Characterization of DNA topoisomerase I from Mycobacterium tuberculosis: DNA cleavage and religation properties and inhibition of its activity

Archives of Biochemistry and Biophysics 528 (2012) 197–203

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Characterization of DNA topoisomerase I from Mycobacterium tuberculosis: DNA cleavage and religation properties and inhibition of its activity Adwait Anand Godbole a, Majety Naga Leelaram a, Anuradha Gopal Bhat a, Paras Jain a, Valakunja Nagaraja a,b,⇑ a b

Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560 064, India

a r t i c l e

i n f o

Article history: Received 16 July 2012 and in revised form 2 October 2012 Available online 17 October 2012 Keywords: Topoisomerase I Mycobacterium tuberculosis Toprim motif DNA relaxation DNA cleavage

a b s t r a c t Type I DNA topoisomerases from bacteria catalyse relaxation of negatively supercoiled DNA in a Mg2+ dependent manner. Although topoisomerases of distinct classes have been subjected for anti-cancer and anti-infective drug development, bacterial type I enzymes are way behind in this regard. Our studies with Mycobacterium smegmatis topoisomerase I (MstopoI) revealed several of its distinct properties compared to the well studied Escherichia coli topoisomerase I (EctopoI) suggesting the possibility of targeting the mycobacterial enzyme for inhibitor development. Here, we describe Mycobacterium tuberculosis topoisomerase I (MttopoI) and compare its properties with MstopoI and EctopoI. The enzyme cleaves DNA at preferred sites in a pattern similar to its ortholog from M. smegmatis. Oligonucleotides containing the specific recognition sequence inhibited the activity of the enzyme in a manner similar to that of MstopoI. Substitution of the acidic residues, D111 and E115 which are involved in Mg2+ co-ordination, to alanines affected the DNA relaxation activity. Unlike the wild type enzyme, D111A was dependent on Mg2+ for DNA cleavage and both the mutants were compromised in religation. The monoclonal antibody (mAb), 2F3G4, developed against MstopoI inhibited the relaxation activity of MttopoI. These studies affirm the characteristics of MttopoI to be similar to MstopoI and set a stage to target it for the development of specific small molecule inhibitors. Ó 2012 Elsevier Inc. All rights reserved.

Introduction Bacterial type I topoisomerases generally belong to the 1A group capable of catalyzing the relaxation of negatively supercoiled DNA. They introduce a transient single strand nick in one of the strands of the DNA and rejoin the DNA backbone after the passage of the intact complementary strand through the cleaved DNA [1,2]. During the process, the enzymes form transient covalent intermediates with 50 end of the cleaved strand. The other end of the strand is held non-covalently forming an enzyme-mediated bridge. Although several anti-bacterial and anti-cancer therapeutics that target different topoisomerases stabilize the covalent complex to accumulate protein–DNA adducts on the genome [3], none of these molecules are known to inhibit the type IA topoisomerases. They target either the type II topoisomerases of bacterial and eukaryotic origin or type IB topoisomerases found mostly in eukaryotes. As such, not many efforts have been documented on the discovery of inhibitors for type IA topoisomerases. A thorough

⇑ Corresponding author at: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India. Fax: +91 80 23602697. E-mail address: [email protected] (V. Nagaraja). 0003-9861/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2012.10.004

understanding of the biochemistry of these enzymes and the factors governing the cleavage-religation reaction are important for designing specific inhibitors for this group of enzymes. In this manuscript, we describe the properties of topoisomerase I from Mycobacterium tuberculosis, a devastating human pathogen that afflicts approximately two million people globally every year. The upsurge of multi-drug resistant (MDR)1 [4] and extremely drug resistant (XDR) [5] strains of the organism demands the identification of new targets and therapeutics. Enzymes such as topoisomerases which participate in the key central processes are obviously well suited targets for new lead molecule discovery studies. The stimulation of DNA cleavage and perturbation of cleavage-religation equilibrium by mutants of various bacterial topoI and the consequent cell killing [6,7] validates this group of enzymes as potential drug targets. Unlike Escherichia coli, which encodes four topoisomerases [8], only two topoisomerases, topoisomerase I and DNA gyrase are encoded by the M. tuberculosis genome [9]. The presence of only a 1 Abbreviations used: MstopoI, Mycobacterium smegmatis topoisomerase I; EctopoI, Escherichia coli topoisomerase I; MttopoI, Mycobacterium tuberculosis topoisomerase I; mAb, monoclonal antibody; STS, strong topoisomerase I site; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; NTD, N-terminal domain; CTD, C-terminal domain.

