A novel active DNA topoisomerase I in Leishmania donovani

Héctor Villa a *, Ana R. Otero Marcos a *, Rosa M. Reguera a, Rafael Balaña-Fouce a ,

Carlos García-Estrada a , Yolanda Pérez-Pertejo a , Babu L. Tekwanib, Peter J. Myler c ,

Kenneth D. Stuart c , Mary-Ann Bjornsti d , David Ordóñez a.

a Departamento de Farmacología y Toxicología, (INTOXCAL) Universidad de León,

Campus de Vegazana s/n, 24071 León, Spain

b National Center for Natural Products Research, School of Pharmacy, University of

Mississippi, MS, 38677 USA

c Seattle Biomedical Research Institute, 4 Nickerson Street, Seattle, WA, 98195, USA

d Molecular Pharmacology, St. Jude Children´s Research Hospital, Memphis, TN,

38105, USA

*Both authors contributed equally to the work

#To whom correspondence and proofs should be sent: Dr. David Ordóñez

Departamento de Farmacología y Toxicología, (INTOXCAL) Universidad de León,

Campus de Vegazana s/n, 24071 León, Spain. Telephone: 34 987 291 257, Fax: 34 987

291 590

Email: dftrbf@unileon.es

Running Title: A novel DNA topoisomerase I in Leishmania

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on November 19, 2002 as Manuscript M203991200 by guest on M

arch 26, 2018http://w

ww

.jbc.org/D

ownloaded from

3

ABSTRACT

A common feature shared by type I DNA topoisomerases is the presence of a “serine,

lysine, X, X, tyrosine” motif as conventional enzyme active site. Preliminary data have

shown that Leishmania donovani DNA topoisomerase I gene (LdTOP1A) lacked this

conserved motif, giving rise to different theories about the reconstitution of an active

DNA topoisomerase I in this parasite. We, herein, describe the molecular cloning of a

new DNA topoisomerase I gene from L. donovani (LdTOP1B) containing the highly

conserved “serine, lysine, X, X, tyrosine” motif. DNA topoisomerase I activity was

detected only when both genes (LdTOP1A and LdTOP1B) were co-expressed in a yeast

expression system, suggesting the existence of a dimeric DNA topoisomerase I in

leishmania parasites.

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

4

INTRODUCTION

DNA topoisomerases are ubiquitous enzymes that catalyze changes in DNA topology

by altering the linkage of DNA strands, solving topological problems caused by cellular

processes such as DNA replication, transcription or recombination (1, 2). These

enzymes are classified on the basis of the number of DNA strands that they cleave, and

the covalent bond formed in the enzyme-DNA intermediate. Unlike type II DNA

topoisomerases, type I enzymes are ATP-independent which transiently break a single

strand of DNA. Type I DNA topoisomerases are classified into two subfamilies: type IA

and type IB. The enzymes of type IA subfamily, including bacterial DNA

topoisomerase I and III, eukaryotic DNA topoisomerase III, and reverse gyrase (3-4),

form a tyrosyl linkage with a 5´-phosphate group of one of the DNA strands generated

due to the enzyme action (2), whereas the enzymes of type IB subfamily, including

eukaryotic and vaccinia virus DNA topoisomerases I (5) and DNA topoisomerase V,

establish the tyrosyl bond with the 3´-phosphate group (2). Type 1A topoisomerases

relax only negatively supercoiled-DNA with Mg2+ requirement, whereas type IB

topoisomerases relax both, negatively and positively supercoiled-DNA even in the

absence of a metallic cofactor, although Mg2+ and Ca2+ stimulate the relaxation activity

(6-7).

Type IB DNA topoisomerases are monomeric enzymes, constituted by four domains (8-

9). The nonconserved amino-terminal domain contains putative signals for nuclear

localization of the enzyme. The largest domain, the core, is essential for enzyme activity

and shows high phylogenetic conservation, particularly in the residues closely

interacting with DNA. The third domain is known as the linker, which is poorly

conserved and highly variable in length and is not essential for the enzyme activity.

Finally, the carboxy-terminal domain is highly conserved and crucial for the catalytic

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

5

activity. This domain contains a tyrosine residue (Tyr723 in the human topoisomerase I)

which interacts with one of the DNA strands, creating a transient covalent

phosphodiester bond between the enzyme and the DNA.

A type I DNA topoisomerase has been purified and characterized from L. donovani

promastigotes, the causative agent for visceral leishmaniasis (10). Topoisomerases have

been shown as the promising targets for new drug development against leishmaniasis

(11). A DNA topoisomerase IB like gene (LdTOP1A) which encodes for a protein

lacking the conventional active site “SKXXY” motif has been characterized in L.

donovani. However, heterologous expression of LdTOP1A gene in E. coli produced an

inactive protein (12).

The present paper describes the molecular cloning and functional expression of a novel

DNA topoisomerase I from L. donovani. Unlike type I DNA topoisomerases from

several other organisms, the leishmanial enzyme is encoded by two different genes

(LdTOP1A and LdTOP1B) located at two different chromosomes, and the polypeptide

encoded by LdTOP1B gene contains the conserved “SKXXY” motif required for

activity. This, to our knowledge, is the first report in which two different genes code for

an active DNA topoisomerase I.

