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Crystallization of the DpnM methylase from the DpnII restriction system of Streptococcus pneumoniae
S Cerritelli1, S W White, S A Lacks
1Department of Biology, Brookhaven National Laboratory, Upton, NY 11973.
Abstract:
Three proteins, two DNA methylases and an endonuclease, from the DpnII restriction system of Streptococcus pneumoniae recognize the DNA sequence 5' GATC 3' but have very different amino acid sequences, which make them interesting subjects for structural determination. A purification procedure was developed that conveniently yields milligram amounts of the DpnM methylase. The DpnM protein tends to precipitate at reduced ionic strength, and this property was exploited to yield well-formed bipyramidal crystals. By X-ray diffraction, the crystals of DpnM were found to be orthorhombic, with cell dimensions a = 56.9 A, b = 68.2 A, c = 84.5 A; systematic absences identify the space group as P2(1)2(1)2(1). Diffraction extends beyond 3 A, so the crystals may allow structural determination at atomic resolution.
Insights
Researchers purified and crystallized the DpnM methylase from Streptococcus pneumoniae, a key protein in the DpnII restriction system. These crystals are suitable for atomic-resolution structural determination, advancing our understanding of DNA modification enzymes.
Area of Science:
- Molecular Biology
- Structural Biology
- Biochemistry
Background:
- The DpnII restriction system in Streptococcus pneumoniae involves three proteins: two DNA methylases and an endonuclease.
- These proteins recognize the specific DNA sequence 5' GATC 3' but exhibit distinct amino acid sequences.
- Understanding the structure of these proteins is crucial for elucidating their distinct functions.
Purpose of the Study:
- To develop a purification procedure for the DpnM methylase.
- To obtain well-formed crystals of DpnM for structural determination.
- To investigate the potential for atomic-resolution structural analysis of DpnM.
Main Methods:
- Development of a protein purification protocol yielding milligram quantities of DpnM.
- Exploitation of DpnM precipitation at reduced ionic strength for crystallization.
- X-ray diffraction analysis of DpnM crystals to determine their crystallographic properties.
Main Results:
- A convenient purification method for DpnM was established.
- Well-formed bipyramidal crystals of DpnM were successfully grown.
- X-ray diffraction revealed an orthorhombic crystal form (space group P2(1)2(1)2(1)) diffracting beyond 3 Å.
Conclusions:
- The developed purification and crystallization methods provide a viable path for structural studies of DpnM.
- The obtained crystals are suitable for high-resolution structural determination.
- This work facilitates the structural elucidation of a key DNA methylase involved in DNA restriction and modification.