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single type I topoisomerase and a DNA gyrase in the genome further highlights the importance of these enzymes in cellular function. Moreover, topoI knock out mutants of M. tuberculosis were not obtained in the genetic screen involving insertional mutagenesis indicating that the enzyme may be essential for cell viability [10]. Further, our studies with topoI from a non-pathogenic mycobacterium, Mycobacterium smegmatis, revealed its unique properties not found with other typeIA topoisomerases. MstopoI binds to both single and double stranded DNA with comparable affinity [11]. The sequence specific binding of the enzyme during its catalytic cycle leads to specific cleavage at the sequence CG/TCT;TC/G (where ; indicates the site of cleavage) [12,13]. Studies carried out with EctopoI [14] and MstopoI [11] have shown that Mg2+ is essential for the relaxation activity. However, mutations in the metal ion co-ordination motif D  D  E in MstopoI showed different properties compared to the mutants in EctopoI [7]. The differences between the two well-studied type IA enzymes also warranted thorough investigation of the properties of MttopoI. In this manuscript, we have compared the properties of MttopoI with MstopoI. The enzyme binds to a specific recognition sequence and cleaves the DNA in a site specific fashion. Importantly, the mutants in the metal ion co-ordination motif cause cell lethality and inhibitors developed against MstopoI inhibit the relaxation activity of the enzyme with equal efficiency.

Materials and methods Cloning of M. tuberculosis topoiosomerase I MttopoI was cloned using two approaches to express the protein with (pAVN1) or without (pAVN2) a C-terminal His-tag. Briefly, the gene was cloned in three fragments, viz. N-terminal domain (NTD)1, NTD2 and C-terminal domain (CTD) in pET20b vector using the strategy followed earlier for cloning MstopoI [15]. Restriction sites, NdeI, EcoRV, BamHI and EcoRI were engineered that could be used to reassemble the gene. Table 1 lists the primers used for cloning.

Site directed mutagenesis Site directed mutagenesis to generate the D111A, D113A and E115A mutants in the metal binding motif, D  D  E, was carried out as described [7]. Expression plasmid harboring the NTD1 was used as a template. Oligonucleotide primers carrying the respective mutant amino acid codon substitutions were used as forward primers (Table 2) and RP1 was used as reverse primer (Table 1). The truncated mutants were subcloned into the full length topoisomerase I using NdeI/EcoRV restriction sites. The mutation in each case was confirmed by restriction digestion followed by sequencing of the plasmid DNA. Table 1 The forward primers (FP) and reverse primers (RP) used for the cloning of MttopoI fragments. Primer

Sequence

FP1

50 -AAAACGATCGATCATATGGCTGACCCGAAAAC GAAG-30 50 -AAGGATATCCCAGTAGGCCGCGCT-30 50 -ATCGATCATATGGATATCCTTGCCAAGCTGGAT GCCAGC-30 50 -GCAGCAAGGATCCACCGGTGCG-30 50 -AAAACGATCGATCATATGGGATCCTTGCTGCGG AGCATGGACCT-30 50 -AGTACGCGGGAATTCTCGCGCTTGGCTG-30 50 -AGTACGCGGGAATTCGCGCTTGGCTG-30

RP1 FP2 RP2 FP3 RP3 (with the His tag) RP3 (without the His-tag)