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

6

MATERIALS AND METHODS

Materials – Media and reagents were purchased from Sigma (St. Louis, MO). Primers

were purchased from Amersham.

Leishmania and Yeast Strains – For protein expression Saccharomyces cerevisiae

strain EKY3 [MAT α ura3-52 his3∆200 leu2 ∆1 trp1 ∆63 top1 ∆::TRP1], deficient in

DNA topoisomerase I activity, was used. Leishmania donovani strain LSB-51.1

(MHOM/SD/00/Khartoum) was maintained as promastigotes at 26ºC in Schneider's

Insect Medium containing 10% foetal bovine serum (BRL Life Technologies),

penicillin (50 U/ml) and streptomycin (50 µg/ml).

Cloning of DNA topoisomerase I - To generate a DNA probe for isolation of the L.

donovani LdTOP1A gene (GenBank accession number AF303577), 100 pmol of two

degenerated primers based on sequence homology alignments: 5´-

GAT/CACGATCGTCGGT/CTGCTG-3´(sense) corresponding to amino acid residues

DTVGCC and 5´-GTAG/CGTA/GCGGAACACCTT-3´(antisense) coding to the

conserved sequence KVFRTY, were added to a reaction mixture containing 100 ng of

leishmania genomic DNA, 200 µM of each dNTP and 2.5 U of Taq DNA polymerase

(Stratagene). A single 264 bp-PCR product was obtained and subcloned into pGEM-T

vector (Promega). A random labeled probe (Random Primed DNA labeling kit,

Boehringer Mannheim) was prepared using this 264-bp PCR product as template to

screen a L. donovani λ−EMBL3 genomic library (a gift from Dr. Meade, University of

Mississippi Medical Center, Jackson, MS) (13). 5 x 104 p.f.u. were plated and screened

by the PCR probe. One positive bacteriophage clone was isolated, which was further

purified through tertiary screening, and sequenced on both strands. Sequence analysis

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

7

was performed by DNAstar), whereas comparisons with other genes of the database

were performed using the search algorithm BLAST (14).

Nucleic acid isolation, pulsed field gel electrophoresis (PFGE), and hybridization

analysis - Genomic DNA was isolated from 2 x 109 L. donovani promastigotes by

standard procedures (15). Total RNA was isolated from 109 cells using a RNA isolation

kit (QIAGEN). Plasmid DNAs were isolated by the alkaline lysis procedure.

Chromosomal localization of the genes was conducted by PFGE. Briefly, L. donovani

LSB-51.1 (MHOM/SD/00/Khartoum) promastigotes were harvested by centrifugation,

washed twice in PBS, resuspended in PBS and mixed with 2% agarose at the ratio of

1:1 (v/v). Processing of the samples was made at 50ºC for 48 h in 10 ml of 0.5 M EDTA

pH 8.0, 1% Sarkosyl, and 150 µl of 2 mg/ml fresh-made proteinase K. Separation of the

chromosomal bands was achieved at 14ºC in 1% agarose gels with 0.5 x TBE running

buffer, using a Clamped Homogeneous Electrical Field electrophoresis (CHEF,

BIORAD) with a 35-120 s ramping pulse at 6 V/cm for 24 h. S. cerevisiae

chromosomes, were used as molecular weight markers. After staining with ethidium

bromide, gels were blotted onto nylon membranes (Sigma) by alkaline transfer. DNA,

RNA and chromosomal blots were hybridized with the randomly primed [α-32P]-labeled

DNA probes. All post-hybridization washes were performed to a final stringency of 0.1

x SSC, 0.1% SDS at 42ºC.

Nuclear run-on assay –Transcription in isolated nuclei was achieved as described

previously by Quijada et al. (16). Briefly, logarithmic phase promastigotes were

harvested by centrifugation and suspended in ice-cold hipotonic buffer. Cells were lysed

by vortexing in the presence of Nonidet P-40 and Triton X-100. Immediately nuclei

washing buffer was added and the nuclei were pelleted (3000 g), washed and stored at –

70ºC until use. In vitro transcription was performed for 10 min at 26ºC in the presence

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

8

of 100 µCi of [α-32P]UTP (3000 Ci/mmol) (Amersham). The reaction was stopped and

the radiolabeled nascent RNA was extracted by phenol:chloroform. Non-incorporated

isotopes were removed on ProbeQuant G-50 microcolumns (Amersham). 3 µg of each

plasmid, to be probed with the nascent RNA, was linearized, denatured, and transferred

to a positively charged nylon membrane. The membrane was then subjected to

hybridization with the purified labeled RNA.

Plasmid constructions – Plasmids were constructed using conventional cloning

techniques (15) and propagated using the E. coli strain TOP10F’ [mcrA ∆(mrr-

hsdRMS-mcrBC) φ80∆lacZ∆M15 ∆lacX74 recA1 deoR araD139 ∆(ara-leu) 7697

galU galK rpsL endA1 nupG (F’: lacYq Tn10 TetR)] (Invitrogen). The sequences were

verified by dideoxy sequencing along both the critical junction sequence sites.

