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Phage T4 DNA [N6-adenine] methyltransferase: kinetic studies using oligonucleotides containing native or modified
V V Zinoviev1, A A Evdokimov, Y A Gorbunov
1Institute of Molecular Biology, State Research Center Vector, Novosibirsk, Russia.
Abstract:
The DNA-[N6-adenine] methyltransferase of T4 phage (T4 Dam MTase) catalyzes methyl group transfer from S-adenosyl-L-methionine (AdoMet) to the N6-position of adenine in the palindromic sequence, GATC. We have investigated the effect of eliminating different structural components of the recognition site on the ability of a substrate to be bound and methylated by T4 Dam. For this purpose, steady state binding (by gel shift assays) and kinetic parameters of methylation (using the methyl donor, [3H-CH3]-AdoMet, at 25 degrees C) were studied using various synthetic duplex oligonucleotides containing some defect in the DNA-target site; e.g., the absence of an internucleotide phosphate or a nucleotide(s) within the recognition site, or a single stranded region. The salient results are summarized as follows: (1) Addition of T4 Dam to a complete reaction mixture (with a 20-mer duplex as substrate) resulted in a 'burst' of 3H-methylated product, followed by a constant rate of product formation that reflected establishment of steady-state conditions. This suggests that the rate-limiting step is release of product methylated DNA from the enzyme [and not the transfer of the methyl group]. (2) A number of the defects in duplex structure had only a weak influence on the binding and Km values, but strongly reduced the kcat. At the same time, several poorly bound duplexes retained good substrate characteristics, especially duplexes having uninterrupted GAT-sequences in both strands. Whereas having only one half of the recognition site element intact was sufficient for stable complex formation, the catalytic turnover process had a strict requirement for an uninterrupted GAT-sequence on both strands. (3) There was no correlation between Km and binding capability; the apparent Kd for some duplexes was 5-70 times higher than Km. This indicates that the T4 Dam methylation reaction can not be explained by a simple Michaelian scheme.
Insights
T4 Dam methyltransferase requires an intact GATC sequence on both DNA strands for efficient methylation. Structural defects weaken binding but severely impair catalytic turnover, indicating product release is rate-limiting.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- DNA methyltransferases play crucial roles in DNA replication and repair.
- T4 Dam methyltransferase specifically recognizes and methylates the GATC sequence.
Purpose of the Study:
- To investigate how structural alterations in the GATC recognition site affect T4 Dam methyltransferase binding and activity.
- To elucidate the rate-limiting step in the T4 Dam methylation reaction.
Main Methods:
- Gel shift assays were used to study DNA binding kinetics.
- Methylation kinetics were measured using radiolabeled S-adenosyl-L-methionine ([3H-CH3]-AdoMet).
- Synthetic oligonucleotides with various structural defects in the GATC site were employed as substrates.
Main Results:
- Product release, not methyl group transfer, is the rate-limiting step in T4 Dam methylation.
- While partial GATC sequence integrity supports binding, both DNA strands require an uninterrupted GATC sequence for efficient catalytic turnover.
- Binding affinity (Kd) and Michaelis constant (Km) showed no direct correlation, deviating from a simple Michaelian model.
Conclusions:
- The catalytic step of T4 Dam methylation is highly sensitive to the integrity of the entire GATC recognition site on both DNA strands.
- The enzyme-DNA interaction is complex, involving more than simple substrate binding and catalysis.