Table 2 The primers used for site directed mutagenesis. Primer

Sequence

Restriction site generated

D111A

50 -TCTGGCCACAGCTGGGGACCGTG-30

PvuII

D113A

50 -CACGGATGGGGCCCGTGAGGGCG-30

ApaI

E115A

50 -GGGACCGTGCCGGCGAAGCTATTGC30

NaeI

Purification of MttopoI and its mutants Purification of MttopoI was carried out from its native source i.e. M. tuberculosis H37Ra cells as well as from E. coli BL21 (DE3) cells expressing the recombinant construct (without His-tag) by following the procedure employed for MstopoI [15]. To purify the protein from its native source, the surface cultures of M. tuberculosis H37Ra were grown in modified YK medium [16] at 37 °C and the cells were harvested after 3 weeks by centrifugation and resuspended in buffer A (50 mM Tris–HCl, pH 7.4, 50 mM KCl, 10 mM EDTA, 5 mM b-mercaptoethanol and 10% (v/v) glycerol). To purify the recombinant protein without the His-tag, E. coli BL21 cells transformed with pAVN2 were induced at 37 °C with 0.3 mM IPTG. The cells were harvested after 3 h by centrifugation and resuspended in buffer A. Both the cell suspensions were processed further in a similar fashion. The cell suspension was sonicated and centrifuged at 100,000g for 3 h. The KCl concentration of the supernatant was brought to 150 mM and polyethylenimine (pH 7.4) was added to a final concentration of 0.5%. The precipitate was removed by centrifugation at 15,000g for 15 min followed by 0–70% ammonium sulfate fractionation. The pellet was dissolved and dialyzed in buffer B (50 mM Tris–HCl, pH 7.4, 50 mM KCl, 1 mM EDTA, 5 mM b-mercaptoethanol and 10% (v/v) glycerol) and purified using a Hitrap-heparin Sepharose followed by Hitrap-SP Sepharose columns (Amersham Pharmacia, UK) using a linear gradient of 50– 700 mM KCl. The active fractions containing the protein near to homogeneity were pooled, dialyzed against buffer C (20 mM Tris–HCl, pH 8.0, 1 mM EDTA, 5 mM b-mercaptoethanol and 20% (v/v) glycerol) and stored at 70 °C. To purify the His-tag variant of MttopoI or its mutants which were expressed in E. coli BL21 (DE3), a similar procedure till resuspension of the ammonium sulphate pellet was followed. The resuspension was loaded onto a Ni– NTA (Qiagen, Germany) column and the proteins were purified as per the manufacturer’s instructions.

Various assays with topoisomerase The DNA relaxation assay of MttopoI and its mutants, MstopoI or EctopoI was carried out as described before [11]. One unit is defined as the amount of enzyme required for 50% relaxation of 500 ng supercoiled pUC18 DNA at 37 °C for 30 min. Reaction products were resolved in a 1.2% (w/v) agarose gel and visualized by staining with 0.5 lg/ml ethidium bromide and documented by Biorad gel documentation system. The supercoiled bands were quantified using ImageGauge software and the concentration at which 50% supercoiled DNA remained is considered 1 Unit and calculated the specific activity. To determine DNA binding, electrophoretic mobility shift assays (EMSA) were carried out with 50 end labeled oligonucleotides having the strong topoisomerase I site (STS) [12]. The oligonucleotides (0.1 pmol) were incubated in buffer containing 40 mM Tris–HCl (pH 8.0), 20 mM NaCl and 1 mM EDTA with indicated concentrations of MstopoI, EctopoI or native MttopoI on ice for 15 min. The DNA–protein non-covalent complexes were resolved from the free DNA in an 8% native polyacrylamide gel (30:0.8) at 4 °C using 1TBE as the running buffer [15]. The

