DNA complexes with human apurinic/apyrimidinic endonuclease 1: structural insights revealed by pulsed dipolar EPR with orthogonal spin labeling

A DNA molecule is under continuous influence of endogenous and exogenous damaging factors, which produce a variety of DNA lesions. Apurinic/apyrimidinic sites (abasic or AP sites) are among the most common DNA lesions. In this work, we applied pulse dipolar electron paramagnetic resonance (EPR) spectroscopy in combination with molecular dynamics (MD) simulations to investigate in-depth conformational changes in DNA containing an AP site and in a complex of this DNA with AP endonuclease 1 (APE1). For this purpose, triarylmethyl (TAM)-based spin labels were attached to the 5′ ends of an oligonucleotide duplex, and nitroxide spin labels were introduced into APE1. In this way, we created a system that enabled monitoring the conformational changes of the main APE1 substrate by EPR. In addition, we were able to trace substrate-to-product transformation in this system. The use of different (orthogonal) spin labels in the enzyme and in the DNA substrate has a crucial advantage allowing for detailed investigation of local damage and conformational changes in AP-DNA alone and in its complex with APE1.

Preferred orientation in an angled intercalation site of a chloro-substituted Λ -[Ru(TAP) 2 (dppz)] 2+ complex bound to d(TCGGCGCCGA) 2

The crystal structure of the ruthenium DNA ‘light-switch’ complex Λ -[Ru(TAP) 2 (11-Cl-dppz)] 2+ (TAP=tetraazaphenanthrene, dppz=dipyrido[3,2- a ′:2′,3′- c ]phenazine) bound to the oligonucleotide duplex d(TCGGCGCCGA) 2 is reported. The synthesis of the racemic ruthenium complex is described for the first time, and the racemate was used in this study. The crystal structure, at atomic resolution (1.0 Å), shows one ligand as a wedge in the minor groove, resulting in the 51 ° kinking of the double helix, as with the parent Λ -[Ru(TAP) 2 (dppz)] 2+ . Each complex binds to one duplex by intercalation of the dppz ligand and also by semi-intercalation of one of the orthogonal TAP ligands into a second symmetrically equivalent duplex. The 11-chloro substituent binds with the major component (66%) oriented with the 11-chloro substituent on the purine side of the terminal step of the duplex.