YCpGAL1-LdTOP1A-URA construction: The 1.9 kb LdTOP1A gene was amplified from

L. donovani genomic DNA, using a sense primer with a flanking BamHI site and a

RGS(His)6tag: 5'-

GCGGATCCGACATGAGAGGATCGCACCACCACCACCACCACAAGGTGGAG

AATAGCAAGATGGGGGTGAAG-3', and an antisense primer with a flanking XbaI

site: 5'-CCTCTAGAGGACTCCGACACCTACAGACGAACAGAGTCACTCG-3'

which correspond respectively to amino-terminal and carboxy-terminal ends of

LdTOP1A gene. Restriction sites are underlined, and the start codon for DNA

topoisomerase I is indicated in bold. The amplified fragment was cloned into the

BamHI-SpeI site of the YCpGAL1-URA vector (a gift from Dr. Wang JC, Harvard

University, Cambridge, Massachusetts). The resultant construct, YCpGAL1-LdTOP1A-

URA contained the LdTOP1A gene driven by the galactose-inducible GAL1 promoter.

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

9

YCpGAL1-LdTOP1B-URA construction: A 838-bp BamHI-ClaI fragment from pSK-

LdTOP1B was subloned into the YCpGAL1-URA vector. This construct contained

LdTOP1B gene also driven by GAL1 promoter.

pESC LdTOP1A-LdTOP1B-URA construction: In a two-fragment ligation reaction, the

construct was created by insertion of LdTOP1A previously cut with BamHI-XhoI from

YCpGAL1-LdTOP1A-URA and LdTOP1B cut with NotI-SpeI from YCpGALI-

LdTOP1B-URA. The resultant construct encodes the full-length L. donovani DNA

topoisomerase I driven by the GAL1 and GAL10 promoters.

YCpGAL1-hTOP-URA described previously (17) was used for expression of human

topoisomerase I. pUC18-rDNA, used in the nuclear run on assay, was kindly provided

by Dr. Requena (Centro de Biología Molecular, Severo Ochoa UAM, Madrid).

pGEM3Z-LdTOP1A and pGEM3Z-LdTOP1B were constructed by insertion of

LdTOP1A and LdTOP1B genes in the BamHI and HindIII restriction sites of

pGEM3Zf(+) vector (Promega).

Protein expression – S. cerevisiae strain, EKY3 was transformed with different

constructs viz., YCpGAL1-LdTOP1A-URA, YCpGAL1-LdTOP1B-URA or pESC

LdTOP1A-LdTOP1B-URA carrying the URA3 selectable marker, by treatment with

lithium acetate (18-20). Transformants were selected on synthetic complete (SC)–uracil

medium. At least four independent clones were selected from each transformation. After

6-h induction with 2 % galactose in SC ura-raffinose medium, the cells were harvested

by centrifugation, washed and resuspended at the ratio of 2 g wet cells/2 ml of TEEG

buffer (50 mM Tris-HCl pH 7,4, 1 mM EDTA, 1 mM EGTA, 10 % glycerol)

supplemented with or without 0.2 M KCl, and a mixture of protease inhibitors [1 x

sodium fluoride, 1 x sodium bisulfite, 2 x Complete Mini® (Roche Laboratories)]. Cell

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

10

extracts were prepared by disruption with acid-washed glass beads according to a

procedure previously described (21,22). Briefly, cells were subjected to one freeze/thaw

cycle at –80ºC, lysed by vortexing with 425-600 µm glass beads and the extracts cleared

by centrifugation at 15000 x g for 30 min at 4ºC.

In vitro Relaxation Assay - DNA topoisomerase I activity was assayed by the

relaxation of negatively supercoiled plasmid DNA. DNA topoisomerase I proteins were

incubated in 20-µl reaction volume containing 0.3 µg of pHC624 DNA (2015 bp,

plasmid substrate), 20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 5 mM DTT, 10 mM

EDTA, 50 mg/ml gelatin, 150 mM KCl. Human (23) and Leishmania enzyme activities

were assayed for 30 min at 37ºC. Reactions were terminated by the addition of 1% SDS,

and the extent of plasmid DNA relaxation was assessed by electrophoresis in a 1%

agarose gel in 0.1 M Tris borate buffer pH 8.0 at 5 V/cm for 4 h. The gels were

visualized under UV illumination after staining with ethidium bromide and

photographed (24).

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

11

RESULTS

Sequence analysis and genomic organization- A single open reading frame (ORF)

consisting of 1908 bp was isolated (LdTOP1A gene, GenBank accession number

AF303577) showing a 50 % identity to Homo sapiens DNA topoisomerase I sequence

(K03077). The ORF encoded for putative polypeptide of 636 amino acids, with a

predicted molecular weight of 73 kDa, which is slightly smaller than human (765 aa)

and S. cerevisiae (769 aa) enzymes. The conserved core domain is present, whereas the

carboxy-terminal domain, which contains the active site, is absent, in this gene (Fig.1).

