Using single-molecule microscopy to investigate macromolecular complexes from NER to Translation

DNA is subjected to many forces within cells. In prokaryotes, those range from the supercoiling of the DNA to the impact of macromolecular complexes like the replisome. Lesions on DNA induce changes in DNA structure and/or flexibility. Some of those are recognized and repaired by Nucleotide Excision Repair (NER). NER is a DNA damage repair pathway composed of a few proteins but can recognize and process a plethora of lesion types. It has been proposed that NER recognizes local changes in the structure or flexibility of the DNA at lesion sites. This thesis aims to assess the effect of structural modifications of the DNA on the ability of NER proteins to recognize the damage, as well as assess the effect of newly found inhibitors on the binding of a NER protein. By using optical traps, we were able to alter the tension applied to a single molecule of double-stranded DNA and assess how DNA distortion affects the kinetics of NER proteins at the single-molecule level. Those changes in the tension altered the binding of fluorescently tagged UvrA and UvrB, the first two responders to damage in NER. The more tension applied to the DNA strand, the faster the attachment and the longer the lifetime of UvrA on the DNA. Similarly, UvrB is recruited more often by UvrA when the tension is increased. This sheds light, for the first time, on how these proteins recognise DNA damage, suggesting these proteins recognize changes in the structure of the DNA in a systemic manner instead of specific alterations for each lesion type. Furthermore, we assessed the effect of various NER inhibitors on the binding of UvrA to the DNA and highlighted potential modes of action. Aprotinin increased the affinity of UvrA to the DNA, while ATBC, Bemcentinib, L-Thyroxine and Dienestrol decreased the affinity of UvrA to the DNA. Interestingly, Dienestrol seems to have a dual mode of action by binding to the UvrA molecule and reducing its affinity for the DNA, as well as binding to the DNA molecule, hindering UvrA binding. Prokaryotic NER inhibitors are of great interest as they could be used as a novel family of antimicrobials and potentially be exploited in cancer therapy in combination with DNA-damaging agents to inhibit the growth of bacterial infections. The discovery of novel antimicrobials is of the foremost importance as antimicrobial resistance is rapidly spreading. With most antibiotics targeting the ribosome, a third aim of this thesis was to deepen the understanding of one understudied aspect of translation which is the impact of near-cognate tRNA in the elongation step of the translation. In this thesis I outline a series of experiments that allowed us to stall ribosomes at a specific codon, the first step to assess codon/near-cognate tRNA interactions.