Abstract
DNA methylation is an essential modification of DNA in mammals that is involved in gene regulation, development, genome defence and disease. In mammals 3 families of DNA methyltransferases (MTases) comprising (so far) 4 members have been found: Dnmt1, Dnmt2, Dnmt3A and Dnmt3B. In addition, Dnmt3L has been identified as a stimulator of the Dnmt3A and Dnmt3B enzymes. In this review the enzymology of the mammalian DNA MTases is described, starting with a depiction of the catalytic mechanism that involves covalent catalysis and base flipping. Subsequently, important mechanistic features of the mammalian enzyme are discussed including the specificity of Dnmt1 for hemimethylated target sites, the target sequence specificity of Dnmt3A, Dnmt3B and Dnmt2 and the flanking sequence preferences of Dnmt3A and Dnmt3B. In addition, the processivity of the methylation reaction by Dnmt1, Dnmt3A and Dnmt3B is reviewed. Finally, the control of the catalytic activity of mammalian MTases is described that includes the regulation of the activity ofDnmt1 by its N-terminal domain and the interaction of Dnmt3A and Dnmt3B with Dnmt3L. The allosteric activation of Dnmt1 for methylation at unmodified sites is described. Wherever possible, correlations between the biochemical properties of the enzymes and their physiological functions in the cell are indicated.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Aapola U, Kawasaki K, Scott HS, Ollila J, Vihinen M, Heino M, Shintani A, Minoshima S, Krohn K, Antonarakis SE, Shimizu N, Kudoh J, Peterson P (2000) Isolation and initial characterization of a novel zinc finger gene, DNMT3L, on 21q22.3, related to the cytosine-5-methyltransferase 3 gene family. Genomics 65:293–298
Aapola U, Liiv I, Peterson P (2002) Imprinting regulator DNMT3L is a transcriptional repressor associated with histone deacetylase activity. Nucleic Acids Res 30:3602–3608
Aoki A, Suetake I, Miyagawa J, Fujio T, Chijiwa T, Sasaki H, Tajima S (2001) Enzymatic properties of de novo-type mouse DNA (cytosine-5) methyltransferases. Nucleic Acids Res 29:3506–3512
Araujo FD, Croteau S, Slack AD, Milutinovic S, Bigey P, Price GB, Zannis-Hajopoulos M, Szyf M (2001) The DNMT1 target recognition domain resides in the N terminus. J Biol Chem 276:6930–6936
Bachman KE, Rountree MR, Baylin SB (2001) Dnmt3a and Dnmt3b are transcriptional repressors that exhibit unique localization properties to heterochromatin. J Biol Chem 276:32282–32287
Bachman KE, Park BH, Rhee I, Rajagopalan H, Herman JG, Baylin SB, Kinzler KW, Vogelstein B (2003) Histone modifications and silencing prior to DNA methylation of a tumor suppressor gene. Cancer Cell 3:89–95
Bacolla A, Pradhan S, Roberts RJ, Wells RD (1999) Recombinant human DNA (cytosine-5) methyltransferase. II. Steady-state kinetics reveal allosteric activation by methylated DNA. J Biol Chem 274:33011–33019
Bacolla A, Pradhan S, Larson JE, Roberts RJ, Wells RD (2001) Recombinanthuman DNA (cytosine-5) methyltransferase. III. Allosteric control, reaction order, and influence of plasmid topology and triplet repeat length on methylation of the fragile X CGG.CCG sequence. J Biol Chem 276:18605–18613
Bestor T, Laudano A, Mattaliano R, Ingram V (1988) Cloning and sequencing of a cDNA encoding DNA methyltransferase of mouse cells. The carboxyl-terminal domain of the mammalian enzymes is related to bacterial restriction methyltransferases. J Mol Biol 203:971–983
