Structural and functional studies of the restriction endonuclease BpuJI ; Restrikcijos endonukleazės BpuJI struktūriniai ir ir funkciniai tyrimai

Sujets

• Biochemistry
• Crystal structure
• Restriction enzyme

Informations

• Publié par: vilnius_university
• Publié le: 01 janvier 2009
• Nombre de lectures: 57
• Poids de l'ouvrage: 21 Mo

Extrait

VILNIUS UNIVERSITY
INSTITUTE OF BIOTECHNOLOGY


Rasa Sukackaitė
STRUCTURAL AND FUNCTIONAL STUDIES OF THE
RESTRICTION ENDONUCLEASE BpuJI
Doctoral dissertation
Physical sciences, biochemistry (04 P), proteins, enzymology (P310)

Vilnius, 2009The work presented in this doctoral thesis was carried out Insat titutethe of
Biotechnology from 2004 to 2009.
Supervisor:
prof .dr. Virginijus Šikšnys (Institute of Biotechnology, physical scie nces,
biochemistry-04 P, proteins, enzymology-P310)
Scientific consultant:
dr. Saulius Gražulis (Institute of Biotechnology, physical sciences,
biochemistry-04 P, proteins, enzymology-P310)
2VILNIAUS UNIVERSITETAS
BIOTECHNOLOGIJOS INSTITUTAS


Rasa Sukackaitė
RESTRIKCIJOS ENDONUKLEAZĖS BpuJI
STRUKTŪRINIAI IR FUNKCINIAI TYRIMAI
Daktaro disertacija
Fiziniai mokslai, biochemija (04 P), baltymai, enzimologija (P310)

