. . "1"^^ . . . "0"^^ . "2010-12-31+01:00"^^ . . "Identifikace aminokyselinov\u00FDch zbytk\u016F podjednotek HsdS a HsdR nutn\u00FDch pro spr\u00E1vn\u00E9 sestaven\u00ED restrik\u010Dn\u011B-modifika\u010Dn\u00EDho enzymu Typu I EcoR124I" . . . . . "restriction-modification; protein-protein interaction; DNA translocation; mutagenesis"@en . "The objective of the project was the analysis of the effect of HsdS and HsdR mutant subunits on the  assembly and function of the complex restriction-modification enzyme EcoR124I. The HsdR motor subunit has amino-acid sequence motifs typical of the superfamily 2 (SF2) of DNA helicases and many endonucleases. We prepared a number of cleavage mutants in the PD-(E/D)xK endonuclease domain of"@en . . "9"^^ . "9"^^ . "We will focus on identification of amino acids residues of both the HsdR and HsdS subunits involved in subunit assembly of the Type I restriction-modification(R-M) enzyme EcoR124I. The HsdS subunit plays a key role in the function of Type I R-M enzymes being responsible for both the specific recognition of the target DNA and interaction with HsdM and HsdR subunits, while the HsdR subunit is representing the motor component of this enzyme being responsible for ATP-binding, ATP- hydrolysis and subsequentDNA translocation. Since the HsdS is the only odd subunit in the multisubunit complex, the opportunity exists to use HsdS assembly mutants for asymmetric assembly with HsdR motor subunit. We intend in the final step to find the optimal combination of cleavage and subunit assembly mutants introduced into single HsdR subunit with HsdS assembly mutants to produce stable R1-complex capable of unidirectional translocation. DNA translocation activity of the mutant enzymes will be investigated using the"@en . "GA204/07/0325" . . . . "2015-02-09+01:00"^^ . "C\u00EDlem  projektu byla anal\u00FDza mutantn\u00EDch podjednotek HsdS a HsdR ovliv\u0148uj\u00EDc\u00EDch sestaven\u00ED a funkci  komplexn\u00EDho enzymu EcoR124I. Motorov\u00E1 podjednotka HsdR obsahuje aminokyselinov\u00E9 sekvence charakteristick\u00E9 jak pro helikasy skupiny SF2, tak pro mnoh\u00E9 endonukleasy. P\u0159ipravili jsme \u0159adu mutac\u00ED v  motivu PD-(E/D)xK endonukleasov\u00E9 dom\u00E9ny neschopn\u00FDch \u0161t\u011Bpit DNA a zjistili jsme, \u017Ee ta"@cs . " DNA translocation" . . " protein-protein interaction" . "Zam\u011B\u0159\u00EDme se\u00A0 na identifikaci aminokyselinov\u00FDch zbytk\u016F podjednotek HsdR a HsdS nutn\u00FDch pro sestaven\u00ED restrik\u010Dn\u011B-modifika\u010Dn\u00EDho enzymu (R-M) Typu I EcoR124I. Kl\u00ED\u010Dov\u00E1 \u00FAloha podjednotky HsdS spo\u010D\u00EDv\u00E1 jednak ve specifick\u00E9m rozpozn\u00E1n\u00ED c\u00EDlov\u00E9 sekvence na DNA, jednak v interakci s podjednotkami HsdM a HsdR, zat\u00EDm co podjednotka HsdR p\u0159edstavuje vlastn\u00ED motor enzym\u016F Typu I, nebo\u0165 je zodpov\u011Bdn\u00E1 za vazbu ATP, jeho hydrol\u00FDzu a n\u00E1slednou translokaci. HsdS je jedin\u00E1 lich\u00E1 podjednotka v tomto komplexn\u00EDm enzymu, nab\u00EDz\u00ED se proto mo\u017Enost vyu\u017E\u00EDt mutanty HsdS se zm\u011Bn\u011Bnou vazbou do komplexu pro asymetrick\u00E9 sestaven\u00ED s motorovou podjednotkou HsdR. Chceme naj\u00EDt optim\u00E1ln\u00ED kombinaci mutac\u00ED eliminuj\u00EDc\u00EDch \u0161t\u011Bpen\u00ED DNA a mutac\u00ED ovliv\u0148uj\u00EDc\u00EDch sestavov\u00E1n\u00ED, vnesen\u00FDch sou\u010Dasn\u011B do podjednotky HsdR, a ve spojen\u00ED s\u00A0mutantn\u00ED podjednotkou HsdS \u00A0p\u0159ipravit stabiln\u00ED komplex R1 schopn\u00FD jednosm\u011Brn\u00E9 translokace. DNA transloka\u010Dn\u00ED aktivita enzymu bude zkoum\u00E1na pomoc\u00ED stopped-flow fluorimetru a magnetick\u00E9 pinzety. T\u00EDm se propoj\u00ED genetick\u00E9" . "http://www.isvav.cz/projectDetail.do?rowId=GA204/07/0325"^^ . "Identification of amino acid residues of the HsdS and HsdR subunits required for correct assembly of Type I restriction-modification enzyme EcoR124I"@en . . . . "2010-04-16+02:00"^^ . "2007-01-01+01:00"^^ . "restriction-modification" . . . "0"^^ .