Multiple DNA damaging agents cause deaminations of the bases of DNA. If not repaired, these can lead to mutations, which in turn can result in dangerous diseases. Endonuclease V (EndoV) is a highly conserved gene from prokaryotes to humans. Prokaryotic EndoV has affinity for deaminated bases in DNA. Escherichia coli EndoV recognizes and binds to deaminated bases, and cleaves the DNA strand at the second phosphodiester bond, 3’ of the lesion. Structural studies of Thermotoga maritima EndoV reveals a DNA strand-separating wedge on the protein surface, containing several fully conserved residues in the EndoV family. The residues making up the DNA binding pocket, as well as the site for catalytic activity are also characterized, and are highly conserved in all EndoV homologues. Until now, structural and biochemical characterization of human EndoV, and its possible role in DNA repair, has not been the subject of any published study. The extraordinary high degree of conservation in the EndoV family suggests an important function also in the eukaryotic cell.
In this thesis, we present the human EndoV (hEndoV) successfully expressed and purified for the first time. Through a series of DNA binding studies we show that human EndoV has specific DNA binding affinity for duplex DNA with distortions – like branching points, loops and flap structures. In contrast to E. coli EndoV, no binding to deaminated inosine was observed. Human EndoV was also tested for endonucleolytic activity, but no such activity has yet been detected. We also present an interaction study between human EndoV and human Slx4, to investigate a possible stimulation of branched DNA binding of hEndoV by Slx4. The results suggest no interaction.
The purified protein was used to verify the binding of commercial hEndoV antibodies to our human EndoV protein. A crystal structure of human EndoV is important in order to study the protein-DNA interaction in detail, and extensive crystallization screening was performed without positive results. Additionally, small angle X-ray scattering experiments were carried out, and the resulting low-resolution structures of a DNA hairpin and a hEndoV-DNA complex are presented.