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radioactivity associated with the free and bound oligonucleotides was visualized by phosphorimager (model BAS 1800; Fuji Film) and quantified using ImageGauge software. The binding affinity was determined by GraphPad Prism Version 5.0 using the equation Y = (Bmax  X)/KD + X where Y = specific binding, X = concentration of enzyme, KD = equilibrium dissociation constant and Bmax = maximum enzyme binding. For DNA cleavage experiments, the radiolabeled oligonucleotides were incubated with indicated concentrations of the enzymes in 40 mM Tris–HCl (pH 8.0), 20 mM NaCl and 1 mM EDTA at 37 °C for 30 min. The reaction products were resolved in 12% denaturing PAGE as described [15]. For religation, the oligonucleotides containing the STS were incubated with 100 nM MttopoI or the mutants at 37 °C for 15 min in reaction buffer as above to form cleavage complex. Religation was initiated with indicated concentrations of Mg2+ at 37 °C for 15 min. The reactions were terminated with 45% formamide and heating at 95 °C for 2 min and the reaction products were resolved in 12% denaturing PAGE as described [7]. Immunoblotting One microgram each of purified MttopoI and EctopoI were resolved on an 8% SDS–PAGE and transferred to PVDF membrane in transfer buffer (50 mM Tris–glycine buffer) for 3 h at 200 mA. The membrane was blocked with 2% BSA in PBS and probed with 2F3G4 mAb. Bound mAb was detected using goat anti-mouse IgG conjugated to horseradish peroxidase and enhanced chemiluminescence (ECL, Millipore, USA).

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the topoI from the two mycobacterial species showed 89% sequence similarity. Such a high degree of similarity (Supplemental Fig. S1B) would suggest that these enzymes could share some of the properties. However, the differences in the primary sequence may also result in subtle differences in their properties. To compare the properties of the topoI from the two related species, MttopoI was purified from M. tuberculosis H37Ra (Supplemental Fig. S2A and S2B) as well as from E. coli BL21 (DE3) expressing a recombinant with or without a C-terminal His-tag (Supplemental Fig. S3A and S3B) as described in Materials and methods. The DNA relaxation activity of the native and the recombinant enzymes was found to be comparable. The specific activity of the native enzyme was found to be 2  105 U/mg while that for the recombinant MttopoI, with or without the His-tag, were 0.5  105 and 0.8  105 U/mg respectively (Supplemental Fig. S3C and S3D). EMSA experiments were carried out to determine whether MttopoI has a similar sequence preference for DNA binding as that of MstopoI. 32-mer oligonucleotides with or without the STS [20] were incubated separately with MstopoI, EctopoI and native MttopoI. The MstopoI and MttopoI formed DNA–protein complexes with the STS-oligonucleotides; complexes with non STS oligonucleotides were not observed indicating that MttopoI also recognizes DNA at specific sites in a manner similar to MstopoI. Recombinant enzymes also bound in similar fashion (not shown). As expected, EctopoI could bind to the oligonucleotides irrespective of the presence or absence of the STS (Fig. 1A). The dissociation constant (KD) of MttopoI to the STS harboring oligonucleotides was comparable to the values obtained for MstopoI (Fig. 1B–D). MttopoI cleaves DNA at preferred sites

Results DNA binding properties of MttopoI Amino acid sequence comparison between EctopoI, MstopoI and MttopoI showed considerable similarity (54%) in their N-terminal two-third region and high degree of variation in the remaining one third CTD (Supplemental Fig. S1A). Zinc fingers found in the CTD of most of the type IA topoisomerases [17,18] were absent in both MstopoI as well as MttopoI [19]. The amino acid sequence of

Bacterial topoI introduces a single strand nick in the DNA during the reaction cycle establishing a 50 phosphotyrosine linkage. The E. coli enzyme is relatively non-specific in its DNA recognition and cleavage. The enzyme cleaves DNA in the sequence context of CXXX; [21]. In contrast, MstopoI was shown to cleave in sequence specific fashion [12]. The DNA cleavage characteristics of the recombinant as well as the native MttopoI were compared with MstopoI and EctopoI. The native MttopoI generated cleavage product similar to MstopoI with STS-oligonucleotide; non-specific DNA

