DNA synthesis with methylated poly ( dA - dT ) templates : possible role of C ^ - methylthymlne

The a l t e r n a t i n g copolymer poly(dA-dT) has been methylated with e i t h e r d i methyl sulphate (DM£) or N-methyl-N-nitrosourea (MNU) and t he l e v e l s of the var ious methylat ion products determined. In addi t ion to the methylated adenines formed by both methylat ing agents , MNU r e s u l t e d a l so i n the forma t ion of 3-nethylthymine, £ -methylthymine and p h o s p h o t r i e s t e r s . The methylated polymers have been used as templates for E . c o l i ENA polymerase I in an in v i t r o assay and the incorpora t ion of complementary and noncomplementary nuc leo t ides determined. With the CMS methylated template no wrong nuc leo t ide incorpora t ion was de t ec t ab l e , but with the MNU methylated polymer the incorpora t ion of dGMP was. observed. The amount of dGMP incorpora ted c o r r e l a t e d with the leve l of 0_ -methylthymine in the template over the range of methylat ion s tud ied . The r e s u l t s i n d i c a t e t h a t (5^methylthymine i e capable of miscoding on a one-to-one b a s i s while the products of DMS methylat ion ( 1 , 3 and 7-methyladenines), and a l so poss ib ly t he phospho t r i e s t e r s , do not lead t o any mis incorporat ion. INTBODUCTION A r o l e for a lky la t ed bases in both mutagenesis and carc inogenes is has been considered for many y e a r s . More r ecen t ly a t t e n t i o n has been focussed on the 0_ -a lkylguanines as poss ib le pro-mutagenic bases and evidence i s accumulating of some r e l a t i o n s h i p between a l k y l a t i o n of the ^ p o s i t i o n of guanine in DNA and t i s s u e s u s c e p t i b i l i t y to tumour induct ion ( r e f s 2 6 ) . A second O-alkylated base , 0_ -methylthymine, has now been detec ted in DNA following r e a c t i o n , both in v i t r o and in vivo , with carcinogenic methylat ing agents and i t has been proposed t h a t t h i s base , c 7 like £ -methylguanine, could also be pro-mutagenic . Whilst bacterial studies have revealed a predominance of GC->AT transitions after treatment with alkylating carcinogens presumably due to the point mutations arising from the 0_ -alkylguanine, a smaller but significant number of AT-XKJ transitions are also observed. These could be due to miscoding by an alkylated adenine or thymine base. Using the alternating copolymer poly(dA-dT) we have been 761 O Information Retrieval Limited 1 Falconberg Court London W1V5FG England Nucleic Acids Research i n v e s t i g a t i n g the p o s s i b i l i t y of miscoding by methylated adenine and thymine bases during DNA syn thes i s . The polymer has been methylated i n v i t r o with e i t h e r dimethyl sulphate (DHS) or the potent carcinogen N-methyl-l*-nitrosourea (MNU) and then used as template for highly pur i f i ed E . c o l i DNA polymeraee I , measuring the incorpora t ion of complementary and noncomplementary nuc leo t ides . HATERIALS AND METHODS Chemicals: Poly(dA-dT), deoxyribonucleoside 5' t r i phospha te s and E . co l i DNA polymerase I (grade I ) were purchased from the Boehringer Corporation (London) Ltd. Radioact ive poly(dA-dT) was prepared according to the method of Schachman et a l using the appropriate l abe l l ed t r iphospha te . Radioact ive monomers were obtained from the Radiochemical Centre, Amersham, England. MNU was prepared in these l a b o r a t o r i e s and was s tored at -20° . DMS was purchased from BDH Chemicals Ltd and r e d i s t i l l e d before u s e . Marker compounds were the g i f t of Dr P . J . O'Connor. p_ -methyl7 thymidine as marker was prepared by the method of Lawley et al . Methylation of Poly(dA-dT): Poly(dA-dT) was methylated at a concentration of l*00nmole nucleotide phosphorus per ml in O.25M sodium cacodylate buffer pH 7.0 and 0.5M NaCl. DMS (0 to 3ul/ml hourly for 3 hours) or MNU (0 to tomg/ml) was added, the solution agitated and the reaction allowed to proceed overnight. The methylated polymer was then extensively dialysed against 0.01M Tris-HCl pH 7.k and 0.02M NaCl at k°. All samples (including non-methylated controls) were handled in the same manner: the only parameter that was altered for differing samples was the methylating agent and the amount. AnalvsiB of Methylation Products: Adenine methylation was determined using [ C-adenine]-poly(dA-dT). The methylated polymer was lyophilised to dryness and hydrolysed with 0.3ml 72# perchloric acid for 1 hour at 100°. The acid hydrolysate was diluted to 10ml and applied to a Dowex-50 column (25 x 1.5cm) equilibrated with O.3M triethylammonium formate pH 6.65The adenine and methyladenine bases were eluted with a linear gradient (0.3 1.0M) triethylammonium formate pH 6.65 collecting 3ml fractions. Phosphate and thymine methylation was measured using [ H-thymineJpoly(dA-dT). The methylated polymer was degraded to the nucleosides (and phoephotriesters) by enzymic hydrolysis with 0.02 units snake venom phosphodiesterase and 3 units alkaline phosphatase in 0.1M Tris-HCl pH 8.9 and the products separated on Dowex-50, ammonium form, column