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Cited by 2Malaria Research and ControlDNA Repair MechanismsBiochemical and Molecular Research

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Molecular characterization of Plasmodium falciparum DNA-3-methyladenine glycosylase

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Pinthong, Nattapon, Limudomporn, Paviga, Vasuvat, Jitlada, Adisakwattana, Poom, Rattaprasert, Pongruj, Chavalitshewinkoon-Petmitr, Porntip·2020·Malaria Journal
DOI 10.1186/s12936-020-03355-wPMC PMC7409487

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Abstract

Background: The emergence of artemisinin-resistant malaria parasites highlights the need for novel drugs and their targets. Alkylation of purine bases can hinder DNA replication and if unresolved would eventually result in cell death. DNA-3-methyladenine glycosylase (MAG) is responsible for the repair of those alkylated bases. Plasmodium falciparum ( Pf ) MAG was characterized for its potential for development as an anti-malarial candidate. Methods: Native Pf MAG from crude extract of chloroquine- and pyrimethamine-resistant P . falciparum K1 strain was partially purified using three chromatographic procedures. From bio-informatics analysis, primers were designed for amplification, insertion into pBAD202/D-TOPO and heterologous expression in Escherichia coli of recombinant Pf MAG. Functional and biochemical properties of the recombinant enzyme were characterized. Results: Pf MAG activity was most prominent in parasite schizont stages, with a specific activity of 147 U/mg (partially purified) protein. K1 PfMAG contained an insertion of AAT (coding for asparagine) compared to 3D7 strain and 16% similarity to the human enzyme. Recombinant Pf MAG (74 kDa) was twice as large as the human enzyme, preferred double-stranded DNA substrate, and demonstrated glycosylase activity over a pH range of 4–9, optimal salt concentration of 100–200 mM NaCl but reduced activity at 250 mM NaCl, no requirement for divalent cations, which were inhibitory in a dose-dependent manner. Conclusion: Pf MAG activity increased with parasite development being highest in the schizont stages. K1 PfMAG contained an indel AAT (asparagine) not present in 3D7 strain and the recombinant enzyme was twice as large as the human enzyme. Recombinant Pf MAG had a wide range of optimal pH activity, and was inhibited at high (250 mM) NaCl concentration as well as by divalent cations. The properties of Pf MAG provide basic data that should be of assistance in developing anti-malarials against this potential parasite target.

