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Objectives and Priorities
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Scheme of Objectives
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The “DNA enzymes” project encompasses both structural and dynamic aspects. This is illustrated in the scheme above.
In order to pursue the project an interdisciplinary and intersectorial network was set up, comprising partners from seven universities, three research institutions and two SMEs, from six different countries (Denmark, Germany, Hungary, Lithuania, Poland, UK), to achieve the following scientific goals:
• to understand the various mechanisms by which proteins or protein complexes locate their target sites on DNA
• to understand how communication is achieved between recognition sites and sites of action, when these are separated by up to thousands of base pairs, and how NTP hydrolysis is used for this purpose
• to understand how the catalytic event is triggered
The nature of these scientific goals is such that the investigative approach to be taken must necessarily be a multidisciplinary one, involving molecular biology, enzymology, bioorganic and biophysical chemistry, as well as computational and structural biology. The range of expertise required cannot be found in academic institutions alone, but rather requires input from specialized research institutions and SMEs with particular expertise in bioinformatics, biotechnology and physico-chemical technology.
List of Deliverables:
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Delivarable No[1] |
Deliverable title |
Del. date[2] |
Nature[3] |
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D1.1 |
Bioinformatic analysis of restriction enzymes and repair proteins |
April 2006 |
R |
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D1.2 |
Single cysteine variants of restriction enzymes (e.g. EcoRV, PvuII) and repair proteins (e.g. MutH, L & S) |
November 2006 |
M |
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D1.3 |
Fluorescently labelled or tagged polymerases (e.g. T7 RNA polymerase) restriction enzymes (e.g. (e.g. EcoRV, PvuII and repair proteins (e.g. MutH, L & S) |
April 2007 |
M |
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D2.1 |
Unmodified and modified oligonucleotides, PCR fragments, plasmid DNAs, bacteriophage DNAs |
April 2006 |
M |
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D3.1 |
Steady-state parameters for cleavage of various DNA substrates by restriction enzymes, repair proteins and their variants (e.g. BsoBI, VSR, MutH) |
April 2007 |
R |
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D3.2 |
Measurement of ATP-consumption by ATP-dependent restriction (e.g. EcoKI, EcoAI and EcoR124I) and repair proteins (MutL & S) |
April 2007 |
R |
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D3.3 |
Comparison of the efficiencies of facilitated diffusion by various restriction enzymes and repair proteins |
April 2007 |
R |
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D3.4 |
Cross-linking of restriction enzyme - DNA complexes and identification of cross-linking sites (e.g. EcoRII, NlaIV) |
November 2007 |
R |
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D3.5 |
Cross-linking of individual components of the MutHLS sytem and identification of cross-linking sites |
November 2007 |
R |
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D3.6 |
Crystallization of restriction enzyme and restriction enzyme - DNA complexes (e.g. EcoRII, SsoII, PspGI) |
November 2008 |
R |
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D4.1 |
Presteady-state kinetic data for the interaction of restriction enzymes, repair proteins and polymerases with DNA |
November 2008 |
R |
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D4.2 |
Hydrodynamic analysis of restriction enzymes (e.g. MspI)and repair proteins (e.g. MutH) in the absence and presence of DNA |
November 2008 |
D |
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D5.1 |
Set-up for multiparameter single molecule fluorescence spectroscopy experiments |
November 2006 |
P |
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D5.2 |
Set-up for optical tweezer and fluorescence microscopy experiments |
November 2006 |
P |
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D5.3 |
Electron and atomic force microscopy of complexes of DNA with restriction enzymes and repair proteins (e.g. MutHLS system) |
November 2008 |
R |
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D5.4 |
Interaction of polymerases, restriction enzymes and repair proteins with DNA measured on the single molecule level |
November 2009 |
R |
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D6.1 |
Modelling of restriction enzyme – DNA complexes (e.g EcoRII). |
April 2007 |
R |
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D6.2 |
Modelling of MutHLS and MutHLS – DNA complexes |
November 2007 |
R |
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D6.3 |
Comparative structure analysis |
November 2008 |
R |
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D6.4 |
Computer simulation of the action of DNA enzymes |
November 2009 |
R |
[1] Deliverable numbers: D.t.1-D.t.n (t is the task number given on the previous page)
[2] Month in which the deliverables will be available. Month 0 marking the start of the project, and all delivery dates being relative to this start date.
[3] The nature of the deliverable is based on one of the following codes: R = Report, M = Material, P = Protocol
23.04.2008 17:57
