title: Mechanistic insights on the inhibition of c5 DNA methyltransferases by zebularine.
abstract: In mammals DNA methylation occurs at position 5 of cytosine in a CpG context and regulates gene expression. It plays an important role in diseases and inhibitors of DNA methyltransferases (DNMTs)--the enzymes responsible for DNA methylation--are used in clinics for cancer therapy. The most potent inhibitors are 5-azacytidine and 5-azadeoxycytidine. Zebularine (1-(beta-D-ribofuranosyl)-2(1H)- pyrimidinone) is another cytidine analog described as a potent inhibitor that acts by forming a covalent complex with DNMT when incorporated into DNA. Here we bring additional experiments to explain its mechanism of action. First, we observe an increase in the DNA binding when zebularine is incorporated into the DNA, compared to deoxycytidine and 5-fluorodeoxycytidine, together with a strong decrease in the dissociation rate. Second, we show by denaturing gel analysis that the intermediate covalent complex between the enzyme and the DNA is reversible, differing thus from 5-fluorodeoxycytidine. Third, no methylation reaction occurs when zebularine is present in the DNA. We confirm that zebularine exerts its demethylation activity by stabilizing the binding of DNMTs to DNA, hindering the methylation and decreasing the dissociation, thereby trapping the enzyme and preventing turnover even at other sites.
authors.affiliation: MNHN CNRS UMR7196, Paris, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Champion
authors.forename: Dominique
authors.initials: D
authors.lastname: Guianvarc'h
authors.forename: Catherine
authors.initials: C
authors.lastname: Sénamaud-Beaufort
authors.forename: Renata Z
authors.initials: RZ
authors.lastname: Jurkowska
authors.forename: Albert
authors.initials: A
authors.lastname: Jeltsch
authors.forename: Loïc
authors.initials: L
authors.lastname: Ponger
authors.forename: Paola B
authors.initials: PB
authors.lastname: Arimondo
authors.forename: Anne-Laure
authors.initials: AL
authors.lastname: Guieysse-Peugeot
mesh_terms: D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D001483:Base Sequence; D003562:Cytidine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D000077209:Decitabine; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D008745:Methylation / Q000187:drug effects; D051379:Mice
chemical_list: D004791:Enzyme Inhibitors; D003562:Cytidine; D000077209:Decitabine; C009131:pyrimidin-2-one beta-ribofuranoside; D004247:DNA; D004248:DNA (Cytosine-5-)-Methyltransferases; D001374:Azacitidine
references.citation: J Mol Biol. 2003 Jun 13;329(4):675-84
references.pmid: 12787669
references.citation: Cancer Chemother Pharmacol. 2009 Feb;63(3):411-6
references.pmid: 18398609
references.citation: Oncogene. 2002 Aug 12;21(35):5483-95
references.pmid: 12154409
references.citation: Biochem J. 1995 Apr 1;307 ( Pt 1):87-92
references.pmid: 7536414
references.citation: J Mol Biol. 1996 Sep 6;261(5):634-45
references.pmid: 8800212
references.citation: Exp Hematol. 2007 Feb;35(2):263-73
references.pmid: 17258075
references.citation: DNA Repair (Amst). 2004 Feb 3;3(2):155-61
references.pmid: 14706349
references.citation: Cell. 1995 Jul 14;82(1):143-53
references.pmid: 7606780
references.citation: Leukemia. 2009 Jun;23(6):1019-28
references.pmid: 19194470
references.citation: Cancer Prev Res (Phila). 2008 Sep;1(4):233-40
references.pmid: 19138966
references.citation: J Mol Biol. 1999 Feb 19;286(2):389-401
references.pmid: 9973559
references.citation: Nature. 2004 May 27;429(6990):457-63
references.pmid: 15164071
references.citation: J Pharm Sci. 1981 Nov;70(11):1228-32
references.pmid: 6170748
references.citation: J Mol Biol. 2002 Aug 23;321(4):591-9
