation
in Leishmania
–
GS complements
wild-type S
RLn complements
wild-type L
–
subunit interaction. The reduced fragment association between
L and S results in a weakly reconstituted enzyme having lower
DNA binding affinity, reduced cleavage and relaxation activity.
The bi-subunit nature of topoisomerase IB in Leishmania allows
functional complementation using truncated constructs. Therefore
functional dissection of these individual subunits will help in
better understanding the molecular architecture of this unusual
heterodimeric type IB topoisomerase of the parasite.
We are grateful to Professor S. Roy, Director, Indian Institute of Chemical Biology, for his
interest in this work. We thank Professor S. M. Beverley and Professor G. A. M. Cross for
the gift of the Leishmania transfection vectors. This work was supported by a grant from
the Department of Biotechnology, Government of India (BT/PR6399/BRB/10/434/05), to
H. K. M. S. B. D. was supported by a Senior Research Fellowship from the CSIR (Council
of Scientific and Industrial Research), Government of India.
REFERENCES
1 Champoux, J. J. (2001) DNA topoisomerases, structure, function and mechanism.
Annu. Rev. Biochem. 70, 369–413
2 Villa, H., Otero Marcos, A. R., Reguera, R. M., Balana-Fouce, R., Garcia-Estrada, C.,
Perez-Pertejo, Y., Tekwani, B. L., Myler, P. J., Stuart, K. D., Bjornsti, M. A. and Ordonez, D.
(2003) A novel active DNA topoisomerase I in Leishmania donovani . J. Biol. Chem. 278,
3521–3526
Minimal fragments of bi-subunit topoisomerase IB of Leishmania
3 Balana Fouce, R., Redondo, C. M., Perez-Pertezo, Y., Diaz-Gonzales, R. and Ruguera, M.
(2006) Targeting atypical trypanosomatid DNA topoisomerase I. Drug Discov. Today 11,
733–740
4 Bakshi, R. and Shapiro, T. A. (2004) RNA interference of Trypanosoma brucei
topoisomerase IB, both subunits are essential. Mol. Biochem. Parasitol. 136, 249–255
5 Ireton, G., Stewart, L., Parker, L. H. and Champoux, J. J. (2000) Expression of human
topoisomerase I with a partial deletion of the linker region yield monomeric and dimeric
enzymes that respond differently to camptothecin. J. Biol. Chem. 275, 25820–25830
6 Yang, Z. and Champoux, J. J. (2002) Reconstitution of enzymatic activity by the
association of the cap and catalytic domains of human topoisomerase I. J. Biol. Chem.
277, 30815–30823
7 Das, B. B., Sen, N., Ganguly, A. and Majumder, H. K. (2004) Reconstitution and functional
characterization of the unusual bi-subunit type I DNA topoisomerase from Leishmania
donovani . FEBS Lett. 565, 81–88
8 Davies, D. R., Mushtaq, A., Interthal, H., Champoux, J. J. and Hol, W. J. (2006) The
structure of the transition state of the heterodimeric topoisomerase I of Leishmania
donovani as a vanadate complex with nicked DNA. J. Mol. Biol. 357,
1202–1210
489
9 Das, B. B., Sen, N., BoseDasgupta, S., Ganguly, A. and Majumder, H. K. (2005) N-terminal
region of the large subunit of Leishmania donovani bi-subunit topoisomerase I is involved
in DNA relaxation and interaction with small subunit. J. Biol. Chem. 280, 16335–16344
10 Wu, L. and Hickson, I. D. (2002) The Bloom’s syndrome helicase stimulates the activity of
topoisomerase IIIα. Nucleic Acids Res. 30, 4823–4829
11 Stewart, L., Ireton, G. C. and Champoux, J. J. (1997) Reconstitution of human
topoisomerase I by fragment complementation. J. Mol. Biol. 269, 355–372
12 Park, S. H. and Raines, R. T. (2004) Fluorescence polarization assay to quantify
protein–protein interactions. Methods Mol. Biol. 261, 161–166
13 Robinson, K. A. and Beverley, S. M. (2003) Improvements in transfection efficiency and
tests of RNA interference (RNAi) approaches in the protozoan parasite Leishmania .
Mol. Biochem. Parasitol. 128, 217–228
14 Goswami, S., Dhar, G., Mukherjee, S., Mahata, B., Chaterjee, S., Home, P. and Adhya, S.
(2006) A bi-functional tRNA import receptor from Leishmania mitochondria. Proc. Natl.
Acad. Sci. U.S.A. 103, 8354–8359
15 Das, B. B., BoseDasgupta, S., Ganguly, A., Mazumder, S., Roy, A. and Majumder, H. K.
(2007) Leishmania donovani bisubunit topoisomerase I gene fusion leads to an active
enzyme with conserved type IB enzyme function. FEBS J. 274, 150–163
Received 3 October 2007; accepted 9 October 2007
Published as BJ Immediate Publication 9 October 2007, doi:10.1042/BJ20071358
c The Authors Journal compilation c 2008 Biochemical Society