The processes of DNA replication, recombination and repair are performed by multi-protein assemblies in a sequence of biochemical steps that are increasingly viewed as integrated events sharing many common features in cellular life. In recent years, considerable effort has been made in developing techniques to investigate the structural mechanism that regulate DNA processing and how they are interconnected are molecular level. From these studies evidences are mounting in favour of a dynamic assembly of the DNA processing partners at the precise time (i.e., in response to damaged DNA and against the existence of pre-formed holoenzyme complexes present inside the cell until they are needed. Integrated in the core of this working model is the concept of transient or weak interactions between partners. This concept of transient interaction has boosted the development of novel biophysical techniques based on single-molecule detection because of their unique ability to provide information about the "history" of a single-assembly on a temporal coordinate, and therefore, the sequence of molecular events associated to a particular DNA-processing pathway. Among these, the Nucleotide Excision Repair mechanism (NER) is a highly conserved and versatile mechanism responsible for the repair of a wide range of helix-distorting DNA lesions.

Organization and function of structure-specific endonucleases XPF and FEN1 involved in DNA repair mechanisms. We are characterising the organization and function at molecular level of the endonucleases XPF and FEN1. The formation of the active complex involves the ordered assembly of several proteins and their interaction with the DNA helix, and undoubtedly requires complex intermediate structures. Our aim is to answer fundamental question such as: a) how do structure-specific DNA-binding proteins find their targets? b) Define the conformational changes that occur in both proteins and DNA during binding, and c) Determine the sequence of events involved in the formation of active complex and d) by making use of single-molecule enzymatic techniques determine the degree of static and dynamic disorder in the cleavage rate constants and its origin. FEN1 and XPF are structure-specific nucleases that recognise a branched DNA structure consisting of a single unpaired nucleotide (3´flap in the case of FEN1 and 5' flap in the case of XPF) overlapping with a variable length region of single-stranded DNA. Both nucleases cleave the substrate after the first base pair preceding the flap to remove the ssDNA flap.

It has been shown that nuclease binding to the DNA double-flap substrate induces a major conformational change in the DNA double helix and also in the enzyme itself. Our aim is to monitor this conformational rearrangements by single-molecule fluorescence resonance energy transfer and use it as a tool to elucidate the binding dynamics and enzymatic cleavage mechanism of FEN1 and XPF at single-molecule level (see figures below) in the presence and absence of PCNA (Proliferating Cell Nuclear Antigen). PCNA forms a trimeric ring with three-fold symmetry perpendicular to the ring plane, and posses a central hole that can be used to clamp DNA and slide in a freely diffusing mode. It has been shown that PCNA stimulates the archaeal and human FEN1 activities by up to 50-fold, due mainly to an increase in binding affinity for substrates. Under the same conditions the activity of Sulfolobus XPF (SsoXPF) is crucially dependent on association with PCNA with a drastic 20000-fold increase in activity (Roberts & White, unpublished), suggesting it may function as an essential cofactor. To date, there is no evidence to clarify if PCNA binding modulates either the affinity or catalytic properties of SsoXPF towards the substrate. Crystal structures of complexes between PCNA and peptides from several PCNA-interacting proteins have been reported, however, the precise molecular mechanisms by which these proteins cooperate with PCNA on the DNA are still not clear. So far, this represents the first attempt to an study at this detail of a DNA:protein complex and the methodology could well be applied to many other biochemical environments. A better understanding on how it is the dynamics of the DNA-protein complex could give a clearer picture for the development of anticancer drugs related with defects on the DNA damage repair mechanisms. This project is in collaboration with Prof. Malcolm White at the Centre for Biomolecular Sciences at St Andrews University http://www.st-and.ac.uk/~mfw2

M. Newman, J. Murray-Rust, J. Lally, J. Rudolf, A. Fadden, P. Knowles, M.F. White and N.Q. McDonald
"Crystal structure of a xeroderma pigmentosum group F endonuclease with and without DNA suggests a model for recognition of branched DNA substrates "
EMBO Journal, 24, 895-905, 2005

J. A. Roberts and M. F. White
"DNA end-directed and processive nuclease activities of the archaeal XPF enzyme "
Nucleic acids Research , 33, 6662-6670, 2005

G.J. Williams, K. Johnson, J. Rudolf, S.A. McMahon, L. Carter, M. Oke, H. Liu, G.L. Taylor, M.F. White & J. H. Naismith
"Structure of the heterotrimeric PCNA from Sulfolobus solfataricus "
Acta Crystallograph F Struct Biol Cryst Commun 62, 944-948, 2006