Bestor TH (1992) Activation of mammalian DNA methyltransferase by cleavage of a Zn binding regulatory domain. EMBO J 11:2611–2617
Bestor TH, Ingram VM (1983) Two DNA methyltransferases from murine erythroleukemia cells: purification, sequence specificity, and mode of interaction with DNA. Proc Natl Acad Sci U S A 80:5559–5563
Biniszkiewicz D, Gribnau J, Ramsahoye B, Gaudet F, Eggan K, Humpherys D, Mastrangelo MA, Jun Z, Walter J, Jaenisch R (2002) Dnmt1 overexpression causes genomic hypermethylation, loss of imprinting, and embryonic lethality. Mol Cell Biol 22:2124–2135
Bourc’his D, Bestor TH (2004) Meiotic catastrophe and retrotransposon reactivation in male germ cells lacking Dnmt3L. Nature 431:96–99
Bourc’his D, Xu GL, Lin CS, Bollman B, Bestor TH (2001) Dnmt3L and the establishment of maternal genomic imprints. Science 294:2536–2539
Cameron EE, Bachman KE, Myohanen S, Herman JG, Baylin SB (1999) Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat Genet 21:103–107
Chedin F, Lieber MR, Hsieh CL (2002) The DNA methyltransferase-like protein DNMT3L stimulates de novo methylation by Dnmt3a. Proc Natl Acad Sci U S A 99:16916–16921
Chen L, MacMillan AM, Chang W, Ezaz-Nikpay K, Lane WS, Verdine GL (1991) Direct identification of the active-site nucleophile in a DNA (cytosine-5)-methyltransferase. Biochemistry 30:11018–11025
Chen T, Li E (2004) Structure and function of eukaryotic DNA methyltransferases. Curr Top Dev Biol 60:55–89
Chen T, Ueda Y, Dodge JE, Wang Z, Li E (2003) Establishment and maintenance of genomic methylation patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b. Mol Cell Biol 23:5594–5605
Cheng X (1995) Structure and function of DNA methyltransferases. Annu Rev Biophys Biomol Struct 24:293–318
Cheng X, Roberts RJ (2001) AdoMet-dependent methylation, DNA methyltransferases and base flipping. Nucleic Acids Res 29:3784–3795
Chuang LS, Ian HI, Koh TW, Ng HH, Xu G, Li BF (1997) Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21 WAF1. Science 277:1996–2000
Datta J, Ghoshal K, Sharma SM, Tajima S, Jacob ST (2003) Biochemical fractionation reveals association of DNA methyltransferase (Dnmt) 3b with Dnmt1 and that of Dnmt 3a with a histone H3 methyltransferase and Hdac1. J Cell Biochem 88:855–864
Dodge JE, Ramsahoye BH, Wo ZG, Okano M, Li E (2002) De novo methylation of MMLV provirus in embryonic stem cells: CpG versus non-CpG methylation. Gene 289:41–48
Dong A, Yoder JA, Zhang X, Zhou L, Bestor TH, Cheng X (2001) Structure of human DNMT2, an enigmatic DNA methyltransferase homolog that displays denaturant-resistant binding to DNA. Nucleic Acids Res 29:439–448
Ehrlich M (2003) Expression of various genes is controlled by DNA methylation during mammalian development. J Cell Biochem 88:899–910
Everett EA, Falick AM, Reich NO (1990) Identification of a critical cysteine in EcoRI DNA methyltransferase by mass spectrometry. J Biol Chem 265:17713–17719
Fahrner JA, Eguchi S, Herman JG, Baylin SB (2002) Dependence of histone modifications and gene expression on DNA hypermethylation in cancer. Cancer Res 62:7213–7218
Fatemi M, Hermann A, Pradhan S, Jeltsch A (2001) The activity of the murine DNA methyltransferase Dnmt1 is controlled by interaction of the catalytic domain with the N-terminal part of the enzyme leading to an allosteric activation of the enzyme after binding to methylated DNA. J Mol Biol 309:1189–1199
