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Covalent Bond Formation, Between a DNA-Cytosine Methyltransferase and DNA Containing 5-Azacytosine (5-Methylcytosine/5-Azacytidine/Hpa II Methylase) DANIEL V

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Covalent Bond Formation, Between a DNA-Cytosine Methyltransferase and DNA Containing 5-Azacytosine (5-Methylcytosine/5-Azacytidine/Hpa II Methylase) DANIEL V Proc. NatI. Acad. Sci. USA Vol. 81, pp. 6993-6997, November 1984 Biochemistry Covalent bond formation, between a DNA-cytosine methyltransferase and DNA containing 5-azacytosine (5-methylcytosine/5-azacytidine/Hpa II methylase) DANIEL V. SANTI, ANNE NORMENT, AND CHARLES E. GARRETT Department of Biochemistry and Biophysics and Department of Pharmaceutical Chemistry, University of California, San Francisco CA 94143 Communicated by Thomas C. Bruice, July 26, 1984 ABSTRACT DNA containing 5-azacytosine (azaC) has the basis of the probable catalytic mechanism of DCMTases previously been shown to be a potent inhibitor of DNA-cyto- and known chemical properties of azaC, we recently specu- sine methyltransferases. In this report, we describe experi- lated that the mechanism of inhibition involves covalent ments which demonstrate that azaC-DNA forms a covalent bond formation between a catalytic nucleophile of the en- complex with Hpa II methylase, a bacterial enzyme that meth- zyme and the reactive 6 position of azaC residues that re- ylates the internal C of C-C-G-G sequences. The complex does placed cytosine methylation sites in DNA (13). In this report not undergo detectable dissociation over at least 3 days and is we describe binding studies of azaC-DNA and Hpa II meth- stable to denaturation with NaDodSO4. After extensive diges- ylase, a bacterial DCMTase that methylates the internal C in tion of the complex with DNase and phosphodiesterase, gel fil- C-C-G-G sequences. The experiments indicate that covalent tration gave the methylase bound to approximately one equiv- complexes are formed that involve azaC residues within the alent of azaC; the digested complex had an apparent molecular recognition sequence of the methylase; however, covalent weight similar to that of the native enzyme. Although prior complexes may also involve azaC residues elsewhere in treatment of azaC-DNA with Hpa II endonuclease had only a DNA. Our results, together with reported kinetic experi- slight effect on binding of the methylase, treatment with Msp I ments (11, 12), explain how incorporation of small amounts endonuclease, which also cleaves at C-C-G-G sequences, re- of azaC into DNA can result in such dramatic decreases in sulted in a significant reduction in binding; this indicates that the methylation of cytosine residues of DNA. azaC residues in the recognition sequence of Hpa II are an important component in the covalent interaction ofthe methyl- MATERIALS AND METHODS ase. However, since there was residual binding it is possible Materials. Hpa II methylase and Msp I endonuclease were that azaC residues elsewhere in DNA also covalently bind to obtained from New England Biolabs. EcoRI methylase was the methylase. These results provide an explanation of why a gift from H. Boyer. Hae III and Hpa II endonucleases azaC-DNA is such a potent inhibitor of cytosine methyltrans- were products of Bethesda Research Laboratories. Bovine ferases and how the incorporation of such low levels of azaC pancreas DNase I was from Sigma, snake venom phosphodi- into DNA can result in dramatic decreases in the methylation esterase was from Boehringer Mannheim, and bacterial alka- of cytosine. Finally, consideration of the probable catalytic line phosphatase was from Worthington. [6_3H]azaCyd (1.6 mechanism of cytosine methylases and the chemical properties Ci/mmol; 1 Ci = 37 GBq) and [methyl-3HJthymidine (125 of azaC suggests that the inhibition is, at least in part, an ac- Ci/mmol) were obtained from Moravek Biochemicals (Brea, tive-site directed process and permits a proposal for the struc- CA) and [methyl-3H]AdoMet (62 Ci/mmol) was from New ture of the covalent complex. England Nuclear. AdoMet and azaCyd were obtained from Calbiochem-Behring. All other reagents were of the purest 5-Methylcytosine, a minor base in the DNA of a variety of grade commercially available. Nitrocellulose BA85 mem- organisms, is formed by postreplicative methylation of DNA branes (0.45-,um pore diameter; 2.4 cm) were obtained from by S-adenosylmethionine (AdoMet) in reactions catalyzed Schleicher & Schuell. by DNA-cytosine methyltransferases (DCMTases). In re- Enzyme Assays. Unless otherwise specified, endonucle- cent years, much evidence has been obtained which indi- ases were used as suggested by the supplier. Hpa II methyl- cates that 5-methylcytosine residues in DNA play an impor- ase was assayed by the method of Yoo and Agarwal (14); 1 tant role in eukaryotic gene expression (for reviews see refs. unit is defined as the amount ofenzyme that catalyzes incor- 1 and 2). Consequently, there has been wide interest in the poration of 1 pmol of