references.pmid: 12206775
references.citation: Oncogene. 2002 Aug 12;21(35):5400-13
references.pmid: 12154403
references.citation: Clin Cancer Res. 2005 Jun 15;11(12):4571-9
references.pmid: 15958643
references.citation: Nat Rev Drug Discov. 2005 Apr;4(4):275-6
references.pmid: 15861567
references.citation: Cancer Chemother Pharmacol. 2009 Jul;64(2):223-32
references.pmid: 18830594
references.citation: J Biol Chem. 1982 Feb 25;257(4):2041-8
references.pmid: 6173384
references.citation: Nat Rev Drug Discov. 2006 Nov;5(11):891-2
references.pmid: 17117522
references.citation: Cancer Chemother Pharmacol. 1992;30(1):7-11
references.pmid: 1375134
references.citation: J Biol Chem. 2002 Jun 7;277(23):20409-14
references.pmid: 11919202
references.citation: Mol Cancer Ther. 2005 Oct;4(10):1505-14
references.pmid: 16227399
references.citation: J Pharm Sci. 1975 Jul;64(7):1148-57
references.pmid: 50433
references.citation: Hum Mol Genet. 2005 Apr 15;14 Spec No 1:R65-76
references.pmid: 15809275
references.citation: Biochim Biophys Acta. 2009 Nov;1794(11):1654-62
references.pmid: 19654054
references.citation: Ann N Y Acad Sci. 2003 Dec;1002:154-64
references.pmid: 14751833
references.citation: Anal Chem. 2005 Jan 15;77(2):504-10
references.pmid: 15649046
references.citation: Proc Natl Acad Sci U S A. 1984 Nov;81(22):6993-7
references.pmid: 6209710
references.citation: Nucleic Acids Res. 1992 Jun 25;20(12):3241-8
references.pmid: 1620620
references.citation: Epigenetics. 2006 Apr-Jun;1(2):63-6
references.pmid: 17965604
references.citation: J Biol Chem. 2002 Apr 5;277(14):11735-45
references.pmid: 11821381
references.citation: J Mol Biol. 1982 Dec 15;162(3):679-92
references.pmid: 6187927
references.citation: J Med Chem. 1986 Aug;29(8):1374-80
references.pmid: 3735306
references.citation: Biochem J. 1993 Apr 15;291 ( Pt 2):493-504
references.pmid: 8484730
references.citation: Anal Biochem. 2003 Oct 1;321(1):50-64
references.pmid: 12963055
references.citation: J Am Chem Soc. 2008 Dec 31;130(52):17634-5
references.pmid: 19108695
references.citation: Nat Rev Drug Discov. 2006 Jan;5(1):37-50
references.pmid: 16485345
references.citation: Cell. 1985 Mar;40(3):485-6
references.pmid: 2578884
references.citation: Mol Cell Biol. 2003 Apr;23(8):2709-19
references.pmid: 12665573
references.citation: Cell Mol Life Sci. 2004 Oct;61(19-20):2571-87
references.pmid: 15526163
references.citation: Cell. 1980 May;20(1):85-93
references.pmid: 6156004
references.citation: Cancer Res. 2006 Mar 1;66(5):2794-800
references.pmid: 16510601
references.citation: Biochemistry. 1988 Jul 12;27(14):5204-10
references.pmid: 3167042
references.citation: Ann N Y Acad Sci. 2005 Nov;1058:246-54
references.pmid: 16394141
references.citation: Mol Cancer Ther. 2006 Feb;5(2):467-75
references.pmid: 16505122
references.citation: Curr Top Microbiol Immunol. 2006;301:203-25
references.pmid: 16570849
references.citation: PLoS Genet. 2009 Mar;5(3):e1000438
references.pmid: 19325872
references.citation: J Med Chem. 1991 Nov;34(11):3280-4
references.pmid: 1956046
references.citation: Biochem Soc Trans. 2004 Dec;32(Pt 6):910-2
references.pmid: 15506921
references.citation: Nucleic Acids Res. 1993 May 25;21(10):2459-64
references.pmid: 8506140
references.citation: J Natl Cancer Inst. 2003 Mar 5;95(5):399-409
references.pmid: 12618505
references.citation: Carcinogenesis. 2000 Mar;21(3):461-7
references.pmid: 10688866
references.citation: Mol Cell Biol. 2004 Feb;24(3):1270-8
references.pmid: 14729971
references.citation: Annu Rev Biochem. 1983;52:93-124
references.pmid: 6311083
references.citation: Biochem Pharmacol. 2009 Sep 15;78(6):633-41
references.pmid: 19467223