Secondary structure of SsoII-like (Cytosine-5)-DNA methyltransferases N-terminal region determined by Circular dichroism spectroscopy

Ryazanova, A. Yu.; Molochkov, Nikolai V.; Abrosimova, L. A.; Alexeevsky, A. V.; Karyagina, A. S.; Protsenko, A. S.; Friedhoff, Peter; Oretskaya, Tatiana S.; Кубарева, Е. А.

doi:10.1134/s0026893310050183

Cited by 7 publications

(15 citation statements)

References 26 publications

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“…Sequence analysis of M.SsoII suggests that only a minor part of the N-terminal region is disordered while the major part (residues 1–55) represents a domain with a pronounced spatial structure. This assumption has been confirmed by circular dichroism spectroscopy combined with gel-shift assay [51]. A deletion mutant representing the N-terminal region of M.Ecl18kI (differs from M.SsoII only by a single residue, Ile56Met) demonstrates a pronounced secondary structure and also retains the ability to bind specifically to the regulatory site, although with a lower affinity.…”

Section: Discussionmentioning

confidence: 75%

Flexibility of the Linker between the Domains of DNA Methyltransferase SsoII Revealed by Small-Angle X-Ray Scattering: Implications for Transcription Regulation in SsoII Restriction–Modification System

et al. 2014

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(Cytosine-5)-DNA methyltransferase SsoII (M.SsoII) consists of a methyltransferase domain (residues 72–379) and an N-terminal region (residues 1–71) which regulates transcription in SsoII restriction–modification system. Small-angle X-ray scattering (SAXS) is employed here to study the low resolution structure of M.SsoII and its complex with DNA containing the methylation site. The shapes reconstructed ab initio from the SAXS data reveal two distinct protein domains of unequal size. The larger domain matches the crystallographic structure of a homologous DNA methyltransferase HhaI (M.HhaI), and the cleft in this domain is occupied by DNA in the model of the complex reconstructed from the SAXS data. This larger domain can thus be identified as the methyltransferase domain whereas the other domain represents the N-terminal region. Homology modeling of the M.SsoII structure is performed by using the model of M.HhaI for the methyltransferase domain and representing the N-terminal region either as a flexible chain of dummy residues or as a rigid structure of a homologous protein (phage 434 repressor) connected to the methyltransferase domain by a short flexible linker. Both models are compatible with the SAXS data and demonstrate high mobility of the N-terminal region. The linker flexibility might play an important role in the function of M.SsoII as a transcription factor.

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Section: Discussionmentioning

confidence: 75%

Flexibility of the Linker between the Domains of DNA Methyltransferase SsoII Revealed by Small-Angle X-Ray Scattering: Implications for Transcription Regulation in SsoII Restriction–Modification System

et al. 2014

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“…Electrophoretic mobility of Nt.BspD6I DNA duplex I complexes in nondenaturing gels containing different amounts of polyacrylamide against the marker proteins was determined to estimate the sto ichiometry of these complexes [20]. The molecular weights of the dominant C1 and C2 complexes were determined (Figs.…”

Section: Resultsmentioning

confidence: 99%

“…Then the slopes of the curves were used. The dependence of the binary logarithm of the opposite sign slope on the common logarithm of molecular weight for each marker protein was plotted [20]. The resulting curve was fitted to a straight line.…”

Section: Resultsmentioning

confidence: 99%

“…The electrophoretic mobility of the marker proteins and protein-DNA complexes in gels containing different amounts of polyacrylamide with respect to the leading dye was determined to estimate the molecular weight of Nt.BspD6I-DNA complexes. The gels was calibrated using the protein markers, as described in [20]. The molecular weights of oligomeric structures of Nt.BspD6I were determined in a similar way, but the sample buffer contained 0.01% Coomassie G 250 solution in 10 mM Tris HCl buffer (pH 7.8), 150 mM KCl, 10 mM CaCl 2 .…”

Section: Enzymes and Proteinsmentioning

confidence: 99%

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Oligomerization of site-specific nicking endonuclease BspD6I at high protein concentrations

et al. 2012

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Ability of site-specific nickase BspD6I (Nt.BspD6I) to oligomerize at concentrations > or = 0.5 microM (> or = 0.035 mg/mL) is studied. Three states of Nt.BspD6I are registered via electrophoretic studies both in the presence and in the absence of DNA. Estimation of their molecular mass allows assigning them as a monomer, a dimer and a trimer. Both dimeric and monomeric Nt.BspD6I are shown to hydrolyze its DNA substrate with the identical specificity. Calculation of the electrostatic potential distribution on the Nt.BspD6I globule surface shows that the protein molecule is a dipole. The Nt. BspD6I oligomeric forms are likely to be the result of ionic protein interactions.

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“…All the specific DNA–protein contacts are located at the regulatory site [ 33 ]. Moreover, two M.SsoII molecules bind to one regulatory site [ 34 ], and the resulting complex has quite unusual structure: the N-terminal domains are bound to the regulatory site, while the C-terminal domains are bound to DNA flanking the regulatory site [ 35 ]. Because the catalytic centre of the MTase domain in such a complex is occupied by non-specific DNA, M.SsoII should lose the ability to bind to the methylation site and to perform DNA methylation.…”

Section: Introductionmentioning

confidence: 99%

Kinetic Basis of the Bifunctionality of SsoII DNA Methyltransferase

et al. 2018

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Type II restriction–modification (RM) systems are the most widespread bacterial antiviral defence mechanisms. DNA methyltransferase SsoII (M.SsoII) from a Type II RM system SsoII regulates transcription in its own RM system in addition to the methylation function. DNA with a so-called regulatory site inhibits the M.SsoII methylation activity. Using circular permutation assay, we show that M.SsoII monomer induces DNA bending of 31° at the methylation site and 46° at the regulatory site. In the M.SsoII dimer bound to the regulatory site, both protein subunits make equal contributions to the DNA bending, and both angles are in the same plane. Fluorescence of TAMRA, 2-aminopurine, and Trp was used to monitor conformational dynamics of DNA and M.SsoII under pre-steady-state conditions by stopped-flow technique. Kinetic data indicate that M.SsoII prefers the regulatory site to the methylation site at the step of initial protein–DNA complex formation. Nevertheless, in the presence of S-adenosyl-l-methionine, the induced fit is accelerated in the M.SsoII complex with the methylation site, ensuring efficient formation of the catalytically competent complex. The presence of S-adenosyl-l-methionine and large amount of the methylation sites promote efficient DNA methylation by M.SsoII despite the inhibitory effect of the regulatory site.

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Secondary structure of SsoII-like (Cytosine-5)-DNA methyltransferases N-terminal region determined by Circular dichroism spectroscopy

Cited by 7 publications

References 26 publications

Flexibility of the Linker between the Domains of DNA Methyltransferase SsoII Revealed by Small-Angle X-Ray Scattering: Implications for Transcription Regulation in SsoII Restriction–Modification System

Flexibility of the Linker between the Domains of DNA Methyltransferase SsoII Revealed by Small-Angle X-Ray Scattering: Implications for Transcription Regulation in SsoII Restriction–Modification System

Oligomerization of site-specific nicking endonuclease BspD6I at high protein concentrations

Kinetic Basis of the Bifunctionality of SsoII DNA Methyltransferase

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