methyl groups into phage X DNA in 1 pyrimidine analog 5-azacytidine (azaCyd), which inhibits hr at 37°C. From the reported molecular weight and specific formation of5-methylcytosine in DNA and results in dramat- activity of the purified enzyme (14), we calculate that 100 ic effects on gene expression and cell differentiation (e.g., units is equivalent to about 1 pmol of enzyme. see refs. 3-9). DNA Preparations. Escherichia coli K-12 strain 993 (F+, Current evidence indicates that the mechanism by which restriction-negative, methionine-requiring), obtained from azaCyd causes decreased DNA methylation involves incor- B. Alberts, was grown in M9 medium containing methionine poration of 5-azacytosine (azaC) into DNA and subsequent at 40 ,ug/ml at 37°C until the OD6w was 0.5-0.6. To individ- inhibition of DCMTase. Incorporation of small amounts of ual 10-ml cultures was added (i) azaCyd at 25 jlg/ml, (ii) 0.3 azaC into DNA of mammalian cells results in a loss of mCi of [6_3H]azaCyd, (iii) 150 ,uCi of [3HJthymidine, or (iv) DCMTase activity in extracts obtained from such cells (5, 150 ,uCi of [3H]thymidine plus azaCyd at 25 ug/ml. The cul- 10). Further, incubation of DNA containing azaC (azaC- ture containing [6-3H]azaCyd was incubated for 30 min and DNA) with mammalian or bacterial DCMTases results in a others for 1 hr prior to harvesting. The DNA preparations very potent inhibition of enzyme activity (11, 12), but kinetic were isolated by a modification of a reported procedure (15) studies have not revealed the mechanism of inhibition. On with care taken to avoid exposure to acidic or basic condi- The publication costs of this article were defrayed in part by page charge Abbreviations: AdoMet, S-adenosylmethionine; DCMTase, DNA- payment. This article must therefore be hereby marked "advertisement" cytosine methyltransferase; azaCyd, 5-azacytidine; azaC, 5-azacy- in accordance with 18 U.S.C. §1734 solely to indicate this fact. tosine; azaC-DNA, DNA containing 5-azacytosine. 6993 Downloaded by guest on September 29, 2021 6994 Biochemistry: Santi et aL Proc. NatL Acad. Sci USA 81 (1984) tions, which would result in degradation of azaC residues LH20 in 25 mM Tris HCl, pH 7.4/30 mM EDTA. The col- (16). Cells were washed with 2 ml of cold 150 mM NaCl/100 umn tube was placed in the carrier tube and centrifuged at mM Na2EDTA/20 mM Tris'HCl, pH 7.4, and resuspended setting 80 in a Clay Adams centrifuge for 2 min, giving a in 1.0 ml of the same buffer. The suspension was treated packed volume of about 0.6 ml. After the carrier tube had with 100 ,ug of lysozyme for 15 min at room temperature and been replaced, up to 60 A.l of sample containing 1% Na- brought to 1% in NaDodSO4. After 5 min, the mixture was DodSO4 was applied to the column tube and centrifuged as extracted three times with equal volumes of phenol/chloro- above, and aliquots of the eluate were analyzed by the nitro- form (1:1, vol/vol) and nucleic acids were precipitated with 2 cellulose binding assay. The recovery of DNA and protein vol of ethanol. The precipitate was dissolved in 0.5 ml of 10 was at least 85%, and control experiments indicated that the mM Tris HCl, pH 7.4/1.0 mM EDTA and treated with previ- NaDodSO4 content in the eluant was less than 0.005% as ously boiled pancreatic RNase at 100 ,ug/ml for 1 hr at 37TC. determined spectrophotometrically (20). The solution was brought to 0.1 M in NaCl and 0.2% in Na- DodSO4 and extracted twice with phenol/chloroform and RESULTS the DNA was precipitated with 2 vol of ethanol. The precipi- Binding of Hpa II Methylase to azaC-DNA. In early experi- tate was redissolved in 10 mM Tris'HCl, pH 7.4/1.0 mM ments, nitrocellulose binding assays were performed with EDTA, and the DNA was precipitated with 2-propanol, re- unmodified radioactive DNAs from E. coli. Although Hpa II dissolved in the same buffer, and stored at -800C. methylase clearly increased the binding of azaC-DNA to ni- The [6-3H]azaC-DNA contained 2.7 x 104 dpm/,g, trocellulose, the background binding of DNAs was unaccept- which, calculated on the basis of the specific activity of the ably high, amounting to 20-30% of the radioactivity applied. analog and the 50% G+C content of E. coli DNA, corre- We subsequently found that treatment of radiolabeled DNAs sponds to 1% substitution of the cytosine residues. To esti- with restriction endonucleases reduced the background mate the azaC content of azaC-DNA prepared with the unla- binding to acceptable levels of about 2-4% of the labeled beled analog, the G+C content was determined by HPLC material applied to the membranes. All nitrocellulose bind- after digestion to the component nucleosides. A solution ing assays described below used DNAs that were previously (300 ,ul) containing 10 Pg of [3H]thymidine-containing azaC- digested with Hae III endonuclease, which cleaves at G-G- DNA ([3H]dT-azaC-DNA), 50 mM Tris HCI at pH 8.2, 10 C-C sequences and should produce fragments of DNA with mM MgCl2, and 70 units of DNase I was incubated at 37°C.
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