Journal of Biochemical and Biophysical Methods, 22 (1991) 19-22 Elsevier

19

JBBM 00844

Microassay for DNA methyltransferase R.L.P. Adams, A. R i n a l d i a n d C. S e i v w r i g h t Department of Biochemistry, Universityof Glasgow, Glasgow, UK

(Received 1 March 1990) (Accepted 17 May 1990)

Summary A microassay for DNA methylase is described which can detect activity in as few as 50 tissue culture cells. The cells are lysed and incubated for 2 h at 37°C with 3/tCi high specific activity [3H]AdoMet and 0.5 #g poly[d(I-C),d0-C)] in a volume of 23 #l. Ribonuclease is present during the assay and the product DNA is isolated by phenol extraction after protease digestion. Key words: DNA; Methyltransferase; Methylase; Microassay

Introduction P a r t i c u l a r l y when s t u d y i n g d e v e l o p m e n t a l p r o b l e m s , it is i m p o r t a n t to b e a b l e to a n a l y s e the activity o f an e n z y m e on a very small n u m b e r of cells. This is true for a tissue available in o n l y small a m o u n t s , b u t is even m o r e a p p l i c a b l e to the analysis of activity in very early m a m m a l i a n e m b r y o s where o b t a i n i n g 100 e m b r y o s d e m a n d s m u c h a r d u o u s a n d skilled application. It was to this e n d that we devised a m i c r o a s s a y for D N A methyltransferase. Results o b t a i n e d with this assay on early m o u s e e m b r y o s will b e r e p o r t e d elsewhere [1]. I n crude cell extracts, R N A a n d p r o t e i n m e t h y l a s e s are likely to b e active and, therefore, a n i m p o r t a n t feature of D N A m e t h y l a s e assays is that p r o d u c t s o t h e r t h a n m e t h y l a t e d D N A m u s t b e excluded. A s e c o n d objective when using c r u d e p r e p a r a t i o n s is to try to ensure the a b s e n c e of i n h i b i t o r y effects. These m a y arise f r o m the b i n d i n g o f the e n z y m e to an alternative ( R N A ) p o l y n u c l e o t i d e or the tight a s s o c i a t i o n of the p r o d u c t to D N A - b i n d i n g proteins.

Correspondence address: R.L.P. Adams, Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, U.K.

0165-022X/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)

20 The normal, in vivo, substrate for D N A methylase is believed to be the hemimethylated D N A arising at replication, and this has been shown to be the best in vitro substrate [4]. An alternative, which was expected to be an equally good substrate, is unmethylated D N A rich in C G dinucleotides. However, the extra stability of such a duplex molecule reduces its capacity to accept methyl groups and the alternating polynucleotide in which guanine is replaced with inosine has proved a far better substrate (Ref 6 and Adams, R.L.P. and Hill, J., unpublished data).

Results and Discussion

Although in somatic cells D N A methylase is a nuclear enzyme, it was clear that we would not be able to analyse nuclear extracts. The most suitable starting material was considered to be a cell lysate as this would entail no loss of material on processing. Cells were harvested (by trypsinisation when necessary), counted and washed in phosphate-buffered saline. The appropriate number of cells were pelleted in microfuge tubes and lysis achieved by freeze thawing the cells four times to - 7 0 ° C in a hypotonic buffer (50 mM Tris-HC1, p H 7.8, 1 m M EDTA, 1 m M dithiothreitol, 0.017o sodium azide, 6 mgTo PMSF, 10% glycerol, 1% Tween 80, 100 /~g/ml RNase A). The ribonuclease was present to digest R N A present in the lysate as R N A is an inhibitor of D N A methylase [2,3]. (Before use, the RNase was dissolved at 10 m g / m l and heated at 100 ° C for 5 min.) The cell lysate was made in 15 #1 of this buffer and the final incubation volume restricted to 23 #l. Following incubation, incorporation of radioactivity was analysed by a modification of the method previously reported for a D N A methylase assay [5]. The reaction was stopped by addition of 300/~1 of a solution containing 1% SDS, 2 m M EDTA, 37o 4-aminosalicylate, 57o butanol, 125 mM NaC1, 0.25 m g / m l carrier salmon testis DNA, and 1 m g / m l protease K; and the incubation continued for a further 30 min at 37 o C. The protease incubation is essential to obtain complete recovery of the D N A which may otherwise be tightly complexed with protein [4]. The solution was vortexed with an equal volume of 887o phenol, 127o m-cresol, 0.17o 8-hydroxyquinoline, and centrifuged in a microfuge for 5 min. Two volumes of absolute ethanol at - 2 0 ° C were added to the aqueous layer and the precipitated D N A pelleted as above, drained and dissolved in 30 /tl 0.3 M N a O H . Any residual RNA, which might be labelled, was removed by incubation at 37 o C for 1 - 2 h and the solution of D N A transferred to paper filters. The D N A was precipitated and washed with 5% trichloracetic acid (five times), ethanol (twice) and ether. The dried filter was transferred to a counting vial with 0.5 ml 0.5 M perchloric acid and heated at 60 ° C for an hour to solubilise the D N A prior to addition of 5 ml scintillation fluid (Ecoscint) and counting. Initial experiments used a hemimethylated D N A substrate obtained either from cells grown in the absence of methionine or from primed, single-stranded M13 D N A replicated in the presence of methyl-dCTP [4]. It is difficult to obtain reproducible samples of such D N A and a more satisfactory substrate was found to be poly [d(I-C)-d0-C)] [6]. As this is a double-stranded molecule it is essential to

