Cofactor induced dissociation of the multifunctional multisubunit EcoR124I investigated using electromobility shift assays, AFM and SPR

0477805 - MBÚ 2018 RIV GB eng J - Článek v odborném periodiku
Youell, J. - Sikora, A.E. - Vejsadová, Štěpánka - Weiserová, Marie - Smith, J.R. - Firman, K.
Cofactor induced dissociation of the multifunctional multisubunit EcoR124I investigated using electromobility shift assays, AFM and SPR.
RSC Advances. Roč. 7, č. 61 (2017), s. 38737-38746. E-ISSN 2046-2069
Grant CEP: GA ČR GA204/07/0325
Institucionální podpora: RVO:61388971
Klíčová slova: ATOMIC-FORCE MICROSCOPY * RESTRICTION-MODIFICATION ENZYMES * I DNA RESTRICTION
Obor OECD: Microbiology
Impakt faktor: 2.936, rok: 2017

We have applied three techniques to the study of subunit assembly of the Type IC Restriction-Modification enzyme EcoR124I. This fully functional enzyme EcoR124I consists of a complex of the three subunits HsdR, HsdM and HsdS in a R2M2S1 stoichiometry, but is known to dissociate readily, releasing free HsdR and producing first an R-1-complex and then the core, DNA-binding methyltransferase (M2S1) complex. Analysis of the assembly pathway of this enzyme has previously employed gel retardation and Surface Plasmon Resonance (SPR), but the studies to date have not included the cofactors required for full enzyme activity. In this paper, we have also used atomic force microscopy (AFM)-based molecular volume measurements, and have analysed the effect of the cofactors ATP and AdoMet on enzyme stability and subunit assembly. We compare the data obtained from all three techniques and we show that they all give consistent results, but inherent differences in the methodologies provide additional information useful for the study of subunit assembly.
Trvalý link: http://hdl.handle.net/11104/0274032