Fig. 1. MttopoI exhibits sequence specific binding. (A) EMSA was carried out using 5 units of MstopoI (lanes 2 and 3), MttopoI (lanes 4 and 5) or EctopoI (lanes 6 and 7) and 50 end labeled 32-mer STS or non-STS oligonucleotides. C, oligonucleotides in absence of topoI; S, STS oligonucleotide; N, non-STS oligonucleotide. (B and C) 32-mer containing STS was incubated with increasing concentrations of MttopoI (B) or MstopoI (C) in a buffer containing containing 40 mM Tris–HCl (pH 8.0), 20 mM NaCl, 1 mM EDTA. Lane 1: control; Lanes 2–10 increasing concentration of MttopoI or MstopoI. (D) The KD values are calculated using GraphPad Prism version 5.0. Ms, MstopoI; Mt, MttopoI and Ec, EctopoI.

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Fig. 2. Cleavage site specificity of MttopoI. (A) Cleavage assays were carried out using 20 units of MstopoI (lanes 2 and 3) or MttopoI (lanes 4 and 5) with 32-mer STS or nonSTS oligonucleotides. C, oligonucleotides in the absence of topoI; S, oligonucleotides with STS; N, oligonucleotides without STS. (B) DNA cleavage of 32-mer STS oligonucleotide with increasing concentrations of MstopoI (Ms, lanes 2–4), MttopoI (Mt) with His-tag (lanes 5–7) or MttopoI without His-tag (lanes 8–10). (C) DNA cleavage of 32-mer STS oligonucleotide with 200 nM EctopoI (Ec, lane 3), MstopoI (lane 4) or MttopoI (lane 5). 50 end labeled 32, 28, 24 and 22-mer were used as marker (lane 1) and indicated towards the left. C, oligonucleotide in the absence of topoI. Arrow indicates the 19-mer cleavage product. MttopoI yielded single major cleavage products similar to MstopoI unlike EctopoI which yielded multiple cleavage products.

Metal binding mutants are compromised in DNA relaxation Sequence alignment of MttopoI with other type IA topoisomerases revealed the presence of a highly conserved acidic triad, D111  D113  E115 within the toprim domain in NTD1 (Supplemental Fig. S4). The role of the motif in Mg2+ co-ordination has been elucidated for both EctopoI [22,23] and MstopoI [7]. To understand the contribution of the individual acidic residues of MttopoI in the metal ion co-ordination, alanine mutants were generated. Since the overall properties viz. DNA relaxation, individual steps like DNA binding and cleavage of the two recombinant proteins were comparable, mutations were generated with the His-tagged variant of the enzyme. D111A and E115A mutants were expressed and purified. The purification profile of both the mutant proteins was similar to that of the wild type enzyme. However, transformation of the D113A into E. coli BL21 pLysS or pLysE cells yielded very few transformants (Supplemental Fig. S5). Upon induction of these transformants with IPTG, protein bands with faster mobility of varied size were observed on SDS–PAGE in each of the colony analyzed (Supplemental Fig. S6A). Western blotting with anti-topoI specific antibody, 2F3G4 [24], confirmed the presence of truncated topoI (Supplemental Fig. S6B). Owing to this problem, the protein could not be purified and characterized further. Mutation at analogous position in MstopoI also did not yield any transformants [7]. Both, D111A and E115A mutants did not show any DNA relaxation activity even at higher enzyme concentrations (Fig. 3A and B). In contrast to these observations, corresponding mutants of MstopoI, D108A and E112A, exhibited 20 and 5-fold reduction in activity respectively [7] while the mutants of EctopoI resulted only in 2- and 5-fold loss of relaxation activity [23]. The activity of the MttopoI mutants could not be rescued either by supplementing with higher Mg2+ concentration or varying other reaction conditions (not shown).

