Contact person: Gert Folkers
(Bloembergen NMR building)

 

Scientific background

During DNA replication, recombination and DNA repair, double-stranded DNA frequently forms three- or four-way junctions, bubbles, flaps or broken ends with single-stranded extensions. These irregular structures must be corrected to maintain genome stability, integrity and fidelity. This task is accomplished by structure-specific endonucleases. Inactivation or malfunctioning of these enzymes causes genetic defects or cancer. The XPF family, is such a structure-specific endonuclease. In humans seven members (XPF, MUS81, ERCC1, EME1, EME2, FANCM and FAAP24) have been characterized by the presence of the ERCC4 nuclease domain. Only two of them however, XPF and MUS81, have nuclease activity mediated by the conserved core nuclease motif (ERKX3D). Their catalytic function however depends on heterodimer formation with the non-catalytic family members. For example XPF forms an obligate heterodimeric complex with ERCC1 but not with other members of the family and functions primarily in the Nucleotide Excision Repair (NER) pathway. This versatile pathway is able to detect and remove a variety of bulky DNA lesions induced by UV light and environmental carcinogens and thereby maintains genome integrity. We have previously determined the structure of various domains within this family and characterized how these proteins heterodimerize, how they bind to DNA but the catalytic reaction remains elusive and what determines the preferential heterodimer formation is also not known. Recently we have implemented an in vitro assay that functionally resembles the DNA repair reaction and assays to study protein-protein and protein-DNA interactions. These assays, in cooperation with the structural information available, permit us to perform structure based drug design by making peptides that inhibit any of the functions of this complex. These studies elucidate the structure-function relationship of these DNA repair proteins.

Research proposal

On the basis of available structural information and functional models design peptides based on the interaction domain between ERCC1 and XPF. Test the effect of these peptides on the DNA binding and catalytic activity of various ERCC1/XPF complexes and determine the effect of the peptides on the kinetics of complex formation using biochemical and biophysical methods including SPR, fluorescence anisotropy, NMR and molecular modeling/MD simulations

Methods

  • PCR
  • Cloning
  • Protein (co-)expression
  • Large scale protein purification using HPLC
  • Radio nuclide experiments
  • Protein-protein interaction assays
  • Protein-DNA interaction assays
  • Site-directed mutagenesis
  • DNA incision assays
  • Biophysical characterization of proteins using NMR spectroscopy
  • Biophysical characterization of proteins using fluorescence techniques