The presence of a tyrosine residue has been described at the enzyme catalytic site of all

DNA topoisomerases I characterized so far, except in leishmania. In order to search for

a new gene encoding a DNA topoisomerase I active site, PCR was performed using

specific oligonucleotides, whose sequence was based on Leishmania Genome

Sequencing Project (AL389894) (which operates with L. major Friendlin strain) (25).

The sense primer sequence was 5´-CGTGAAAGGCAAGTCTGAGG-3´ and the

antisense primer was 5´-AGGCGGCATGTGAATTAAAG-3´. Genomic DNA from L.

donovani LSB-51.1 (MHOM/SD/00/Khartoum) was used as a template. A single 826

bp-PCR product was obtained, cloned and sequenced, revealing a 95% identity with the

L. major sequence (LdTOP1B GenBank accession number AY062908). This fragment

contained the highly conserved “SKXXY” motif with the tyrosine as the active site

residue, and the sequence alignment analysis displayed ≈ 50% homology with the

carboxy-terminal domain of other eukaryotic DNA topoisomerases I (Fig 1).

To determine the LdTOP1A gene copy number, Southern blot studies were performed as

described in the Materials and Methods using the 264-bp PCR as a probe. A single band

was obtained (Fig 2A) revealing that it is a single copy gene confirming the earlier

results (12). The same experiment was performed for the second gene (LdTOP1B),

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

12

using the 826-bp PCR fragment described above. Figure 2B showed a single

hybridizing band, thus suggesting that this gene is also present as a single copy in the

Leishmania genome. Chromosomal location analysis revealed that LdTOP1B gene is

placed at a single chromosomal band of ≈ 0.4 Mb. These data concur with the

Leishmania Genome Sequencing Project findings, according to which the LdTOP1B

gene EST has been identified on chromosome 4 (0.46 Mb) in L. major

(http://www.ebi.ac.uk/parasites/LGN/chromo4.html). These results show clearly that

LdTOP1A and LdTOP1B genes are located on different chromosomes, since LdTOP1A

gene was located on a chromosomal band of 1.6-1.9 Mb (Fig. 2C).

Transcription analysis- Northern Blot analysis was conducted to explore the

possibility that the two genes may undergo post-transcriptional processing, which may

lead to sharing of a common mRNA. A single hybridizing band of ~2.3 kb was

observed with the LdTOP1B gene probe, which differs from the ~3.6 kb mRNA band

observed for LdTOP1A gene (see Fig. 3A). In order to test whether these two mRNAs

are transcribed at a similar rate, nuclear run-on experiments were conducted using

nuclei isolated from logarithmic phase promastigotes. The rate of transcription of each

gene was determined relative to the rate of rDNA transcription. Genes whose relative

rates of transcription were measured include the LdTOP1A, LdTOP1B and rDNA genes,

pGEM-3Zf(+) and pUC-18 plasmids (3 µg of double-stranded, linearized and denatured

plasmids). Results of figure 3B show the transcription of LdTOP1A and LdTOP1B

genes relative to the rDNA transcription. The results of three independent experiments

are shown in Table I. The hybridization signal to the rDNA gene was arbitrary chosen as

1.0 and the other signals were reported relative to that value. Despite LdTOP1A and

LdTOP1B genes are located at different genomic clusters, their transcription rates,

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

13

quantified in a phosphorimager, were similar approximately 10-13 fold lower respect

the rDNA signal.

DNA topoisomerase I activity- As shown in Fig 4. DNA topoisomerase I activity was

reconstituted using a deficient S. cerevisiae strain (EKY3, see Materials and Methods).

LdTOP1A and LdTOP1B genes were co-expressed together (Fig. 4A) in a pESC-URA

vector which contains the GAL1 and GAL10 yeast promoters in opposing orientation.

Co-expression of the two genes cloned in this vector guarantees protein-protein

interactions after induction in the yeast host strain. Nevertheless, when LdTOP1A (Fig.

4B) and LdTOP1B (Fig. 4C) genes were individually expressed (each one in a different

experiment), the resulting proteins did not show topoisomerase activity. Expression of

Human TOP 1 gene under similar conditions produced a functional protein, which

catalyzed the plasmid relaxation activity in vitro (Fig 4D).

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

14

DISCUSSION

This paper describes the molecular cloning and characterization of a new gene

(LdTOP1B) encoding the carboxy-terminal domain of DNA topoisomerase I in L.

donovani. The results suggest that two different proteins, codified by two different

genes located on different chromosomes, were required to reconstitute a catalytically

active DNA topoisomerase I in leishmania. Similar intensities of the hybridization

signals obtained with LdTOP1A and LdTOP1B genes in the nuclear run-on assays

indicate that the abundance of nascent RNA transcripts derived from both genes was

similar and an interaction, probably at post-translational level, should occur to

reconstitute an active DNA topoisomerase I.

A type I DNA topoisomerase of 67 kDa has been purified from Leishmania donovani

promastigotes nuclear extracts by Chakratorty et al. (10). Das et al. (25) have recently

described that this enzyme harbors a serine in place of the usual catalytic tyrosine. In

addition a theoretical protein model for L. donovani topoisomerase was presented,

suggesting that the serine 553 acts as the reactive nucleophile for enzyme catalysis.

