DNA, the carrier of genetic information, is very susceptible to damage. This is because of the inherent instability of the DNA itself and the presence of certain cellular metabolites such as activated oxygen which are important sources of lesions. In addition, damage to DNA can result from external sources, such as (man-made) chemicals, ionising radiation and ultra-violet light. UV-damage may arise from (over)-exposure to sunlight (sunbathing) resulting in an elevated susceptibility to skin cancer.
DNA damage is hence a direct threat to the integrity of the genetic information. Because of its ability to remove a wide variety of structurally different lesions, excision repair of DNA is by far the most important repair mechanism to be found in prokaryotes and eukaryotes. Even in a relatively simple organism as the gut bacterium Escherichia coli the excision repair process is quite complex and involves multi-subunit complexes. The core of the reaction, which involves damage recognition, formation of the pre-incision complex, and dual incision of the damaged DNA is performed by three factors, UvrA, UvrB and UvrC. The gap in the DNA duplex is then filled by UvrD, DNA Polymerase I with the nick being closed by DNA Ligase. To date the E. coli UvrABC endonuclease is the best biochemically characterised repair mechanism. There is an overall understanding of the mechanism of DNA excision repair, but on a molecular, truly mechanistic level, not much is known.
The main objective of this proposal is to understand the broad substrate specificity and the molecular mechanism of DNA excision repair. We are focusing specifically on the UvrABC endonuclease, because of the relative simplicity of the system; the understanding of the enzymology; the possibility of isolating specific reaction intermediates; and the availability of ultra-pure protein preparations in sufficient quantities to allow a biophysical approach. In the future we shall also be working on higher eukaryotic systems. We shall be using a multi-disciplinary approach to study the structure of the subunits and the reaction intermediates by engaging expertise in molecular genetics, biochemistry (Prof. Pieter van de Putte's group, Dept. of Molecular Genetics, University of Leiden, NL); X-ray crystallography by using both convential "in-house" X-ray crystal structure solution using Multiple Isomporphous Relacement MIR phasing (Dr. Mark Sanderson's group, The Randall Institute, King's College London, GB) and Multiple Anomalous Dispersion MAD phasing using multi-wave-length synchrotron radiation from the ESRF (Dr. Juan Fontecilla-Camps' group, Institut de Biologie Structurale Jean-Pierre Ebel, Grenoble, FR); nucleic acid organic chemistry to synthesise defined DNA lesions (Prof. Jacques van Boom's group, Dept. of Organic Chemistry, University of Leiden, The Netherlands) and computational chemistry (Prof. Modesto Orozco's group, Dept. of Chemistry, University of Barcelona, ES). This programme will also generate a variety of DNA substrates containing site-specific lesions, so that the structures of these DNAs may be studied on their own by X-ray diffraction (Dr. Mark Sanderson's group, King's College London) and by Nuclear Magnetic Resonance spectroscopy (Prof. Jacques van Boom's group and Dr. Mark Sanderson's group). These studies will help to assess the structural changes induced by protein binding and will provide more information on the way a lesion is recognized. Computational studies will provide a full understanding of the energetics, electrostatics and dynamics of the relevant protein-DNA interactions. This programme should make a significant contribution to the understanding of DNA excision repair.
Funding SchemeCSC - Cost-sharing contracts
2300 RA Leiden