Fatemi M, Hermann A, Gowher H, Jeltsch A (2002) Dnmt3a and Dnmt1 functionally cooperate during de novo methylation of DNA. Eur J Biochem 269:4981–4984
Flynn J, Reich N (1998) Murine DNA (cytosine-5-)-methyltransferase: steady-state and substrate trapping analyses of the kinetic mechanism. Biochemistry 37:15162–15169
Flynn J, Glickman JF, Reich NO (1996) Murine DNA cytosine-C5 methyltransferase: pre-steady-and steady-state kinetic analysis with regulatory DNA sequences. Biochemistry 35:7308–7315
Fuks F, Hurd PJ, Deplus R, Kouzarides T (2003) The DNA methyltransferases associate with HP1 and the SUV39H1 histone methyltransferase. Nucleic Acids Res 31:2305–2312
Ge YZ, Pu MT, Gowher H, Wu HP, Ding JP, Jeltsch A, Xu GL (2004) Chromatin targeting of de novo DNA methyltransferases by the PWWP domain. J Biol Chem 279:25447–25454
Glickman JF, Pavlovich JG, Reich NO (1997) Peptide mapping of the murine DNA methyltransferase reveals a major phosphorylation site and the start of translation. J Biol Chem 272:17851–17857
Goedecke K, Pignot M, Goody RS, Scheidig AJ, Weinhold E (2001) Structure of the N6-adenine DNA methyltransferase M. TaqI in complex with DNA and a cofactor analog. Nat Struct Biol 8:121–125
Gowher H, Jeltsch A (2001) Enzymatic properties of recombinant Dnmt3a DNA methyltransferase from mouse: the enzyme modifies DNA in a non-processive manner and also methylates non-CpG sites. J Mol Biol 309:1201–1208
Gowher H, Jeltsch A (2002) Molecular enzymology of the catalytic domains of the Dnmt3a and Dnmt3b DNA methyltransferases. J Biol Chem 277:20409–20414
Gowher H, Jeltsch A (2004) Mechanism of inhibition of DNA methyltransferases by cytidine analogs in cancer therapy. Cancer Biol Ther 3
Gowher H, Liebert K, Hermann A, Xu G, Jeltsch A (2005) Mechanism of stimulation of catalytic activity of Dnmt3a and Dnmt3b DNA-(cytosine C5)-methyltransferaes by Dnmt3L. J Biol Chem 280:13341–13348
Gruenbaum Y, Cedar H, Razin A (1982) Substrate and sequence specificity of a eukaryotic DNA methylase. Nature 295:620–622
Gruenbaum Y, Szyf M, Cedar H, Razin A (1983) Methylation of replicating and post-replicated mouse L-cell DNA. Proc Natl Acad Sci U S A 80:4919–4921
Hanck T, Schmidt S, Fritz HJ (1993) Sequence-specific and mechanism-based crosslinking of Dcm DNA cytosine-C5 methyltransferase of E. coli K-12 to synthetic oligonucleotides containing 5-fluoro-2′-deoxycytidine. Nucleic Acids Res 21:303–309
Handa V, Jeltsch A (2005) Profound flanking sequence preference of Dnmt3a and Dnmt3b mammalian methyltransferases shape the human epigenome. JMol Biol 348:1103–1112
Hansen RS, Wijmenga C, Luo P, Stanek AM, Canfield TK, Weemaes CM, Gartler SM (1999) The DNMT3B DNA methyltransferase gene is mutated in the ICF immunodeficiency syndrome. Proc Natl Acad Sci U S A 96:14412–14417
Hata K, Okano M, Lei H, Li E (2002) Dnmt3L cooperates with the Dnmt3 family of de novo DNA methyltransferases to establish maternal imprints in mice. Development 129:1983–1993
Hermann A, Schmitt S, Jeltsch A (2003) The human Dnmt2 has residual DNA-(cytosine-C5) methyltransferase activity. J Biol Chem 278:31717–31721
Hermann A, Gowher H, Jeltsch A (2004a) Biochemistry and biology of mammalian DNA methyltransferases. Cell Mol Life Sci 61:2571–2587
Hermann A, Goyal R, Jeltsch A (2004b) The Dnmt1 DNA-(cytosine-C5)-methyltransferase methylates DNA processively with high preference for hemimethylated target sites. J Biol Chem 279:48350–48359
