A directed evolution design of target specificity and kinetic analysis of conformational transitions in the HhaI methyltransferase ; DNR metiltransferazės HhaI atpažįstamos sekos inžinerija ir konformacinių virsmų kinetikos tyrimas

Ruta Svarinskaite

Sujets

• Biochemistry
• DNA methyltransferase
• Specificity

Informations

• Publié par: vilnius_university
• Publié le: 01 janvier 2011
• Nombre de lectures: 42
• Poids de l'ouvrage: 6 Mo

Extrait

        VILNIUS UNIVERSITY INSTITUTE OF BIOTECHNOLOGY             Rūta Gerasimaitė       A DIRECTED EVOLUTION DESIGN OF TARGET SPECIFICITY AND KINETIC ANALYSIS OF CONFORMATIONAL TRANSITIONS IN THE HhaI METHYLTRANSFERASE                   Summary of doctoral dissertation Physical science, biochemistry (04 P)                   Vilnius, 2011

This study has been carried out at the Institute of Biotechnology during 200332009. Dissertation is maintained by extern. Scientific consultant Prof. habil. dr.Saulius Klimašauskas (Institute of Biotechnology, Vilnius University; physical sciences, biochemistry – 04 P) Evaluation board of dissertation of Biochemistry trend Chairman: Prof. habil. dr.Vida Mildažienė(Vytautas Magnus University; physical sciences, biochemistry – 04 P) Members: Prof. dr.Vilmantė Borutaitė(Institute for Biomedical Research, Lithuanian University of Health Sciences; physical sciences, biochemistry – 04 P) Dr.Daumantas Matulis(Institute of Biotechnology, Vilnius University; physical sciences, biochemistry – 04 P) Dr.Rolandas Meškys (Institute of Biochemistry, Vilnius University; physical sciences, biochemistry – 04 P) Dr.Gintautas Tamulaitis (Institute of Biotechnology, Vilnius University; physical sciences, biochemistry – 04 P) Official opponents: Habil. dr.arimantas Čėnas (Institute of Biochemistry, Vilnius University; physical sciences, biochemistry – 04 P) Dr.Arūnas Lagunavičius(Thermo Fisher Scientific; physical sciences, biochemistry – 04 P) The thesis defence will take place at 11 a.m. on 2 nd  May, 2011, at the Institute of of Biotechnology. Address: V. A. Graičiūno 8, Vilnius LT302241, Lithuania The abstract of thesis was distributed on 2 nd of April, 2011. The thesis is available at the Library of Vilnius University and at the Library of Institute of Biotechnology.

 

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        VILNIAUS UNIVERSITETAS BIOTECHNOLOGIJOS INSTITUTAS             Rūta Gerasimaitė       DNR METILTRANSFERAZĖS HhaI ATPAŽĮSTAMOS SEKOS INŽINERIJA IR KONFORMACINIŲ VIRSMŲ KINETIKOS TYRIMAS                   Daktaro disertacijos santrauka Fiziniai mokslai, biochemija (04 P)                   Vilnius, 2011

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Disertacija rengta 2003–2009 metais Biotechnologijos Institute. Disertacija ginama eksternu. Mokslinis konsultantas: Prof. habil. dr.Saulius Klimašauskas(Biotechnologijos Institutas; fiziniai mokslai, biochemija – 04 P) Disertacija ginama Vilniaus universiteto Biochemijos mokslo krypties taryboje. Pirmininkė: Prof. habil. dr.Vida Mildažienė(Kauno Vytauto Didžiojo universitetas; fiziniai mokslai, biochemija 04 P) – Nariai: Prof. dr.Vilmantė Borutaitė(Lietuvos sveikatos mokslų universitetas, Biomedicininių tyrimų institutas; fiziniai mokslai, biochemija – 04 P) Dr.Daumantas Matulis(Vilniaus universitetas, Biotechnologijos institutas; fiziniai mokslai, biochemija – 04 P) Dr.Rolandas Meškys(Vilniaus universitetas, Biochemijos institutas; fiziniai mokslai, biochemija – 04 P) Dr.Gintautas Tamulaitis(Vilniaus universitetas, Biotechnologijos institutas; fiziniai mokslai, biochemija – 04 P) Oponentai: Habil. dr.arimantas Čėnas(Vilniaus universitetas, Biochemijos institutas; fiziniai mokslai, biochemija – 04 P) Dr.Arūnas Lagunavičius(Thermo Fisher Scientific; fiziniai mokslai, biochemija – 04 P) Disertacija bus ginama viešame Biochemijos mokslo krypties tarybos posėdyje 2011 m. gegužės mėn. 2 d. 11 val. Biotechnologijos instituto aktų salėje. Adresas: Graičiūno 8, Vilnius, LT–02241, Lietuva. Disertacijos santrauka išsiuntinėta 2011 m. balandžio mėn. 2 d. Disertaciją galima peržiūrėti Biotechnologijos instituto ir Vilniaus universiteto bibliotekose.

 

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COTETS Contents..........................................................................................................................................5  Introduction .................................................................................................................................... 6 Materials and methods.................................................................................................................10  Materials....................................................................................................................................... 10 Plasmids, strains and protein expression...................................................................................... 11 Molecular cloning........................................................................................................................12  Protein purification and characterization.....................................................................................12  Characteriztion of Methyltransferasesin vitro........... ..13................................................................ Enzymology methods ................................................................................................................... 15 Results and discussion..................................................................................................................17  1. Engineering MTases with new target specificity ..................................................................... 17 1.1. Sequence specificity of L2Bspin vivo................1. 9............................................................. 1.2. Strategy for designing catalytically efficient GCG3specific methyltransferase................ 20 1.3. Catalytic efficiency and sequence3specificity of selected MTases ................................... 22 1.4. Kinetic characterization of selected MTases..................................................................... 26 1.5. DNA transalkylation using synthetic AdoMet analogs..................................................... 27 1.6. Discussion.........................................................................................................................29  2. Kinetic analysis of conformational transitions during the M.HhaI3catalyzed reaction............ 32 2.1. Direct observation of cytosine flipping and covalent bond formation.............................. 32 2.2. Simultaneous sensing of the catalytic loop motion with built3in fluorophores ................ 35 2.3. Interaction with mismatched DNA substrates...................................................................40  2.4. Ternary complexes with AdoMet: the complete reaction cycle........................................ 42 2.5. Discussion.........................................................................................................................44  Conclusions .................................................................................................................................. 47 List of publications....................................................................................................................... 48 Acknowledgements ...................................................................................................................... 49 Curriculum vitae9 ...4....................................................................................................................... Reziumė........................................................................................................................................50  References .................................................................................................................................... 51    

 

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