Shedding light on DNA-protein interactions involved in the nucleotide excision repair pathway

Abstract

DNA, or deoxyribonucleic acid, contains the molecular blueprint of organisms. Although, very stable, DNA can be damaged by genotoxic agents such as UV-light, reactive oxygen species or chemotherapeutic drugs, amongs others. Organisms have evolved several biological pathways to repair different types of damage and nucleotide excision repair (NER) is one of them. NER repairs mainly lesions inducing structural distortion of the double helix. Briefly, the lesion is detected, unwinding takes place to create a 30 nucleotides long bubble, the damage-containing single strand is then excised; using the opposite strand as a template, the DNA is resynthesized and ligated. Nucleotide Excision Repair is a non-mutagenic repair system. Single-strand DNA binding proteins (SSB) are involved in NER and many other pathways. Their role is to coat and protect the ssDNA from degradation and re-hybridisation. Here, we studied SSBs from Saccharolobus solfataricus and Sulfolobus acidocaldarius as well as a chimeric protein containing DNA binding domains from both organisms. We showed that the chimeric protein had better resistance to extreme conditions of temperature, ionic strength or acidic pH. We also proved that our chimeric SSB can be used as a tool to image single strand DNA or RNA using super resolution microscopy. The first step of NER is damage recognition by XPC. We studied XPC binding to a DNA substrate and showed the possibility of two XPC binding to a damage whilst demonstrating that the presence of a nick on the DNA backbone does not constitute a substrate for XPC. Furthermore, we used single molecule FRET technique to show that XPC could bind a three nucleotides bubble in two different modes, showing a strong preference for one of these binding modes. The second step is the unwinding by the XPD helicase, we studied the impact of the purine/pyrimidine composition on the unwinding activity and investigated a possible damage sensor role of XPD, using Archaean models. To complete our work, we demonstrated a cooperative effect on the distortion of the repair bubble of XPA and RPA.