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Fig. 1 Partial purification of native PfMAG. Purification of parasite crude extract using HiTrap Capto Q column where unbound fractions were collected (a). Active fractions from HiTrap Capto Q column were purified using HiTrap Capto S column (b). Pooled active fractions from HiTrap Capto S column were purified using HiTrap Heparin HP column (c)
Fig. 1 Partial purification of native PfMAG. Purification of parasite crude extract using HiTrap Capto Q column where unbound fractions were collected (a). Active fractions from HiTrap Capto Q column were purified using HiTrap Capto S column (b). Pooled active fractions from HiTrap Capto S column were purified using HiTrap Heparin HP column (c)CC BY · PubMed Central
Fig. 2 Expression of PfMAG during asexual stage development. a PCR cycle threshold (Ct) values of expression levels of PfMAG and serine-tRNA ligase gene are shown as mean (horizontal line), 25th to 75th percentile (box) and range (vertical) for ring, trophozoite and schizont samples. b Fold change in PfMAG expression of ring, trophozoite and schizont are shown with their standard deviations
Fig. 2 Expression of PfMAG during asexual stage development. a PCR cycle threshold (Ct) values of expression levels of PfMAG and serine-tRNA ligase gene are shown as mean (horizontal line), 25th to 75th percentile (box) and range (vertical) for ring, trophozoite and schizont samples. b Fold change in PfMAG expression of ring, trophozoite and schizont are shown with their standard deviationsCC BY · PubMed Central
Fig. 3 Amplification of PfMAG and expression of recombinant PfMAG. a PCR amplification of P. falciparum DNA-3-methyladenine glycosylase gene. Lane M, DNA size ladder; lane 1, PfMAG amplicon. b SDS-PAGE and c western blotting of recombinant PfMAG heterologously expressed in E. coli LMG 194 at 15 °C for 16 h. Recombinant protein was purified using HisTrap HP column and eluted with a 10–500 mM imidazole linear gradient. Lane M, standard protein size markers; lane 1, purified PfMAG eluted at 500 mM imidazole
Fig. 3 Amplification of PfMAG and expression of recombinant PfMAG. a PCR amplification of P. falciparum DNA-3-methyladenine glycosylase gene. Lane M, DNA size ladder; lane 1, PfMAG amplicon. b SDS-PAGE and c western blotting of recombinant PfMAG heterologously expressed in E. coli LMG 194 at 15 °C for 16 h. Recombinant protein was purified using HisTrap HP column and eluted with a 10–500 mM imidazole linear gradient. Lane M, standard protein size markers; lane 1, purified PfMAG eluted at 500 mM imidazoleCC BY · PubMed Central
Fig. 4 Structural comparison of PfMAG with hAAG. a Binding of hAAG (219 residues) with double-strand DNA containing pyrrolidine (PDB 1f6O). Tyr162 intercalates into DNA duplex, flipping pyrrolidine into the active site. b Simulated 3D structure of PfMAG (502 residues) using I-TASSER server. Tyr171 intercalates into DNA strand flipping pyrrolidine into the active site. cPfMAG structure is superimposed on that of hAAG showing similarity of catalytic domain of both proteins and the extended C-terminus of the parasite protein
Fig. 4 Structural comparison of PfMAG with hAAG. a Binding of hAAG (219 residues) with double-strand DNA containing pyrrolidine (PDB 1f6O). Tyr162 intercalates into DNA duplex, flipping pyrrolidine into the active site. b Simulated 3D structure of PfMAG (502 residues) using I-TASSER server. Tyr171 intercalates into DNA strand flipping pyrrolidine into the active site. cPfMAG structure is superimposed on that of hAAG showing similarity of catalytic domain of both proteins and the extended C-terminus of the parasite proteinCC BY · PubMed Central
Fig. 5 Substrate preference of PfMAG compared to human hAAG in presence of 0.5 μM oligo duplex substrate and 1.5 μM enzyme in the assay reaction. PfMAG cleaves εA in both double-stranded and single-stranded DNA
Fig. 5 Substrate preference of PfMAG compared to human hAAG in presence of 0.5 μM oligo duplex substrate and 1.5 μM enzyme in the assay reaction. PfMAG cleaves εA in both double-stranded and single-stranded DNACC BY · PubMed Central
Fig. 6 Effects of different buffer conditions on glycosylase activity of recombinant PfMAG. a Assay reaction was carried out under standard conditions except buffer used was 50 mM citrate-sodium citrate pH 3-5, phosphate-citrate pH 6-7 and Tris–Cl pH 7–9. b Effects of NaCl on glycosylase activity of recombinant PfMAG. Assay reaction was carried out under standard conditions but with 0–500 mM NaCl. c Effects of divalent cation on PfMAG activity. The 27-mer double-stranded DNA of εA–T was used as a substrate, and negative control contained no enzyme (− ve)
Fig. 6 Effects of different buffer conditions on glycosylase activity of recombinant PfMAG. a Assay reaction was carried out under standard conditions except buffer used was 50 mM citrate-sodium citrate pH 3-5, phosphate-citrate pH 6-7 and Tris–Cl pH 7–9. b Effects of NaCl on glycosylase activity of recombinant PfMAG. Assay reaction was carried out under standard conditions but with 0–500 mM NaCl. c Effects of divalent cation on PfMAG activity. The 27-mer double-stranded DNA of εA–T was used as a substrate, and negative control contained no enzyme (− ve)CC BY · PubMed Central

