Shape and Subunit Organisation of the DNA Methyltransferase M.AhdI by Small-angle Neutron Scattering

Type I restriction-modification (R-M) systems encode multisubunit/multidomain enzymes. Two genes (M and S) are required to form the methyltransferase (MTase) that methylates a specific base within the recognition sequence and protects DNA from cleavage by the endonuclease. The DNA methyltransferase...

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Bibliographic Details
Published in:Journal of molecular biology 2007-05, Vol.369 (1), p.177-185
Main Authors: Callow, P., Sukhodub, A., Taylor, James E.N., Kneale, G.G.
Format: Article
Language:English
Subjects:
Aeromonas hydrophila - enzymology
Amino Acid Sequence
contrast variation
Crystallography, X-Ray
DNA Modification Methylases - chemistry
Hydrogenation
Kinetics
Models, Molecular
Molecular Sequence Data
multi-subunit enzymes
Neutron Diffraction
perdeuteration
Protein Subunits - chemistry
restriction-modification
Scattering, Small Angle
Sequence Alignment
type I DNA methyltransferase
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Summary:Type I restriction-modification (R-M) systems encode multisubunit/multidomain enzymes. Two genes (M and S) are required to form the methyltransferase (MTase) that methylates a specific base within the recognition sequence and protects DNA from cleavage by the endonuclease. The DNA methyltransferase M.AhdI is a 170 kDa tetramer with the stoichiometry M2S2 and has properties typical of a type I MTase. The M.AhdI enzyme has been prepared with deuterated S subunits, to allow contrast variation using small-angle neutron scattering (SANS) methods. The SANS data were collected in a number of 1H:2H solvent contrasts to allow matching of one or other of the subunits in the multisubunit enzyme. The radius of gyration (Rg) and maximum dimensions (Dmax) of the M subunits in situ in the multisubunit enzyme (50 Å and 190 Å, respectively) are close of those of the entire MTase (51 Å and 190 Å). In contrast, the S subunits in situ have experimentally determined values of Rg=35 Å and Dmax=110 Å, indicating their more central location in the enzyme. Ab initio reconstruction methods yield a low-resolution structural model of the shape and subunit organization of M.AhdI, in which the Z-shaped structure of the S subunit dimer can be discerned. In contrast, the M subunits form a much more elongated and extended structure. The core of the MTase comprises the two S subunits and the globular regions of the two M subunits, with the extended portion of the M subunits most probably forming highly mobile regions at the outer extremities, which collapse around the DNA when the MTase binds.
ISSN:0022-2836
1089-8638
DOI:10.1016/j.jmb.2007.03.012