-

wt

100

200

300 nM N/R

D111A

(A)

S

(B)

N/R E115A

sequences were refractory to cleavage (Fig. 2A). However, an additional minor cleavage product was observed with the recombinant MttopoI (with and without the His-tag) indicating minor differences in the cleavage characteristics compared to MstopoI (Fig. 2B). However, the additional minor cleavage product was also observed in assays with MstopoI when the enzyme was used in excess with some DNA substrates (not shown). DNA cleavage with EctopoI using the same substrate yielded multiple cleavage products (Fig. 2C) – a pattern expected based on its well established properties.

S 1

2

3

4

5

Fig. 3. DNA relaxation assay. 500 ng of supercoiled pUC18 was incubated with increasing concentrations of D111A (A, lanes 3–5) or E115A (B, lanes 3–5) in assay buffer and processed as described in Materials and methods. 25 nM wt MttopoI was used as control (lane 2). S, supercoiled DNA; N/R, nicked or relaxed DNA.

For bacterial type IA enzymes, DNA cleavage is Mg2+ independent but religation requires the metal ion [25]. This prompted us to check the effect of the mutations in the metal binding motif in MttopoI on DNA cleavage and religation. When the cleavage reactions were carried out in the absence of Mg2+, D111A was compromised in DNA cleavage activity (Fig. 4A), while E115A showed DNA cleavage comparable to wild type (Fig. 4B). Similar to D108A mutant of MstopoI, D111A mutant of MttopoI required Mg2+ for DNA cleavage; with increasing concentrations of Mg2+, the mutant exhibited stimulated DNA cleavage (Fig. 4C). To check the effect of the mutations on religation, assays were carried out as described for MstopoI earlier ([7], Materials and methods). The 50 end labeled oligonucleotides were incubated with the enzyme to yield a 19mer cleavage product and a 13-mer-enzyme covalent complex. Religation of the cleaved strand regenerates the 32-mer substrate which is assessed by the decrease in the 19-mer cleavage product. While the cleavage product decreased with wild type MttopoI in the presence of Mg2+ (Fig. 5, compare lanes 2 and 3), it remained unaltered with the mutants (compare lanes 4 with 5 and 6 with 7), indicating that both D111A and E115A are compromised in religation.

Inhibition of DNA relaxation activity of topoisomerase I Site specific recognition and cleavage of the substrate DNA by MttopoI suggests that oligonucleotide substrates containing the

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(A) wt C

1

25

2

50

3

100

4

(B)

D111A 25

5

50

6

wt C

1

(C)

wt

100 nM

C

0

7

1

2

5

3

D111A 10

0

5

4

5

6

10

mM Mg2+

7

E115A

25

50

100

25

50

2

3

4

5

6

100 nM

7

Fig. 4. DNA cleavage by metal binding mutants. Increasing concentration of MttopoI (A and B, lanes 2–4), D111A (A, lanes 5–7) or E115A (B, lanes 5–7) were incubated with 32-mer STS oligonucleotide. MttopoI (lanes 2–4) or D111A (C, lanes5–7) were incubated with a 32-mer harboring an STS for 30 min at 37 °C in increasing concentration of Mg2+. Arrow indicates the cleavage product. C, oligonucleotide in the absence of topoI.

Fig. 5. Intramolecular religation by D111A and E115A 32-mer harboring STS was incubated with wt, D111A or E115A at 5 mM Mg2+ for DNA cleavage (lanes 2, 4 and 6 respectively) and the concentration was increased to 10 mM to initiate religation (lanes 3,5 and 7) (left panel). The bands were quantified by ImageGauge software version 2.0 and plotted by GraphPad Prism version 5.0 (right panel). The amount of cleavage by each enzyme at 5 mM Mg2+ was normalized to 100%. Arrow indicates the cleavage product. An additional minor cleavage product was observed with the mutants similar to the recombinant wild type enzyme.