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Fig. 1. Standardisation of microassay. In sections a and b, 5 #g of a crude fraction of mouse DNA methylase [3] were used in a standard microassay with varying amounts of (a), poly[d(I-C).d(l-C)]; or (b), [3H]methyl S-adenosyl methionine (AdoMet). (c) Time course of the reaction using 200 mouse L929 cells. (d) Linear response in the standard microassay to increasing numbers of L929 cells.

m a i n t a i n as low a n ionic strength as p o s s i b l e as even 50 m M NaC1 inhibits the r e a c t i o n b y over 80% (results n o t shown). P r e l i m i n a r y e x p e r i m e n t s with a c r u d e s a m p l e of D N A m e t h y l a s e from K r e b s 2 ascites cells i n d i c a t e d that a c o n c e n t r a t i o n o f s u b s t r a t e of 4 0 / x g / m l was s a t u r a t i n g (Fig. l a ) a n d in the m i c r o a s s a y 0.5/~g was used. This is a similar c o n c e n t r a t i o n to that f o u n d s a t u r a t i n g in the s t a n d a r d assay. A m e r s h a m I n t e r n a t i o n a l ( A m e r s h a m , U . K . ) have recently i n t r o d u c e d a very high specific activity f o r m of tritiated S - a d e n o s y l m e t h i o n i n e ( T R K 581, 87 C i / m m o l ) . A l t h o u g h this is s u p p l i e d in dilute acid, the buffering c a p a c i t y of the Tris-HC1 r e a d i l y copes with the a d d i t i o n of 20% the r e a c t i o n v o l u m e c o m i n g f r o m the isotope. Fig. l b indicates that a c o n c e n t r a t i o n of 1.5 /~M was s a t u r a t i n g a n d this was e m p l o y e d in the microassay. Again, this is a similar c o n c e n t r a t i o n to that used in

22 the s t a n d a r d assay but, b y decreasing the volume of the assay, this c o n c e n t r a t i o n can b e achieved b y using 3/LCi of the highest specific activity A d o M e t . Fig. l c shows a time course of the reaction using 200 m o u s e L929 cells. A f t e r a slight lag, the r e a c t i o n was linear for 2 h. I n c o r p o r a t i o n was p r o p o r t i o n a l to cell n u m b e r , a n d the assay was able to detect the activity p r e s e n t in as few as 50 L929 cells (Fig. l d ) . L i n e a r i t y is m a i n t a i n e d up to 20000 cells [1]. In a survey of a n u m b e r of different cell types the most active we have f o u n d so far are K r e b s 2 ascites cells, with r a p i d l y growing L929 cells a l m o s t as active; whereas n o n - g r o w i n g L929 cells (0.05 c p m / c e l l ) , l y m p h o c y t e s isolated from fresh b l o o d (0.14 c p m / c e l l ) a n d h e p a t o c y t e s directly isolated f r o m rat liver (0.03 c p m / c e l l ) showed very little activity. T h e m e t h o d d e s c r i b e d is a p p l i c a b l e to tissue culture cells a n d to small pieces of tissue where the cells can be d i s r u p t e d b y freeze thawing. It is n o t necessary to use such a sensitive assay when s t u d y i n g the isolated enzyme. A l t h o u g h the s u b s t r a t e s are unlikely to b e c o m e limiting, even if purified D N A m e t h y l a s e is used, their cost (especially that of the isotope) renders this m o r e expensive t h a n the s t a n d a r d assay.

Acknowledgements W e w o u l d like to a c k n o w l e d g e the financial assistance from the M e d i c a l Research C o u n c i l a n d the s u p p o r t of Prof. H o u s l a y a n d the U n i v e r s i t y of G l a s g o w which m a d e this w o r k possible.

References 1 Monk, M., Adams, R.L.P. and Rinaldi, A. (1990) Decrease in DNA methylase activity during mouse early development; submitted. 2 Bolden, A., Ward, C., Siedlecki, J.A. and Weissbach, A. (1984) Inhibition of de novo and maintenance methylation in vitro by RNA and synthetic polynucleotides. J. Biol. Chem. 259, 12437-12443. 3 Adams, R.L.P., Gardiner, K., Rinaldi, A., Bryans, M., McGarvey, M. and Burdon, R.H. (1986) Mouse ascites DNA methylase: characterisation of size, proteolytic breakdown and nucleotide recognition. Biochim. Biophys. Acta 868, 9-16. 4 Adams, R.L.P. (1990) DNA methylation: the effect of minor bases on DNA-protein interactions. Biochem. J. 265, 309-320. 5 Turnbull, J.F. and Adams, R.L.P. (1976) DNA methylase: purification from ascites cells and the effect of various DNA substrates on its activity. Nucl. Acids Res. 3, 677-695. 6 Pfeifer, G.P. and Drahovsky, D. (1986) Preferential binding of DNA methyltransferase and increased de novo methylation of deoxyinosine containing DNA. FEBS Lett. 207, 75-78.

Microassay for DNA methyltransferase.

A microassay for DNA methylase is described which can detect activity in as few as 50 tissue culture cells. The cells are lysed and incubated for 2 h ...
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