Poster Presentation The 48th Lorne Conference on Protein Structure and Function 2023

A novel interaction between the MRN and the SOSS1 complex plays an important role in DNA repair (#117)

Serene El-Kamand 1 , Mar-Dean Du Plessis 1 , Jacqueline Matthews 2 , Mark N Adams 3 , Roland Gamsjaeger 1 2 , Liza Cubeddu 1 2
  1. School of Science, Western Sydney University, Penrith, New South Wales , Australia
  2. School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, Australia
  3. School of Biomedical Research, Institute of Health and Biomedical Innovation at the Translational Research Institute, Queensland University of Technology, Woolloongabba, QLD, Australia

The repair of double stranded DNA breaks (DSBs) is crucial in the maintenance of genome integrity. The sensor of single-stranded DNA (SOSS1) protein complex promotes DSB repair by rapidly localising at the site of the break and recruiting the Mre11-Rad50-Nbs1 (MRN) complex. While the mechanism by which the two complexes interact remains unconfirmed, Nbs1 has previously been shown to interact with proteins of the SOSS1 complex. Nbs1 contains an N-terminal FHA domain (pThr binding), coupled to two tandem BRCT domains (pSer binding). The presence of phosphorylation sites within the C-terminal domain of INTS3, a component of the SOSS1 complex, suggests the potential for MRN-SOSS1 interaction to occur via Nbs1-INTS3 binding. In this study, mass spectrometry was used to confirm phosphorylation at INTS3 sites of interest and isothermal titration calorimetry (ITC) was utilised to analyse the interaction of Nbs1 with INTS3 phospho-peptides. ITC data reveal that the Nbs1 FHA domain interacts with INTS3 via phosphorylation-dependent binding to the C-terminus of INTS3 at pT592 with contributions of pS590. Further, functional experiments reveal that mutations of INTS3 at these positions impairs cell survival after exposure to ionising radiation (IR) triggering dsDNA breaks to a degree comparable to INTS3 depleted cells. Our data uncover a novel phosphorylation-dependent interaction between two important DNA repair complexes.