Hsieh CL (1999) In vivo activity of murine de novo methyltransferases, Dnmt3a and Dnmt3b. Mol Cell Biol 19:8211–8218
Hurd PJ, Whitmarsh AJ, Baldwin GS, Kelly SM, Waltho JP, Price NC, Connolly BA, Hornby DP (1999) Mechanism-based inhibition of C5-cytosine DNA methyltransferases by 2-H pyrimidinone. J Mol Biol 286:389–401
Jackson JP, Lindroth AM, Cao X, Jacobsen SE (2002) Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase. Nature 416:556–560
Jeltsch A (2002) Beyond Watson and Crick: DNA methylation and molecular enzymology of DNA methyltransferases. Chembiochem 3:274–293
Kaneda M, Okano M, Hata K, Sado T, Tsujimoto N, Li E, Sasaki H (2004) Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting. Nature 429:900–903
Kim GD, Ni J, Kelesoglu N, Roberts RJ, Pradhan S (2002) Co-operation and communication between the human maintenance and de novo DNA (cytosine-5) methyltransferases. EMBO J 21:4183–4195
Kim M, Trinh BN, Long TI, Oghamian S, Laird PW (2004) Dnmt1 deficiency leads to enhanced microsatellite instability in mouse embryonic stem cells. Nucleic Acids Res 32:5742–5749
Kimura H, Shiota K (2003) Methyl-CpG-binding protein, MeCP2, is a target molecule for maintenance DNA methyltransferase, Dnmt1. J Biol Chem 278:4806–4812
Klimasauskas S, Kumar S, Roberts RJ, Cheng X (1994) HhaI methyltransferase flips its target base out of the DNA helix. Cell 76:357–369
Klinman DM, Yi AK, Beaucage SL, Conover J, Krieg AM (1996) CpG motifs present in bacteria DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon gamma. Proc Natl Acad Sci U S A 93:2879–2883
Krieg AM (2002) CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol 20:709–760
Kunert N, Marhold J, Stanke J, Stach D, Lyko F (2003) A Dnmt2-like protein mediates DNA methylation in Drosophila. Development 130:5083–5090
Lehnertz B, Ueda Y, Derijck AA, Braunschweig U, Perez-Burgos L, Kubicek S, Chen T, Li E, Jenuwein T, Peters AH (2003) Suv39h-mediated histone H3 lysine 9 methylation directs DNA methylation tomajor satellite repeats at pericentric heterochromatin. Curr Biol 13:1192–1200
Leonhardt H, Page AW, Weier HU, Bestor TH (1992) A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei. Cell 71:865–873
Li E, Bestor TH, Jaenisch R (1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69:915–926
Liang G, Chan MF, Tomigahara Y, Tsai YC, Gonzales FA, Li E, Laird PW, Jones PA (2002) Cooperativity between DNA methyltransferases in the maintenance methylation of repetitive elements. Mol Cell Biol 22:480–491
Lin IG, Han L, Taghva A, O’Brien LE, Hsieh CL (2002) Murine denovomethyl transferase Dnmt3a demonstrates strand asymmetry and site preference in the methylation of DNA in vitro. Mol Cell Biol 22:704–723
Lin MJ, Tang LY, Reddy MN, Shen CK (2004) DNA methyltransferase gene dDnmt2 and longevity of Drosophila. J Biol Chem 280:861–864
Liu K, Wang YF, Cantemir C, Muller MT (2003) Endogenous assays of DNA methyltransferases: evidence for differential activities of DNMT1, DNMT2, and DNMT3 in mammalian cells in vivo. Mol Cell Biol 23:2709–2719
Liu Z, Fisher RA (2004) RGS6 interacts with DMAP1 and DNMT1 and inhibits DMAP1 transcriptional repressor activity. J Biol Chem 279:14120–14128
Maga G, Hubscher U (2003) Proliferating cell nuclear antigen (PCNA): a dancer with many partners. J Cell Sci 116:3051–3060
