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Characterization of the interaction between the restriction endonuclease McrBC from E. coli and its cofactor GTP
U Pieper1, T Brinkmann, T Krüger
1Institut für Biochemie (FB 15), Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 58, Giessen, D-35392, Germany.
Abstract:
McrBC, a GTP-dependent restriction enzyme from E. coli K-12, cleaves DNA containing methylated cytosine residues 40 to 80 residues apart and 3'-adjacent to a purine residue (PumCN40-80PumC). The presence of the three consensus sequences characteristic for guanine nucleotide binding proteins in one of the two subunits of McrBC suggests that this subunit is responsible for GTP binding and hydrolysis. We show here that (i) McrB binds GTP with an affinity of 10(6) M-1 and that GTP binding stabilizes McrB against thermal denaturation. (ii) McrB binds GDP about 50-fold and ATP at least three orders of magnitude more weakly than GTP. (iii) McrB hydrolyzes GTP in the presence of Mg2+ with a steady-state rate of approximately 0.5 min-1. (iv) McrC stimulates GTP hydrolysis 30-fold, but substrate DNA has no detectable effect on the GTPase activity of McrB, neither by itself nor in the presence of McrC. (v) Substitution of N339 and N376 with alanine allowed us to identify NTAD (339 to 342) rather than NKKA (376 to 379) as the equivalent of the third consensus sequence motif characteristic for guanine nucleotide binding proteins, NKXD.
Insights
The restriction enzyme McrBC utilizes GTP binding and hydrolysis for DNA cleavage. McrB binds GTP strongly, while McrC enhances GTP hydrolysis, crucial for its enzymatic activity.
Area of Science:
- Molecular Biology
- Enzymology
- Bacterial Genetics
Background:
- McrBC is a GTP-dependent restriction enzyme in E. coli K-12.
- It cleaves methylated DNA at specific sites (PumCN40-80PumC).
- Consensus sequences suggest GTP-binding capability in McrBC subunits.
Purpose of the Study:
- To investigate the GTP-binding and hydrolysis activities of McrBC subunits.
- To identify the specific subunit responsible for nucleotide binding and hydrolysis.
- To characterize the role of McrC in McrBC's enzymatic function.
Main Methods:
- GTP binding affinity measurements using purified McrB.
- Thermal denaturation assays to assess protein stability upon GTP binding.
- GTP hydrolysis rate assays in the presence of Mg2+.
- Enzyme kinetics studies with and without McrC and substrate DNA.
- Site-directed mutagenesis to identify key residues in nucleotide binding motifs.
Main Results:
- McrB exhibits high affinity for GTP (10^6 M-1), stabilizing it against denaturation.
- GTP binding is significantly stronger than GDP or ATP binding.
- McrB hydrolyzes GTP at a rate of ~0.5 min-1, enhanced 30-fold by McrC.
- Substrate DNA does not affect GTPase activity.
- Mutagenesis identified NTAD as the critical motif for guanine nucleotide binding.
Conclusions:
- McrB is the subunit responsible for GTP binding and hydrolysis in McrBC.
- McrC acts as a positive regulator, stimulating the GTPase activity of McrB.
- The NTAD motif is essential for the guanine nucleotide-binding function of McrBC.