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Citations

Cites

Research works printed but not linked: 53

  1. Sachs J, Malaney P. The economic and social burden of malaria. Nature. 2002;415:680–5. · DOI · Paper page
  2. Noedl H, Se Y, Schaecher K, Smith BL, Socheat D, Fukuda MM, et al. Evidence of artemisinin-resistant malaria in western Cambodia. N Engl J Med. 2008;359:2619–20. · DOI
  3. Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, Tarning J, et al. Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med. 2009;361:455–67. · DOI · Paper page
  4. Suksangpleng T, Leartsakulpanich U, Moonsom S, Siribal S, Boonyuen U, Wright GE, et al. Molecular characterization of Plasmodium falciparum uracil-DNA glycosylase and its potential as a new anti-malarial drug target. Malar J. 2014;13:149. · DOI
  5. Vasuvat J, Montree A, Moonsom S, Leartsakulpanich U, Petmitr S, Focher F, et al. Biochemical and functional characterization of Plasmodium falciparum DNA polymerase δ. Malar J. 2016;15:116. · DOI
  6. Limudomporn P, Moonsom S, Leartsakulpanich U, Suntornthiticharoen P, Petmitr S, Weinfeld M, et al. Characterization of Plasmodium falciparum ATP-dependent DNA helicase RuvB3. Malar J. 2016;15:526. · DOI
  7. Haltiwanger BM, Matsumoto Y, Nicolas E, Dianov GL, Bohr VA, Taraschi TF. DNA base excision repair in human malaria parasites is predominantly by a long-patch pathway. Biochemistry. 2000;39:763–72. · DOI
  8. Lau AY, Schärer OD, Samson L, Verdine GL, Ellenberger T. Crystal structure of a human alkylbase-DNA repair enzyme complexed to DNA: mechanisms for nucleotide flipping and base excision. Cell. 1998;95:249–58. · DOI
  9. Dosanjh MK, Chenna A, Kim E, Fraenkel-Conrat H, Samson L, Singer B. All four known cyclic adducts formed in DNA by the vinyl chloride metabolite chloroacetaldehyde are released by a human DNA glycosylase. Proc Natl Acad Sci USA. 1994;91:1024–8. · DOI
  10. Saparbaev M, Kleibl K, Laval J. Escherichia coli, Saccharomyces cerevisiae, rat and human 3-methyladenine DNA glycosylases repair 1, N6-ethenoadenine when present in DNA. Nucleic Acids Res. 1995;23:3750–5. · DOI
  11. Santerre A, Britt AB. Cloning of a 3-methyladenine-DNA glycosylase from Arabidopsis thaliana. Proc Natl Acad Sci USA. 1994;91:2240–4. · DOI
  12. Wyatt MD, Allan JM, Lau AY, Ellenberger TE, Samson LD. 3-methyladenine DNA glycosylases: structure, function, and biological importance. Bioassays. 1999;21:668–76. · DOI
  13. Engelward BP, Dreslin A, Christensen J, Huszar D, Kurahara C, Samson L. Repair-deficient 3-methyladenine DNA glycosylase homozygous mutant mouse cells have increased sensitivity to alkylation-induced chromosome damage and cell killing. EMBO J. 1996;15:945–52. · DOI
  14. Paik J, Duncan T, Lindahl T, Sedgwick B. Sensitization of human carcinoma cells to alkylating agents by small interfering RNA suppression of 3-alkyladenine-DNA glycosylase. Cancer Res. 2005;65:10472–7. · DOI
  15. Racine JF, Zhu Y, Mamet-Bratley MD. Mechanism of toxicity of 3-methyladenine for bacteriophage T7. Mutat Res. 1993;294:285–98. · DOI
  16. Tolentino JH, Burke TJ, Mukhopadhyay S, McGregor WG, Basu AK. Inhibition of DNA replication fork progression and mutagenic potential of 1, N6-ethenoadenine and 8-oxoguanine in human cell extracts. Nucleic Acids Res. 2008;36:1300–8. · DOI
  17. Boiteux S, Laval J. Imidazole open ring 7-methylguanine: an inhibitor of DNA synthesis. Biochem Biophys Res Commun. 1983;110:552–8. · DOI
  18. Pollack Y, Katzen AL, Spira DT, Golenser J. The genome of Plasmodium falciparum. I: DNA base composition. Nucleic Acids Res. 1982;10:539–46. · DOI
  19. Pollack Y, Kogan N, Golenser J. Plasmodium falciparum: evidence for a DNAmethylation pattern. Exp Parasitol. 1991;72:339–44. · DOI
  20. Choi SW, Keyes MK, Horrocks P. LC/ESI-MS demonstrates the absence of 5-methyl-2′-deoxycytosine in Plasmodium falciparum genomic DNA. Mol Biochem Parasitol. 2006;150:350–2. · DOI
  21. Hammam E, Ananda G, Sinha A, Scheidig-Benatar C, Bohec M, Preiser PR, et al. Discovery of a new predominant cytosine DNA modification that is linked to gene expression in malaria parasites. Nucleic Acids Res. 2020;48:184–99. · DOI
  22. Ponts N, Fu L, Harris EY, Zhang J, Chung DW, Cervantes MC, et al. Genome-wide mapping of DNA methylation in the human malaria parasite Plasmodium falciparum. Cell Host Microbe. 2013;14:696–706. · DOI
  23. Yanow SK, Purcell LA, Lee M, Spithill TW. Genomics-based drug design targets the AT-rich malaria parasite: implications for antiparasite chemotherapy. Pharmacogenomics. 2007;8:1267–72. · DOI
  24. Thaithong S, Beale GH, Chutmongkonkul M. Susceptibility of Plasmodium falciparum to five drugs: an in vitro study of isolates mainly from Thailand. Trans R Soc Trop Med Hyg. 1983;77:228–31. · DOI
  25. Trager W, Jensen JB. Human malaria parasites in continuous culture. Science. 1976;193:673–5. · DOI
  26. Lambros C, Vanderberg JP. Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol. 1979;65:418–20. · DOI