Malone T, Blumenthal RM, Cheng X (1995) Structure-guided analysis reveals nine sequence motifs conserved among DNA amino-methyltransferases, and suggests a catalytic mechanism for these enzymes. J Mol Biol 253:618–632
Margot JB, Aguirre-Arteta AM, Di Giacco BV, Pradhan S, Roberts RJ, Cardoso MC, Leonhardt H (2000) Structure and function of the mouse DNA methyltransferase gene: Dnmt1 shows a tripartite structure. J Mol Biol 297:293–300
Margot JB, Ehrenhofer-Murray AE, Leonhardt H (2003) Interactions within the mammalian DNA methyltransferase family. BMC Mol Biol 4:7
Okano M, Xie S, Li E (1998) Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nat Genet 19:219–220
Okano M, Bell DW, Haber DA, Li E (1999) DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99:247–257
Osterman DG, DePillis GD, Wu JC, Matsuda A, Santi DV (1988) 5-Fluorocytosine in DNA is a mechanism-based inhibitor of HhaI methylase. Biochemistry 27:5204–5210
Pradhan S, Esteve PO (2003a) Allosteric activator domain of maintenance human DNA (cytosine-5) methyltransferase and its role in methylation spreading. Biochemistry 42:5321–5332
Pradhan S, Esteve PO (2003b) Mammalian DNA (cytosine-5) methyltransferases and their expression. Clin Immunol 109:6–16
Pradhan S, Kim GD (2002) The retinoblastoma gene product interacts with maintenance human DNA (cytosine-5) methyltransferase and modulates its activity. EMBO J 21:779–788
Pradhan S, Talbot D, Sha M, Benner J, Hornstra L, Li E, Jaenisch R, Roberts RJ (1997) Baculovirus-mediated expression and characterization of the full-length murine DNA methyltransferase. Nucleic Acids Res 25:4666–4673
Pradhan S, Bacolla A, Wells RD, Roberts RJ (1999) Recombinant human DNA (cytosine-5) methyltransferase. I. Expression, purification, and comparison of de novo and maintenance methylation. J Biol Chem 274:33002–33010
Ramsahoye BH, Biniszkiewicz D, Lyko F, Clark V, Bird AP, Jaenisch R (2000) Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a. Proc Natl Acad Sci U S A 97:5237–5242
Reinisch KM, Chen L, Verdine GL, Lipscomb WN (1995) The crystal structure of HaeIII methyltransferase convalently complexed to DNA: an extrahelical cytosine and rearranged base pairing. Cell 82:143–153
Reither S, Li F, Gowher H, Jeltsch A (2003) Catalytic mechanism of DNA-(cytosine-C5)-methyltransferases revisited: covalent intermediate formation is not essential for methyl group transfer by the murine Dnmt3a enzyme. JMol Biol 329:675–684
Rhee I, Bachman KE, Park BH, Jair KW, Yen RW, Schuebel KE, Cui H, Feinberg AP, Lengauer C, Kinzler KW, Baylin SB, Vogelstein B (2002) DNMT1 and DNMT3b cooperate to silence genes in human cancer cells. Nature 416:552–556
Roberts RJ, Cheng X (1998) Base flipping. Annu Rev Biochem 67:181–198
Roberts RJ, Belfort M, Bestor T, Bhagwat AS, Bickle TA, Bitinaite J, Blumenthal RM, Degtyarev S, Dryden DT, Dybvig K, et al (2003) A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes. Nucleic Acids Res 31:1805–1812
Robertson KD, Ait-Si-Ali S, Yokochi T, Wade PA, Jones PL, Wolffe AP (2000) DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters. Nat Genet 25:338–342
Rountree MR, Bachman KE, Baylin SB (2000) DNMT1 binds HDAC2 and a new corepressor, DMAP1, to form a complex at replication foci. Nat Genet 25:269–277
Rui L, Vinuesa CG, Blasioli J, Goodnow CC (2003) Resistance to CpG DNA-induced autoimmunity through tolerogenic B cell antigen receptor ERK signaling. Nat Immunol 4:594–600