However, it is difficult to understand how a serine residue can stand-in for

phosphodiesterase activity, since some experiments in which active yeast DNA

topoisomerase I was mutated at their active site (Tyr727) to Ser or Phe, resulted in

enzyme inactivation (23). On the other hand, a DNA topoisomerase I-like gene, lacking

the sequence corresponding to a conventional active site motif, has been described in L.

donovani. Heterologous expression of LdTOP1A) gene in E. coli resulted in production

of a catalytically inactive protein (12).

In previous studies with human DNA topoisomerase I, Stewart et al. (26) were able to

reconstitute the enzyme activity by mixing a 58 kDa recombinant core domain with

series of different recombinant carboxy-terminal fragments, which bind tightly to the

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

15

core domain forming 1:1 complex probably through non-covalent interactions. This

model hypothesizes that the core and carboxy-terminal domains of topoisomerase I are

folded independently and then are simply associated with each other to form an active

enzyme (26). A similar mechanism may be suggested for independent refolding of

LdTOP1A and LdTOP1B gene products resulting in reconstitution of an active

topoisomerase I in L. donovani. The results therefore suggest the presence of a novel

type of dimeric topoisomerase I in L. donovani. Understanding of distinct molecular

characteristics of the leishmanial topoisomerase I and regulation of expression of the

enzyme during parasite growth may be useful for development of selective inhibitors of

leishmanial topoisomerase I as promising leishmanicidal agents.

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

16

ACKNOWLEDGMENTS

We thank William Colley and John Vance (St. Jude Children´s Research Hospital,

Memphis, TN), Santiago Martinez-Calvillo (Seattle Biomedical Research Institute,

Seattle, WA), José María Requena and his group (Centro de Biología Molecular Severo

Ochoa, Universidad Autónoma de Madrid, Spain) for their help in molecular

techniques. We also want to thank Francisco Fierro (Universidad de León, Spain) and

Iris Segura for technical support in PFGE, and John Chris Meade (University of

Mississippi Medical Center, Jackson, MS) for the Leishmania genomic library. This

research was supported by Comisión Interministerial de Ciencia y Tecnología (grants

PM98/0036 and PB96/0159) and Junta de Castilla y León (grants LE05/01 and

LE06/02) and by a grand from the National Institute of Health, CA 58755 and ALSAC.

Fellowships awards to HV and AROM from the Ministerio de Ciencia y Tecnología,

Spain, is gratefully acknowledged.

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

17

REFERENCES

1- Champoux, J. J. (2001) Annu. Rev. Biochem. 70, 369-413

2- Wang, J. C. (1996) Annu. Rev. Biochem. 65, 635-692

3- Jaxel, C., Nadal, M., Mirambeau, G., Forterre, P., Takahashi, M., and Duguet, M.

(1989) EMBO J. 8, 3135-3139

4- Brahms, S., Nakasu, S., Kikuchi, A., and Brahms, J. G. (1989) Eur. J. Biochem. 184,

297-303

5- Cheng, C., and Shuman, S. (1998) J. Biol.Chem. 273, 11589-11595

6- Liu, L. F., and Miller, K. G. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 3487-3491

7- Goto, T., Laipis, P., and Wang, J. C. (1984) J. Biol. Chem. 259, 10422-10429

8- Stewart, L., Ireton, G. C., and Champoux, J. J. (1996) J. Biol. Chem. 271, 7602-7608

9- Stewart, L., Ireton, G. C., Parker, L. H., Madden, K. R., and Champoux, J. J. (1996)

J. Biol. Chem. 271, 7593-7601

10- Chakraborty, A. D., Gupta, A., and Majumder, H. K. (1993) Ind. J. Biochem.

Biophys. 30, 257-263

11- Chessman, S. J. (2000) Parasitol. Today 16, 277-281

12.- Broccoli, S., Marquis, J. F., Papadopoulou, B., Oliver, M., and Drolet, M (1999)

Nucleic Acids Res. 27, 2745-2752

13- Meade, J. C., Shaw, J., Lemaster, S., Gallagher, G., and Stringer, J. R. (1987) Mol.

Cell. Biol. 11, 3937-3946

14- Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W., and

Lipman, D. J. (1997) Nucleic Acids Res. 25, 3389-3402

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

18

15- Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning A

Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

16-.Quijada, L., Soto, M., Alonso, C., and Requena, J. M. (1997) J. Biol. Chem. 272,

4493-4499.

17- Woo, M. H., Vance, J. R., Otero Marcos, A. R., Bailly, C., and Bjornsti, M.-A.

(2002) J. Biol. Chem. 277, 3813-3822

18- Burke, D., Dawson, D., and Stearns, T. (2000) Methods in Yeast Genetics, Edition

Ed. A Cold Spring Harbor Lab Course Manual, Cold Spring Harbor Laboratory Press.

19- Gietz, R. D., and Schiestl, R. H. (1991) Yeast 7, 253-263

20- Gietz, D., St Jean, A., Woods, R. A., and Schiestl, R. H. (1992) Nucleic Acid. Res.