Vilnius, 2009
3Disertacija parengta Biotechnologijos institute 2004-2009 metais.
Mokslinis vadovas:
prof .dr. Virginijus Šikšnys (Biotechnologijos instituta fsiz,inia i moksla i,
biochemija-04 P, baltymai, enzimologija-P310)
Mokslinis konsultantas:
dr. Saulius Gražulis(Biote chnologijos institutas, fiziniai mok slai,
biochemija-04 P, baltymai, enzimologija-P310)
4Table of Contents
List of abbreviations ................................................................................. 7 ...................
Introduction............................................................................................................ 8 ......
1. Literature overview ..................................................................... 12 ..........................
1.1 The PD-(D/E)XK domai............................................................n 12 .....................
1.1.1 The common structural core 12 ..........
1.1.2 The catalytic.................................................................................. motif 13 ...
1.1.3 The mechanism of DNA cleava ........................................................ge 15 ....
1.2 The helix-turn-helix doma.................................................in 16 ............................
1.2.1 The tri-helical core 16 ...........................
1.2.2 DNA binding by HTH domains ...................................... 17 ..........................
1.3 Structure-function relationships-( iD/n PE)XD K nuclease........s ...18..................
1.3.1 λ exonucle...........................................................................................ase 18 ..
1.3.2 Type II restriction endonuc ....................................................leases 20 ..........
1.3.2.1 Single-domain REases 21 ...............
1.3.2.2 REases with an extra structural........................................... domain 26 ..
1.3.2.3 REases with an effector dom..................................ain 27 ......................
1.3.2.4 REases with separate domains for recognition and c.a. t.al.y.29s.is......
1.3.2.5 REases with two catalytic dom..........................ains 32 .........................
1.3.2.6 REases with methyltransferase.............................. activity 33 ...............
1.3.3 Mismatch repair endonucle.............................................................ases 35 ....
1.3.4 Other sequence-specific nuc.......................................leases 38 .....................
1.3.5 Structure-specific nuc..............................................leases 40 ........................
1.3.5.1 Holliday junction res......................................................olvases 41 ........
1.3.5.2 The XPF/Mus81 family enzy....................................mes 44 ..................
1.3.6 NTP-driven molecular ma.....................................................chines 48 ..........
1.3.6.1 Type I R .....................................................................Eases 48 ...............
1.3.6.2 Type III R ........................................................Eases 51 .........................
1.3.6.3 Mc......................................................................rBC 52 .........................
1.3.6.4 Rec.................................................................................BCD 53 ............
2 Materials and Methods ..................................................................................... 58 ......
2.1 Plasmids and bacterial s..................................................trains 58 .........................
2.2 Oligonucleotides and radiolabe.....................................lling 58 ............................
2.3 Chromatography.......................................................................................... 59 .....
2.4 Expression and purification of BpuJ...........................................................I 59 .....
2.5 Limited proteoly...........................................................................................sis 60 .
2.5.1 Preparation of t - ahend C N -terminal proteolytic fragm..............ents 61 .......
2.5.2 Preparation of the N-domain/DNA compl..........................................ex 61 ...
2.6 Bioinformatics analysis of the BpuJI seque....................nce 62 ............................
2.7 Crystallization and diffraction data col...............................lection 62 ...................
2.7.1 Crystallization of B..........................................................................puJI 63 ...
2.7.2 Crystallization of the BpuJ-domI N ain/DNA complex ................ 63 .............
2.7.3 Crystallization of the B-puJdom I Cain ...............................................64 .......
2.7.4 Data collec...................................................................................tion 64 .......
52.8 Structure determination and anal..........................................ysis 65 ......................
2.9 Cloning and mutagenes.........................................................is 66 .........................
2.9.1 Site-specific mutagenesis of the active sit......................e residues 67 ...........
2.9.2 Cloning of the BpuJI N domain .................................. 67 ..............................
2.9.3 Mutagenesis of the specifity-determining re..................sidues 67 .................
2.9.4 Expression and purification of mutant prot .........................................eins 68
2.10 DNA cleavage ass..........................................................................ays 69 ............
2.10.1 Plasmid and phage DNA cleavage................................................. assay 69
2.10.2 PCR fragment cleavage...................................... assay 70 ...........................
2.10.3 Oligoduplex cleavage assay 71 ...........................
2.11 Gel mobility shift assay for DNA bindi...................................ng 71 ..................
2.12 Analytical gel-fil........................................................................tration 72 ...........
2.13 Analytical ultracentrifuga................................................tion 72 .........................
3 Results and discussion ...................................................................... 74 ......................
3.1 Functional characterization of B..........................................puJI 74 ......................
3.1.1 Variability of the BpuJI cleavage............................... position 75 .................
3.1.2 BpuJI needs two recognition sites for its optima.l. a.ct.ivi..ty... 77...............
3.1.3 The 3’-end-directed nucleolytic....................... activity 78 ...........................
3.1.4 BpuJI binds two DNA copies simultaneous...................................ly 80 .......
3.2 Structural organization of B .....................................................puJI 82 ..................
3.2.1 BpuJI is a dimer in sol.................................................................ution 82 ......
3.2.2 The modular architecture of BpuJI 82 .......
3.2.3 The C-domain possesses the 3’-end-directed nucleolytic act.i.vi.ty..85.......
3.2.4 The- Cdomain has a PD-(D/E)XK fold .......................................87 .............
3.2.5 The- Ndomain binds a single DNA copy as a monomer ....................89 .......
3.2.6 Crystallization of BpuJI and its proteolytic....................... fragments 90 .......
3.2.7 The crystal structure of t-domheai Nn/DNA complex ...........91...................
3.2.8 DNA recognition by the BpuJI N -domain .................. 93 ..............................
3.2.9 Mutational analysis of the DNA binding interfa....................................ce 96
3.3 A model of BpuJI action and its similarity to other proteins 98..................
3.3.1 A model of BpuJI ac............................................tion 98 ..............................
3.3.2 The place for BpuJI in the classification of restriction............ enzym100es .
3.3.3 Domains, similar to BpuJ-Idom Cain, are present in diverse seque nce
contexts...................................................................................................... 102 ......
3.3.4 Structural similarity of the- domBpuaJIin Nto other DNA binding
domains ..............................................................................105 ..............................
3.3.5 Comparison of BpuJI, BspD6I and FokI ......................................108 ..........
3.3.6 A model of the Nt.BspD6I/DNA com.................................plex 110 ......