How does hSSB1 recruit the MRN complex to the site of double stranded DNA breaks?

Abstract

DNA is susceptible to degradation and damage from both the external and internal environments. It is therefore essential to maintain and repair DNA to conserve vital genetic information to prevent the accumulation of mutations that may alter normal function and potentially lead to the development of disease such as cancer. Cancer treatment methods that cause DNA damage, such as some chemotherapies and radiotherapy, are effective against cancer cells; however, cancer cells can repair the DNA damage caused by these treatments. Elucidating the specific interactions of DNA repair pathways (e.g., homologous recombination) increases our knowledge of how these cells repair their damaged DNA. This in turn can allow us to design ways to inhibit these DNA repair mechanisms. The DNA binding protein hSSB1 is known to initiate homologous recombination by recruiting the MRN repair complex to the site of double stranded DNA breaks (DSBs) via the Nbs1 component. Published pull-down data shows that the flexible C-terminal region of hSSB1 interacts with the N-terminal region of Nbs1 (residues 1-334) that contains three phosphopeptide binding domains (one Fork-head associated domain and two Breast Cancer C-terminal repeats). Published in vivo data suggest that hSSB1 binding to MRN (i.e., hSSB1:Nbs1) is enhanced by ionising radiation (creating DSBs), which may indicate the presence of post-translational modifications (PTMs). This thesis examines the molecular mechanism of the hSSB1 and Nbs1 interaction which may be mediated by the presence of PTMs. Quantitative binding methods demonstrated no significant interaction occurring between hSSB1 and Nbs11-334. The data presented here may indicate that the Nbs1 component may not be responsible for the hSSB1 interaction. However, the N-terminal region of Nbs1 contains a FHA and two BRCT domains which are phosphopeptide binding domains. Therefore, to determine if PTMs facilitate the hSSB1:Nbs1 interaction, the two known phosphorylation sites within the C-terminal extension of hSSB1 (T117 and S134) were investigated. No significant interaction between Nbs1 and these hSSB1 phosphorylation sites was found, although, several putative FHA and BRCT phosphorylation sites in the hSSB1 C-terminal extension have been identified which may elucidate a potential new phosphorylation-dependent interaction. Discovering the molecular mechanisms that drive the hSSB1 and Nbs1 interaction can lead to the development of new cancer treatment strategies administered in conjunction with current cancer treatment methods, such as radiotherapy or some chemotherapies, to increase their effectiveness and efficiency.

Date of Award	2017
Original language	English