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

摘要

The DNA-[N-6-adenine]-methyltransferase (Dam MTase) of phage T4 catalyzes methyl group transfer from S-adenosyl-L-methionine (AdoMet) to the NG-position of adenine in the palindromic sequence, GATC, We have used a gel shift assay to monitor complex formation between T4 Dam and various synthetic duplex oligonucleotides, either native or modified/defective, The results are summarized as follows, (i) T4 Dam bound with similar to 100-fold higher affinity to a 20mer specific (GATC-containing) duplex containing the canonical palindromic methylation sequence, GATC, 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 complexes with T4 Dam, (iii) No stable complex was formed with a synthetic 12mer specific (GATC-containing) duplex, although T4 Dam can methylate it, This indicates that there is no relation between formation of a catalytically competent 12mer-Dam complex and one stable to gel electrophoresis. (iv) Formation of a stable complex did not require that both strands be contiguous or completely complementary, Absence of a single internucleotide phosphate strongly reduced complex formation only when missing between the T and C residues, This suggests that if T4 Dam makes critical contact(s) with a backbone phosphate(s), then the one between T and C is the only likely candidate, Having only one half of the recognition site intact on one strand was sufficient for stable complex formation provided that the 5' G.C base-pairs be present at both ends of the palindromic, GATC, Since absence of either a G or C abolished T4 Dam binding, we conclude that both strands are recognized by T4 Dam.