Molecular characterisation of proteins involved in DNA repair

Abstract

While the protective layer of chromatin shields the human genome to some extent, endogenous and exogenous degrading events can still lead to damage. For example, UV exposure, reactive oxygen species (ROS) contact or errors in replication can result in the formation of pyrimidine dimers, oxidised bases or DNA strand breaks. To overcome this, cells have evolutionarily conserved DNA damage repair pathways that involve many different proteins most of which work together in complexes. Brd8 is a member of the bromodomain family of proteins that are able to bind acetylated lysine residues. Brd8 is part of different complexes including the p400/Brd8 histone exchange complex and the Tip60 nucleosome remodelling and acetylase complex, both of which are also involved in double stranded DNA break repair. The structure of Brd8 has not been solved to date however it is proposed to be similar to bromodomains that are able to bind proteins that contain di-acetylated lysine residues. Within the Tip60 complex Brd8 is thought to work as an anchor for the complex by binding acetylated lysines that are located at the carboxyl-tail of histone 4 (H4), however, no experimental evidence is available to date. Single-stranded DNA-binding proteins (SSBs) are ubiquitous across all forms of life and participate in DNA repair. These proteins bind single strands of DNA (via their oligonucleotide binding OB domain) that arise as a product of DNA damage in order to protect the DNA from further degradation. Human single-stranded binding protein 1 and 2, hSSB1 and hSSB2, respectively, are SSBs that contain a single OB domain and in contrast the well-known RPA, were only discovered in 2008. hSSB1 has been implicated in homologous recombination (HR) and base excision repair (BER), while hSSB2 has been shown to be important in nucleotide excision repair (NER). Most recent studies have focused on the molecular details of hSSB1 binding to DNA as well the involvement of hSSB1 in the MRN and SOSS complexes. However, significantly less is known about the mechanism and action of hSSB2 in DNA repair. This thesis consists of three main parts: The first part contains preliminary structural data on the binding of Brd8 to acetylated histone 4 lysines. The second shows the major contributions that have been made to a review (first author) that summarises the structural details of all known hSSB1 interactions. Finally, the third part of the thesis contains experimental data on the interaction of the less well-studied hSSB2 protein with DNA as part of a (first author) publication.

Date of Thesis submission	2019
Original language	English
Awarding Institution	Western Sydney University
Supervisor	Roland Gamsjaeger (Supervisor) & Liza Cubeddu (Supervisor)