Mechanism interactions and function of the structure specific nuclease XPF

The archaea are a group of microbes, often found in extreme environments such as volcanic pools and salt pans. With the advent of DNA sequencing, it was recognised that the archaea are not closely related to bacteria such as the ones that make us sick, but rather are more similar to the eukarya (organisms with a nucleus), such as yeast, worms and humans. This relationship is reflected in similarities between the archaea and eukarya in the machinery that makes copies of the genetic material and transfers information (the information processing pathways). For this reason archaea have been studied as a useful model system, but they are also important and interesting in their own right, as they make up a big proportion of the living world. There is still a lot we don't know about information processing in the archaea. We have studied these pathways in archaea for the last 7 years and have discovered and characterised a lot of new proteins. We have been studying the archaeal nuclease XPF (a protein that cuts DNA strands) that is similar to a human protein important for repair of DNA damage. We know the structure of the protein and we wish to carry out studies of its structure, function, mechanism and interactions.

In eukaryotes, the nuclease XPF-ERCC1 is involved in several DNA repair pathways. We have cloned, expressed and purified the archaeal orthologue, SsoXPF from the hyperthermophilic archaeon Sulfolobus solfataricus. The domain organisation of XPF is unusual / with a dimeric nuclease domain linked to a dimeric DNA binding (HhH2) domain by flexible linkers, and a PCNA interaction motif at the C-terminus. We have carried out extensive single turnover kinetic studies of substrate preference of SsoXPF, which suggest a close functional relationship to the Mus81 enzyme. However the archaeal enzyme also has features in common with eukaryal XPF-ERCC1, and may represent the ancestral form of this protein family. We have demonstrated an interaction between SsoXPF and the sliding clamp PCNA that is highly unusual in that the nuclease has little or no activity in the absence of PCNA. This suggests the interaction with PCNA may allow a conformation change to activate the nuclease. PCNA also interacts with the helicase XPB in Sulfolobus. There is thus the possibility of a ternary complex between XPF, XPB and PCNA that may be relevant for DNA repair. Our collaborator Neil McDonald has solved the structure of the closely related Aeropyrum pernix XPF in complex with a DNA duplex. This structure suggests a mechanism for DNA binding, distortion and cleavage. We now wish to follow up the biochemical and structural data to investigate the molecular details of substrate recognition and catalysis by the XPF protein, and the nature of its interaction with PCNA. Our main objectives are: 1) Characterise the way in which XPF binds, distorts and cleaves DNA substrates at a molecular level using a variety of biochemical and biophysical techniques including enzyme kinetics, fluorescence spectroscopy and site directed mutagenesis. 2) Investigate the observed interaction with the cofactor PCNA in more detail, using enzyme kinetics, isothermal titration calorimetry and binding studies. 3) Make heterodimeric and chimeric XPF proteins to test the roles of the different domains and protein interaction motifs. 4) Look for evidence of a physical and functional interaction of XPF-PCNA-XPB helicase, and probe its role in vivo, using protein interaction studies, kinetics. We have all the necessary resources in place to achieve these objectives. We have demonstrable expertise in the main biochemical and molecular techniques required. We have a large volume of published and unpublished data to build on. All the equipment and infrastructure required for this work is available in the Centre for Biomolecular Sciences in St Andrews. We also have all of the materials required: cloned and purified archaeal proteins for DNA repair and transcription, DNA substrates and constructs, and antisera. In summary, this is an ambitious proposal with clear, achievable aims. We will build on our prior research to deliver important new insights to fundamental pathways in archaeal information processing. We expect to generate significant research findings that will be published in high impact journals.