Santi DV, Norment A, Garrett CE (1984) Covalent bond formation between a DNA-cytosine methyltransferase and DNA containing 5-azacytosine. Proc Natl Acad Sci U S A 81:6993–6997
Sarraf SA, Stancheva I (2004) Methyl-CpG binding protein MBD1 couples histone H3 methylation at lysine 9 by SETDB1 to DNA replication and chromatin assembly. Mol Cell 15:595–605
Suetake I, Shinozaki F, Miyagawa J, Takeshima H, Tajima S (2004) DNMT3L stimulates the DNA methylation activity of Dnmt3a and Dnmt3b through a direct interaction. J Biol Chem 279:27816–27823
Tamaru H, Selker EU (2001) Ahistone H3 methyltransferase controls DNA methylation in Neurospora crassa. Nature 414:277–283
Tang LY, Reddy MN, Rasheva V, Lee TL, Lin MJ, Hung MS, Shen CK (2003) The eukaryotic DNMT2 genes encode a new class of cytosine-5 DNA methyltransferases. J Biol Chem 278:33613–33616
Tariq M, Saze H, Probst AV, Lichota J, Habu Y, Paszkowski J (2003) Erasure of CpG methylation in Arabidopsis alters patterns of histone H3 methylation in heterochromatin. Proc Natl Acad Sci U S A 100:8823–8827
Tollefsbol TO, Hutchison CA 3rd (1995) Mammalian DNA (cytosine-5-)-methyltransferase expressed in Escherichia coli, purified and characterized. J Biol Chem 270:18543–18550
Tollefsbol TO, Hutchison CA 3rd (1997) Control of methylation spreading in synthetic DNA sequences by the murine DNA methyltransferase. J Mol Biol 269:494–504
Wang KY, James Shen CK (2004) DNA methyltransferase Dnmt1 and mismatch repair. Oncogene 23:7898–7902
Wu JC, Santi DV (1985) On the mechanism and inhibition of DNA cytosine methyltransferases. Prog Clin Biol Res 198:119–129
Wu JC, Santi DV (1987) Kinetic and catalytic mechanism of HhaI methyltransferase. J Biol Chem 262:4778–4786
Wyszynski MW, Gabbara S, Bhagwat AS (1992) Substitutions of a cysteine conserved among DNA cytosine methylases result in a variety of phenotypes. Nucleic Acids Res 20:319–326
Wyszynski MW, Gabbara S, Kubareva EA, Romanova EA, Oretskaya TS, Gromova ES, Shabarova ZA, Bhagwat AS (1993) The cysteine conserved among DNA cytosine methylases is required for methyl transfer, but not for specific DNA binding. Nucleic Acids Res 21:295–301
Xu GL, Bestor TH, Bourc’his D, Hsieh CL, Tommerup N, Bugge M, Hulten M, Qu X, Russo JJ, Viegas-Pequignot E (1999) Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene. Nature 402:187–191
Yoder JA, Soman NS, Verdine GL, Bestor TH (1997) DNA (cytosine-5)-methyltransferases in mouse cells and tissues. Studies with a mechanism-based probe. J Mol Biol 270:385–395
Zimmermann C, Guhl E, Graessmann A (1997) Mouse DNA methyltransferase (MTase) deletion mutants that retain the catalytic domain display neither de novo nor maintenance methylation activity in vivo. Biol Chem 378:393–405
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Springer-Verlag Berlin Heidelberg
About this chapter
Cite this chapter
Jeltsch, A. (2006). Molecular Enzymology of Mammalian DNA Methyltransferases. In: Doerfler, W., Böhm, P. (eds) DNA Methylation: Basic Mechanisms. Current Topics in Microbiology and Immunology, vol 301. Springer, Berlin, Heidelberg. https://doi.org/10.1007/3-540-31390-7_7
Download citation
DOI: https://doi.org/10.1007/3-540-31390-7_7
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-540-29114-5
Online ISBN: 978-3-540-31390-8
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)