INTERACTION OF THE PHAGE-T4 DAM DNA-[N-6-ADENINE] METHYLTRANSFERASE WITH OLIGONUCLEOTIDES CONTAINING NATIVE OR MODIFIED (DEFECTIVE) RECOGNITION SITES

The DNA-[N-6-adenine]-methyltransferase (Dam MTase) of phage T4 cataly zes methyl group transfer from S-adenosyl-L-methionine (AdoMet) to the NG-position of adenine in the palindromic sequence, GATC, We have use d a gel shift assay to monitor complex formation between T4 Dam and va rious synthetic duplex oligonucleotides, either native or modified/def ective, The results are summarized as follows, (i) T4 Dam bound with s imilar to 100-fold higher affinity to a 20mer specific (GATC-containin g) duplex containing the canonical palindromic methylation sequence, G ATC, than to a non-specific duplex containing another palindrome, GTAC , (ii) Compared with the unmethylated duplex, the hemimethylated 20mer specific duplex had a slightly increased (-2-fold) ability to form co mplexes with T4 Dam, (iii) No stable complex was formed with a synthet ic 12mer specific (GATC-containing) duplex, although T4 Dam can methyl ate it, This indicates that there is no relation between formation of a catalytically competent 12mer-Dam complex and one stable to gel elec trophoresis. (iv) Formation of a stable complex did not require that b oth strands be contiguous or completely complementary, Absence of a si ngle internucleotide phosphate strongly reduced complex formation only when missing between the T and C residues, This suggests that if T4 D am makes critical contact(s) with a backbone phosphate(s), then the on e between T and C is the only likely candidate, Having only one half o f the recognition site intact on one strand was sufficient for stable complex formation provided that the 5' G.C base-pairs be present at bo th ends of the palindromic, GATC, Since absence of either a G or C abo lished T4 Dam binding, we conclude that both strands are recognized by T4 Dam.