The repair of DNA damage is crucial for the maintenance of genome stability. In base excision repair, lesion-specific DNA glycosylases recognize and cleave a variety of base lesions. The human Nei-like (NEIL) DNA glycosylases NEIL1, NEIL2 and NEIL3 have specificity for oxidized pyrimidines, in particular when present in singlestrand
DNA or bubble structures. Even though their substrate specificity is known, the role of these enzymes remains unclear. Whereas the expression of NEIL1 seems to be cell-cycle dependent, NEIL2 is expressed during the entire cell-cycle and NEIL3 expression has been shown in connection with embryonic development. To date, only the 3D X-ray structure of NEIL1 has been solved.
In this thesis, truncated versions of NEIL1 have been designed for co-crystallization and cross-linking with DNA for 3D structure determination by X-ray crystallography. We show that a truncated version of NEIL1, in combination with the crucial selection of an appropriate cryo-protectant, results in a much improved diffraction
of a crystal containing a NEIL1-DNA complex. Since no crystal structures of NEIL2 and NEIL3 have been reported, we have screened for crystallization conditions of the free protein of these two enzymes. In relation to this, truncated versions of NEIL3 have been designed to improve the protein stability and expression. We further show that for both NEIL1 and NEIL3, a few residues in difference between truncated versions can be essential for the stability and expression of the enzymes. So far, no crystals have been obtained for NEIL2 and NEIL3.
DNA repair enzymes are interesting drug targets in relation to cancer therapy. Therefore, potential DNA glycosylase inhibitors have been tested on NEIL2 to study their effect on the NEIL2 activity. In this thesis, we show that the same compounds that inhibit other human DNA glycosylases such as OGG1, NTH1 and NEIL1 also seem to inhibit NEIL2.