20, 1425

21- Bjornsti, M.-A, and Fertala, J. Methods Mol. Biol. Humana Press Inc; Totowa, N.J.

22- Jazwinski, S. M. (1990) Method Enzymol. 182, 154-174.

23- Lynn, R. M., Bjornsti, M.-A, Caron, P. R., and Wang, J. C. (1989) Proc. Natl. Acad.

Sci. USA. 86, 3559-3563

24- Ivens, A. C., Lewis, S. M., Bagherzadeh, A., Zhang, L., Chan, H. M., and Smith, D.

F. (1998) Genome Research 8, 135-145

25- Das, A., Mandal, C., Dasgupta, A., Sengupta, T., and Majumder, H. K. (2002)

Nucleic Acid. Res. 30, 794-802

26- Stewart, L., Ireton, G. C., and Champoux, J. J. (1997) J. Mol. Biol. 269, 355-372

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

19

FIGURE LEGENDS

Figure 1. Alignment of LdTOP1B gene product with other eukaryotic type I DNA

topoisomerases. The sequence was translated to protein and aligned with other DNA

topoisomerase I proteins from different organisms, including L. donovani topoisomerase

I-like gene reported previously (11). GeneBank accession numbers are as follows:

LdTOP1B gene, AY062908; LdTOP1A gene, AF145121; Plasmodium falciparum,

X83758; Sacharomyces cerevisiae, J03250; Homo sapiens, K03077.

Figure 2. Genomic organization of LdTOP1A and LdTOP1B genes encoding DNA

topoisomerase I in L. donovani. A, Southern analysis of L. donovani LdTOP1A gene

(filter probed with 264-bp fragment). B, Southern analysis of L. donovani LdTOP1B

gene (filter probed with 826-bp fragment). C, Pulsed field gel electrophoresis (PFGE)

analysis of L. donovani indicating localization of LdTOP1A and LdTOP1B genes on

different chromosomal bands.

Figure 3. Expression analysis of LdTOP1A and LdTOP1B genes encoding DNA

topoisomerase I in L. donovani. A, Northern analysis of mRNA from L. donovani

promastigotes (day 2 culture) Lane 1: MWM, lane 2: total RNA, lane 3: filter probed

with 264-bp fragment; lane 4: filter probed with 826-bp fragment. B, Levels of nascent

LdTOP1A and LdTOP1B transcripts in isolated nuclei of L. donovani by nuclear run-on

assay.

Figure 4. In vitro plasmid DNA relaxation assay – A, Yeast extracts co-expressing

LdTOP1A-LdTOP1B genes. B, yeast extracts overexpressing only LdTOP1A gene. C,

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

20

yeast extracts overexpressing only LdTOP1B gene. D, yeast extracts overexpressing

hTOP1 gene. a, b, c, d lanes are respectively serial dilutions of the yeast extracts (1/1;

1/10; 1/50 and 1/100), incubated with negatively supercoiled plasmid DNA in reaction

buffer for 30 min at 37ºC as detailed under “Materials and Methods”. Reactions were

terminated by the addition of SDS and the products were resolved in agarose gels,

followed by ethidium bromide staining. Supercoiled (Sc) and relaxed (R) plasmid DNA

topoisomers are as indicated.

Table I. Quantification of relative transcription rates for various genes in L. donovani

promastigotes. Nuclear run-on transcripts from promastigotes were hybridized to

plasmid DNAs immobilized on nylon filter, and the hybridization signals were

quantified on a phosphorimager. Values are relative signal intensities normalized for the

rDNA plasmid value (mean + standard deviation of three independent experiments).

