Interaction of the phage T4 Dam DNA-[N6-adenine] methyltransferase with oligonucleotides containing native or modified (defective) recognition sites

作者：Ernst G. Malygin, N A Petrov, Yu. A. Gorbunov, Valeri G. Kossykh, Stanley Hattman · 发表于：Nucleic Acids Research · 年份：1997 · DOI：10.1093/nar/25.21.4393 · 被引用次数：27 · 研究领域：Epigenetics and DNA Methylation、RNA modifications and cancer、DNA and Nucleic Acid Chemistry

The DNA-[N 6-adenine]-methyltransferase (Dam MTase) of phage T4 catalyzes methyl group transfer from S-adenosyl-l-methionine (AdoMet) to the N6-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 approximately 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 ( approximately 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 recogni...