  27. Chavalitshewinkoon P, Wilairat P. A simple technique for large scale in vitro culture of Plasmodium falciparum. Southeast Asian J Trop Med Public Health. 1991;22:544–7.
  28. Baker J, Gatton ML, Peters J, Ho MF, McCarthy JS, Cheng Q. Transcription and expression of Plasmodium falciparum histidine-rich proteins in different stages and strains: implications for rapid diagnostic tests. PLoS ONE. 2011;6:e22593. · DOI
  29. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-∆∆Ct method. Methods. 2001;25:402–8. · DOI · Paper page
  30. Zhang Y. I-TASSER server for protein 3D structure prediction. BMC Bioinform. 2008;9:40. · DOI · Paper page
  31. Roy A, Kucukural A, Zhang Y. I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc. 2010;5:725–38. · DOI
  32. Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y. The I-TASSER Suite: protein structure and function prediction. Nat Methods. 2015;12:7–8. · DOI · Paper page
  33. Rydberg B, Lindahl T. Nonenzymatic methylation of DNA by the intracellular methyl group donor S-adenosyl-l-methionine is a potentially mutagenic reaction. EMBO J. 1982;1:211–6. · DOI
  34. Asaeda A, Ide H, Asagoshi K, Matsuyama S, Tano K, Murakami A, et al. Substrate specificity of human methylpurine DNA N-glycosylase. Biochemistry. 2000;39:1959–65. · DOI
  35. Singer B, Antoccia A, Basu AK, Dosanjh MK, Fraenkel-Conrat H, Gallagher PE, et al. Both purified human 1, N6-ethenoadenine-binding protein and purified human 3-methyladenine-DNA glycosylase act on 1, N6-ethenoadenine and 3-methyladenine. Proc Natl Acad Sci USA. 1992;89:9386–90. · DOI
  36. Lee CYI, Delaney JC, Kartalou M, Lingaraju GM, Maor-Shoshani A, Essigmann JM, et al. Recognition and processing of a new repertoire of DNA substrates by human 3-methyladenine DNA glycosylase (AAG). Biochemistry. 2009;48:1850–61. · DOI
  37. Alseth I, Osman F, Korvald H, Tsaneva I, Whitby MC, Seeberg E, et al. Biochemical characterization and DNA repair pathway interactions of Mag1-mediated base excision repair in Schizosaccharomyces pombe. Nucleic Acids Res. 2005;33:1123–31. · DOI
  38. Lau AY, Wyatt MD, Glassner BJ, Samson LD, Ellenberger T. Molecular basis for discriminating between normal and damaged bases by the human alkyladenine glycosylase, AAG. Proc Natl Acad Sci USA. 2000;97:13573–8. · DOI
  39. Foth BJ, Zhang N, Mok S, Preiser PR, Bozdech Z. Quantitative protein expression profiling reveals extensive post-transcriptional regulation and post-translational modifications in schizont-stage malaria parasites. Genome Biol. 2008;9:R177. · DOI
  40. Le Roch KG, Johnson JR, Florens L, Zhou Y, Santrosyan A, Grainger M, et al. Global analysis of transcript and protein levels across the Plasmodium falciparum life cycle. Genome Res. 2004;14:2308–18. · DOI
  41. Shi L, Kent R, Bence N, Britt AB. Developmental expression of a DNA repair gene in Arabidopsis. Mutat Res. 1997;384:145–56. · DOI
  42. Song S, Xing G, Yuan L, Wang J, Wang S, Yin Y, et al. N-methylpurine DNA glycosylase inhibits p53 mediated cell cycle arrest and coordinates with p53 to determine sensitivity to alkylating agents. Cell Res. 2012;22:1285–303. · DOI
  43. Samson L, Derfler B, Boosalis M, Call K. Genetics Cloning and characterization of a 3-methyladenine DNA glycosylase cDNA from human cells whose gene maps to chromosome 16. Proc Natl Acad Sci USA. 1991;88:9127–31. · DOI
  44. Bjelland S, Seeberg E. Different efficiencies of the Tag and AlkA DNA glycosylases from Escherichia coli in the removal of 3-methyladenine from single-stranded DNA. FEBS Lett. 1996;397:127–9. · DOI
  45. Eftedal I, Guddal PH, Slupphaug G, Volden G, Krokan HE. Consensus sequences for good and poor removal of uracil from double stranded DNA by uracil-DNA glycosylase. Nucleic Acids Res. 1993;21:2095–101. · DOI
  46. Haushalter KA, Todd Stukenberg MW, Kirschner MW, Verdine GL. Identification of a new uracilDNA glycosylase family by expression cloning using synthetic inhibitors. Curr Biol. 1999;9:174–85. · DOI
  47. O’Connor Timothy R. Purification and characterization of human 3-methyladenine-DNA glycosylase. Nucleic Acids Res. 1993;21:5561–9. · DOI
  48. Memisoglu A, Samson L. Cloning and characterization of a cDNA encoding a-methyladenine DNA glycosylase from the fission yeast Schizosaccharomyces pombe. Gene. 1996;177:229–35. · DOI
  49. Warren JC, Cheatum SG. Effect of neutral salts on enzyme activity and structure. Biochemistry. 1966;5:1702–7. · DOI
  50. Park C, Raines RT. Quantitative analysis of the effect of salt concentration on enzymatic catalysis. J Am Chem Soc. 2001;123:11472–9. · DOI
  51. Izumi T, Wiederhold LR, Roy G, Roy R, Jaiswal A, Bhakat KK, et al. Mammalian DNA base excision repair proteins: their interactions and role in repair of oxidative DNA damage. Toxicology. 2003;15:43–65. · DOI
  52. Adhikari S, Toretsky JA, Yuan L, Roy R. Magnesium, essential for base excision repair enzymes, inhibits substrate binding of N-methylpurine-DNA glycosylase. J Biol Chem. 2006;281:29525–32. · DOI
  53. Wang P, Guliaev AB, Hang B. Metal inhibition of human N-methylpurine-DNA glycosylase activity in base excision repair. Toxicol Lett. 2006;166:237–47. · DOI