Relative intensities

LdTOP1A 0.11 + 0.05

LdTOP1B 0.08 + 0.03

rDNA 1.0

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

L.donovani subunit B ------------------------------------------------------------ L.donovani subunit A ------------------------------------------------------------ P.falciparum ------------------------------------MQSMEINDNNSIKNESTSDDDILI 24 S.cerevisiae --------------------------------------MTIADASKVNHELSSDDDDDVP 22 H.sapiens MSGDHLHNDSQIEADFRLNDSHKHKDKHKDREHRHKEHKKEKDREKSKHSNSEHKDSEKK 60 L.donovani subunit B ------------------------------------------------------------ L.donovani subunit A ------------------------------------------------------------ P.falciparum NKIKQNLGNNKSCNSRSSKKESIKKQKSNSELGIKKNTKKSLGIKKEEEKKKQISKRKSN 84 S.cerevisiae LSQTLKKRKVASMNSASLQDEAEPYDSDEAISKISKKKTKKIKTEPVQSSSLPSP----- 77 H.sapiens HKEKEKTKHKDGSSEKHKDKHKDRDKEKRKEEKVRASGDAKIKKEKENGFSSPPQIKDEP 120 L.infantum subunit B ------------------------------------------------------------ L.donovani subunit A -------------------------------MKVENSKMGVKR-----------EQSHSN 18 P.falciparum ELKEKNNLKEGKKKYVEKKSRTVKDETKLTNVIKKETQNNKKP-----------KKLLKK 133 S.cerevisiae -------PAKKSATSKPKKIKKEDGDVKVKTTKKEEQENEKKK-----------REEEEE 119 H.sapiens EDDGYFVPPKEDIKPLKRPRDEDDVDYKPKKIKTEDTKKEKKRKLEEEEDGKLKKPKNKD 180 L.donovani subunit B ------------------------------------------------------------ L.donovani subunit A EDEEINEED-------------LNWWEQENLRIAMKGERRWETLAHNGVLFPPEYEP--H 63 P.falciparum SEENFEPIN--------------RWWEKIDDQTDIQ----WNYLEHRGLIFSPPYVQ--H 173 S.cerevisiae EDKKAKEEE-----------EEYKWWEKENEDDTIK----WVTLKHNGVIFPPPYQPLPS 164 H.sapiens KDKKVPEPDNKKKKPKKEEEQKWKWWEEERYPEGIK----WKFLEHKGPVFAPPYEPLPE 236 L.donovani subunit B ------------------------------------------------------------ L.donovani subunit A GIPIFYDGREFKMTPEEEEVATMFAVMKEHDYYRMEVFRRNFFESWR-EILDK------- 115 P.falciparum HVPIFYKSIKIELNAKSEELATYWCSAIGSDYCTKEKFILNFFKTFINSLENDNIIKQEN 233 S.cerevisiae HIKLYYDGKPVDLPPQAEEVAGFFAALLESDHAKNPVFQKNFFNDFLQVLKESGGP---- 220 H.sapiens NVKFYYDGKVMKLSPKAEEVATFFAKMLDHEYTTKEIFRKNFFKDWRKEMTNE------- 289 L.donovani subunit B ------------------------------------------------------------ L.donovani subunit A ----RQHPIRRLELCDFEPIYQWHLVQREKKLSRTKEEKKAIKEKQDAEAEPYRYCVWDG 171 P.falciparum ETKLKKGDISNFKFIDFMPIKDHLLKLREEKLNKTKEEKEEEKKMRMEKELPYTYALVDW 293 S.cerevisiae ---LNGIEIKEFSRCDFTKMFDYFQLQKEQKKQLTSQEKKQIRLEREKFEEDYKFCELDG 277 H.sapiens ----EKNIITNLSKCDFTQMSQYFKAQTEARKQMSKEEKLKIKEENEKLLKEYGFCIMDN 345 L.donovani subunit B -MQPVQSPPVAPP-------SVPVAAPKKTPIDISALKLKMSPSVRATLAAAG------- 45 L.donovani subunit A RREQVANFRVEPPGLFRGRGKHPLMGKLKVRVQPEDITINIGETAEVPVPPA---GHKWA 228 P.falciparum IREKISSNKAEPPGLFRGRGEHPKQGLLKKRIFPEDVVINISKDAPVPRLYDNMCGHNWG 353 S.cerevisiae RREQVGNFKVEPPDLFXGRGAHPKTGKLKRRVNPEDIVLNLSKDAPVPPAPE---GHKWG 334 H.sapiens HKERIANFKIEPPGLFRGRGNHPKMGMLKRRIMPEDIIINCSKDAKVPSPPP---GHKWK 402 : : . ** * . * : . : :: . . .. L.donovani subunit B -------------------------VLGQCPRPIEAADEDALLKLKPMKTMVPSVKKVVA 80 L.donovani subunit A AVQHDHTVTWLAMWRDSVAGNMKYVMLAPSSSVKGQSDMVKFEKARKLKDKVDDIRASYM 288 P.falciparum DIYHDNKVTWLAYYKDSINDQIKYTFLSAQSKFKGYKDLMKYENARKLKSCVHKIREDYK 413 S.cerevisiae EIRHDNTVQWLAMWRENIFNSFKYVRLAANSSLKGQSDYKKFEKARQLKSYIDAIRRDYT 394 H.sapiens EVRHDNKVTWLVSWTENIQGSIKYIMLNPSSRIKGEKDWQKYETARRLKKCVDKIRNQYR 462 * . * . : :* : :: L.donovani subunit B P------VATTAPPPKVRRVESSSSSSSDSSSSSSDDDSS-------------------- 114 L.donovani subunit A EDFKSNDLHVAQRAVAMYFIDRLALRVGNEKGEDE-ADTVGCCSLRVEHIQLMPDN---- 343 P.falciparum NKMKNKNIIDKQLGTAVYLIDFLALRVGGEKDIDEEADTVGCCSLRVEHISFAHDIPFKS 473 S.cerevisiae RNLKSKVMLERQKAVAIYLIDVFALRAGGEKSEDE-ADTVGCCSLRYEHVTLKPPN---- 449 H.sapiens EDWKSKEMKVRQRAVALYFIDKLALRAGNEKEEGETADTVGCCSLRVEHINLHPELDGQE 522 : : :: : .... .. *: L.donovani subunit B --------------------------------TDDSSGSDSSSYSS-------------- 128 L.donovani subunit A ------------------------------IVRFDFLGKDSIRYQNDVAVLPEVYALLQR 373 P.falciparum VDSKEQKTNDEKVNKIPLPTNLESISSEDCYITLDFLGKDSIRYFNTVKIDKQAYINIII 533 S.cerevisiae ------------------------------TVIFDFLGKDSIRFYQEVEVDKQVFKNLTI 479 H.sapiens Y-----------------------------VVEFDFLGKDSIRYYNKVPVEKRVFKNLQL 553 * *.