Structural and dynamical basis of NusA´s role in transcription-coupled DNA repair

Abstract

DNA repair processes involves a host of repair machineries that are able to recognize and repair the damaged DNA. Nucleotide excision repair (NER) pathway is one of these processes exploited by cells to remove bulky DNA lesions. In Escherichia coli, transcription-repair coupling factors (TRCF) such as Mfd, NusA, and UvrD, mediate coupling of transcription to DNA repair by recruiting necessary repair machineries. NusA is a flexible 55 kDa protein composed of six domains. Isolated free NusA in solution undergoes autoinhibitory intramolecular interdomain interaction which reduces its nascent RNA binding activity. The role of NusA in transcription regulation as well as DNA repair have been widely reported, however, structural characterization of NusA autoinhibition and interaction complex with repair enzymes has so far not been studied. Furthermore, the structural dynamics that underline the DNA-substrate binding activity of one of the repair enzymes, translesion DNA polymerase IV (DinB), is still missing. The aim of this thesis is to unravel the structural dynamics behind NusA autoinhibitory phenomenon as well as the role of the DinB-Thumb in translesion DNA repair. I also aim to further characterize the transcription-coupled repair complex of NusA and DinB, alongside the UvrD helicase, using both biochemical and biophysical techniques. In this thesis, I reported for the first combined backbone and methyl groups resonance assignment of full-length NusA and confirms that the wild-type NusA and “open” NusA mutant are in different state. Relaxation data reports on a low population of the open state in solution and shows a clear trend of released autoinhibition for the open NusA state. For DinB, I also for the first time completed the sequence resonance assignment and elucidate the structural dynamics governing its DNA substrate accommodation in the active site. Relaxation data reveal that the DNA-binding Thumb domain is structurally flexible. I showed for the first time that the stalled RNAP makes contact with DinB. I also observed that the self-autoinhibitory phenomenon of NusA diminishes its affinity to DinB in solution. On the other hand, DinB affinity to NusA further stabilizes in the presence of additional domains. For the NusA:UvrD interaction, I identified an additional binding interface within the central NusA S1-KH1-KH2 region, which is required to stabilize the interaction.