recognition sequence could be competitive inhibitors of the relaxation activity of the enzyme. In the DNA relaxation assay, the enzyme was competed with increasing concentrations of STS or non-STS oligonucleotides. While the DNA relaxation activity of MttopoI and MstopoI was inhibited at a 10-fold excess of STS-oligonucleotides compared to pUC18 DNA, no inhibition was observed with non-specific oligonucleotides. In contrast, activity of the EctopoI was inhibited by both the types of oligonucleotides. The STS harboring oligonucleotides inhibit the relaxation activity of the enzymes by competitively denying access to the supercoiled substrate (Fig. 6 and Supplemental Fig. S7). Next, we tested the effect of another inhibitor on MttopoI. Among a panel of mAbs generated against MstopoI, one of the mAbs, 2F3G4 specifically recognized and inhibited the activity of MstopoI but not of EctopoI [24]. The mAb recognized MttopoI as determined by immunoblotting (Fig. 7A and B). To assess the inhibitory potential of 2F3G4, MttopoI was incubated with the supercoiled DNA substrate in the presence of the mAb. The relaxation activity was inhibited with increasing concentrations of the mAb with complete inhibition at 25 nM (Fig. 7C) which is comparable to the inhibition of MstopoI by this mAb [24].

Discussion In this manuscript, we have examined the characteristics of the single type IA topoisomerase encoded by the M. tuberculosis genome. The enzyme recognizes and cleaves DNA in a site specific fashion similar to its ortholog from M. smegmatis. Generally, topoisomerases are not known to exhibit a high degree of sequence specificity and a vast majority of them do not recognize specific sequences to carry out their respective transesterification reactions. For instance, EctopoI and Micrococcus luteus topoI cleave the DNA at CXXX; [21]. This is in accordance with their function of catalyzing topological alterations by binding to DNA wherever required irrespective of the sequence. However, a few enzymes have been shown to exhibit a higher degree of sequence specificity; Vaccinia topoisomerase I binds and cleaves the sequence at (C/T)CCT;T [26] and E. coli topoisomerase III at CTT; [27]. The only other topoI having a high degree of sequence specificity comparable to Vaccinia topoI is from M. smegmatis characterized by us earlier [11]. The enzyme recognizes the hexameric sequence CG/TCT;TC/G [12,13]. In the present study, we demonstrate that topoI from M. tuberculosis also exhibits similar sequence specificity. In this context, it was

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Fig. 6. Inhibition of MttopoI relaxation activity by specific oligonucleotides. DNA relaxation assays were carried out using 500 ng of pUC18 DNA as substrate and 5 units of (A) MstopoI, (B) MttopoI or (C) EctopoI in presence of STS or non-STS oligonucleotides. Lane 1: pUC18 DNA. Lane 2: DNA relaxation with topoisomerase I. Lanes 3–7: DNA relaxation in presence of 0.5, 1.0, 2.0, 5.0, 10.0 M excess of oligonucleotides with STS. Lanes 8–12: DNA with topoisomerase I in presence of 0.5, 1.0, 2.0, 5.0, 10.0 M excess of oligonucleotides without STS. S, supercoiled DNA; N/R, nicked or relaxed DNA.

Fig. 7. 2F3G4 interacts with MttopoI. 1 lg each of purified MttopoI and EctopoI was resolved on a 8% SDS–PAGE. The gels were (A) silver stained and (B) probed with 2F3G4 hybridoma supernatant. Arrow indicates the MttopoI band. (C) Inhibition of relaxation activity of MttopoI by 2F3G4. Lane 1: pUC18 DNA, lane 2: DNA relaxation with MttopoI, lanes 3–6: DNA relaxation activity in the presence of 5, 10, 25 and 50 nM of 2F3G4. C, DNA in the absence of topoI. S, supercoiled DNA; N/R, nicked or relaxed DNA.