Metadata

Authors
Pinthong, N., Limudomporn, P., Vasuvat, J., Adisakwattana, P., Rattaprasert, P., & Chavalitshewinkoon-Petmitr, P.
Affiliations
  • Mahidol University (TH)
  • Kasetsart University (TH)
Corresponding author
Chavalitshewinkoon-Petmitr, Porntip
ORCID
Venue
Malaria Journal
Publisher
Springer Science and Business Media LLC
Published
2020-08-06
Volume
19
Issue
1
Pages
284-284
ISSN
1475-2875 (ISSN-L)
DOI
10.1186/s12936-020-03355-w
PMC
PMC7409487
OpenAlex
W3047367396
Cited by
2
References
2
Citations by year
2026: 1 · 2024: 1
FWCI
0.00
Type
journal article
Language
en
Open access
Licence
Locations
  • Malaria Journal (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Mahidol University (MU.2558-2559)
Topics
  • Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Malaria Research and Control
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Molecular Research
Keywords
Plasmodium falciparum, base excision repair, artemisinin resistance, antimalarial drug targets, recombinant enzyme characterization, schizont stage, K1 strain, 3D7 strain, insertion/deletion polymorphism, divalent cation inhibition, pH optimum
MeSH terms
Plasmodium falciparum, Protozoan Proteins, DNA Glycosylases

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