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Interaction of the phage T4 Dam DNA-[N6-adenine] methyltransferase with oligonucleotides containing native or
E G Malygin1, N A Petrov, Y A Gorbunov
1Institute of Molecular Biology, State Research Center of Virology, and Biotechnology 'Vector', Koltsovo, Novosibirsk Region 633159, Russia and Department of Biology, University of Rochester, Rochester, NY 14627-0211, USA.
Abstract:
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 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.
Insights
Phage T4 DNA-[N6-adenine]-methyltransferase (Dam MTase) binds specifically to GATC sequences. Stable complex formation requires intact GATC sites and critical phosphate contacts, indicating recognition of both DNA strands.
Area of Science:
- Molecular Biology
- Enzymology
- Structural Biology
Background:
- DNA methyltransferases (MTases) are crucial enzymes in DNA metabolism.
- Phage T4 Dam MTase methylates the N6-position of adenine within the GATC sequence.
- Understanding enzyme-DNA interactions is key to elucidating biological functions.
Purpose of the Study:
- To investigate the binding interactions of T4 Dam MTase with various DNA substrates.
- To identify the specific DNA features required for stable T4 Dam MTase complex formation.
- To determine the role of DNA sequence, methylation status, and backbone integrity in binding.
Main Methods:
- Gel shift assay to monitor DNA-protein complex formation.
- Utilized synthetic duplex oligonucleotides with native, modified, or defective sequences.
- Systematically altered DNA structure to assess binding requirements.
Main Results:
- T4 Dam MTase exhibited 100-fold higher affinity for specific GATC duplexes compared to non-specific GTAC duplexes.
- Hemimethylated GATC duplexes showed a modest 2-fold increase in complex formation.
- Stable complex formation was independent of full complementarity but sensitive to specific phosphate deletions, particularly between T and C.
- Binding required the presence of both G and C bases, indicating recognition of both DNA strands.
Conclusions:
- Phage T4 Dam MTase displays high specificity for the GATC recognition site.
- Specific backbone phosphate interactions, especially between T and C, are critical for stable complex formation.
- The enzyme recognizes and interacts with both strands of the DNA duplex.