Defining the structural details of protein interactions in nucleic acid replication and repair

Abstract

The maintenance of genomic integrity across all domains of life is reliant on a complex series of protein-nucleic acid and protein-protein interactions that occur during the fundamental processes of replication and repair. Determining the structural and functional details of these interactions is crucial in understanding the molecular mechanisms of disease, including cancer progression and the proliferation of viral infections. This thesis presents 5 publications which elucidate the structural and functional details of the interactions of nucleic acid binding proteins, which are pertinent to their roles in human DNA repair, and in the replication of viral genomes. In chapter 2, the interaction between two human DNA damage response (DDR) complexes involved in the repair of double strand breaks (DSBs), sensor of single-stranded DNA (SOSS) and MRE11-RAD50-Nbs1 (MRN), is investigated using mass spectrometry, isothermal titration calorimetry (ITC) and functional experiments. The study reveals the details of a novel phosphorylation dependent interaction between SOSS protein, integrator complex subunit 3 (INTS3), with MRN component, nijmegen breakage syndrome protein 1 (Nbs1). In chapter 3, techniques including nuclear magnetic resonance (NMR) and biolayer interferometry (BLI) are utilised to determine the structural mechanism by which another constituent of the SOSS complex, human single stranded DNA binding protein 2 (hSSB2), binds to UV-damaged DNA. A similar methodology is subsequently employed in chapter 4 to uncover the nucleic acid binding interface and binding affinity of a virally encoded RNA binding protein, non-structural protein 9 (nsp9), from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Finally, a second study presented in chapter 5 reveals that the RNA binding interface of SARS-CoV-2 nsp9 is conserved in the nsp9 from middle east respiratory syndrome coronavirus (MERS-CoV).

Date of Award	2023
Original language	English
Awarding Institution	Western Sydney University
Supervisor	Roland Gamsjaeger (Supervisor) & Liza Cubeddu (Supervisor)