** : . L.donovani subunit B -----DDRSSSSAESVVSGEATLFHIAQSQGLVNKEVLTQ-------------------- 163 L.donovani subunit A FTRR-KSPGMDIFDQLNPTQLNDHLKSFMDGLSAKVFRTYNASITLDRWFKEKPVDPKWS 432 P.falciparum FCKN-KNRDEGVFDQITCSKLNEYLKEIMPTLSAKVFRTYNASITLDQQLKRIKEVYGKT 592 S.cerevisiae FKRPPKQPGHQLFDRLDPSILNKYLQNYMPGLTAKVFRTYNASKTMQDQLDLI--PNKGS 537 H.sapiens FMEN-KQPEDDLFDRLNTGILNKHLQDLMEGLTAKVFRTYNASITLQQQLKEL-TAPDEN 611 .. : : . . * * . * L.donovani subunit B ------------------------------------------------------------ L.donovani subunit A ------------------------------------------------------------ P.falciparum TYSLYSGETELHKSKKRKSSHLTSDTNILSDASDSTINDVNNEYDENGINKKLSYATTVG 652 S.cerevisiae ------------------------------------------------------------ H.sapiens ------------------------------------------------------------ L.donovani subunit B ----------------------------EEEEVPTLVPPRPPVVR--------------- 180 L.donovani subunit A ------------------TADKLAYFNKANTEVAILCNHQKSVSK--------------- 459 P.falciparum KENDVDDKNSPIEVDVSNINELINFYNNANREVAILCNHQRSIPK--------------- 697 S.cerevisiae ------------------VAEKILKYNAANRTVAILCNHQRTVTKGHAQTVEKANNRIQE 579 H.sapiens ------------------IPAKILSYNRANRAVAILCNHQRAPPK--------------- 638 : *. * : . : L.donovani subunit B ------------------------------------------------------------ L.donovani subunit A ------NFKLQMMQLT-----------------TKSEYTRKTIELLEKAEVTAKKKSVEE 496 P.falciparum ------QHDTTMSKIKK-------------QIELYNEDIKEYKKYLQHLKKNSDKKFIFV 738 S.cerevisiae LEWQKIRCKRAILQLDKDLLKKEPKYFEEIDDLTKEDEATIHKRIIDREIEKYQRKFVRE 639 H.sapiens ------TFEKSMMNLQ-----------------TKIDAK--------KEQLADARRDLKS 667 L.donovani subunit B -----------SFPNDIGKALERYR----------------------------------- 194 L.donovani subunit A AAKEFLEEQ----DRMQREWLESYGTEEQ---------------------------KKEF 525 P.falciparum SKVSTLDGTLR--PNKVKENMKEESCKKK--------------------------LITLI 770 S.cerevisiae NDKRKFEKEELLPESQLKEWLEKVDEKKQEFEKELKTGEVELKSSWNSVEKIKAQVEKLE 699 H.sapiens AKADAKVMK----DAKTKKVVESKKKAVQ---------------------------RLE- 695 : :: L.donovani subunit B ERLNREENIIRIKDDNKAVSLGTSKINYIDPRIICSWAKAQD------------------ 236 L.donovani subunit A EEIVAKRAAPRVRSEKKKSTSGAKKAESASGKKRAAKKKKSAKKGGKVLSKKAASKSSKK 585 P.falciparum KKVELLNNQMKVRDDNKTIALGTSKINYMDPRITVAFCKKFE------------------ 812 S.cerevisiae QRIQTSSIQLKDKEENSQVSLGTSKINYIDPRLSVVFCKKYD------------------ 741 H.sapiens EQLMKLEVQATDREENKQIALGTSKLNYLDPRITVAWCKKWG------------------ 737 :.: :.::. : *:.* : . : * L.donovani subunit B ------------VPINKIFSATIQKK-----FPWAMNAENFDF-------- 262 L.donovani subunit A APKKLKEEDEDDVPLVSMAAKTKKTAGVKRQRANKVVSDDDDVPLAALRVM 636 P.falciparum ------------IPIEKVFNRSLRLK-----FPWAMFATKN-FTF------ 839 S.cerevisiae ------------VPIEKIFTKTLREK-----FKWAIESVDENWRFM----- 770 H.sapiens ------------VPIEKIYNKTQREK-----FAWAIDMADEDYEFM----- 766 :*: .: : : : .

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Stuart, Mary-Ann Bjornsti and David OrdóñezGarcía-Estrada, Yolanda Pérez-Pertejo, Babu L. Tekwani, Peter J. Myler, Kenneth D. Héctor Villa, Ana R. Otero Marcos, Rosa M. Reguera, Rafael Balaña-Fouce, Carlos

A novel active DNA topoisomerase I in Leishmania donovani

published online November 19, 2002J. Biol. Chem. 

  10.1074/jbc.M203991200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from