surprising to note that the DNA cleavage characteristics described for MttopoI in a previous report by Annamalai et al. is similar to that of EctopoI and not MstopoI [28]. From the bioinformatic analysis and the detailed characterization presented in this manuscript, it is evident that the topoI from two related species of mycobacteria share several properties. The differences in the properties of MttopoI observed in the present and earlier work could be due to the differences in the strains used for expression, purification and other experimental conditions employed vis-à-vis the buffer composition and/or the substrates used for the assays. It may also be noted that the three different MttopoI preparations described here have higher specific activity compared to the ones reported previously from two different groups [28,29]. The site specificity of MstopoI is imparted by the transesterification domain located in NTD [15]. From the high degree of amino acid sequence similarity between MstopoI and MttopoI in the NTD, we surmise that this domain is responsible for site specificity of MttopoI as well. The site directed mutagenesis of the metal binding motif affected MttopoI differently from that of EctopoI and MstopoI. First, the single amino acid replacements with EctopoI retained a high degree of activity when compared to the mutants of MstopoI [23]. Only a combination of double or triple mutants completely affected the activity of EctopoI [22]. In contrast, even single amino acid substitutions in MstopoI, especially mutation in the first residue of the acidic triad, D108A, led to highly reduced enzyme activity [7]. Second, the single mutants of the acidic triad in EctopoI cleave DNA independent of Mg2+. D108A of MstopoI, on the other hand was dependent on Mg2+ for DNA cleavage and religation is severely affected in the mutant. The other mutant, E112A, was not dependent on Mg2+ for cleavage and did not show severe reduction in religation [7]. From the results presented in the manuscript, it is apparent that the mutations in MttopoI have different characteristics. Both the single mutants studied, D111A and E115A, had

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completely lost the relaxation activity and were compromised in religation step. These studies, thus reveal a more critical role for Mg2+ binding residues of MttopoI in enzyme function when compared to the two other enzymes. The drastic impairment of enzyme activity in acidic triad mutants indicates the potential to target the domain with small molecule inhibitors. Such inhibitors when bound to the enzyme may affect one or the other steps of DNA relaxation cycle of the enzyme. Eubacterial topoI has a central role in maintaining supercoiling homeostasis in the genome and thus influence the essential functions. In our pursuit to mark MttopoI as a target for inhibitory lead molecules, we employed two approaches. The sequence specific binding of the enzyme to the DNA opened up avenues for the design of oligonucleotides harboring the STS which can act as an inhibitor of the enzyme activity. Based on this rationale, previous studies with MstopoI showed the specific inhibitory potential of oligonucleotides harboring the STS [30]. Inhibitors which act on MstopoI should be active on MttopoI as well considering the high degree of similarity between the two proteins. Indeed, as per the prediction, the oligonucleotides having the recognition sequence inhibited the activity of MttopoI. The differences between EctopoI and mycobacterial topoI can thus be exploited to develop inhibitors specific to mycobacterial topoI. With this in view, a panel of anti-MstopoI monoclonal antibodies was generated [31]. One of the mAbs, 2F3G4, which specifically recognized and inhibited MstopoI, was shown to inhibit MttopoI with comparable efficiency. Although, these candidates by themselves are unlikely to be developed as lead molecules, their inhibitory potential opens up avenues for designing similar molecules against MttopoI. To conclude, the single type I topoisomerase of M. tuberculosis is markedly distinct from EctopoI. Although it is similar to MstopoI in most respects, it exhibits subtle differences in the metal binding properties which could be exploited for specific inhibitor development. While inhibition of the enzyme activity by mAbs and oligonucleotides serve as a ‘proof of principle’ and provide strategies to target the enzyme activity, much larger efforts may be required to hit the vulnerability of the enzyme. Screening for such small molecule inhibitors is currently underway.

Appendix A. Supplementary data

Acknowledgments

[25] [26] [27] [28] [29] [30] [31]

The authors acknowledge the members of the laboratory for critical reading of the manuscript and useful suggestions. B. Mallick and S.M. Hegde are acknowledged for technical assistance. V.N. is a J.C. Bose fellow of Department of Science and Technology, and a recipient of the Centre for Excellence grant from Department of Biotechnology, Government of India and partner 14 in an EU consortium project, MM4TB.

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