Characterisation of selected post-translational modifications mediating the response to stress in mammalian cells

Baldock, Robert (2016) Characterisation of selected post-translational modifications mediating the response to stress in mammalian cells. Doctoral thesis (PhD), University of Sussex.

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Abstract

Eukaryotic initiation factors (eIFs) govern assembly of ribosomes on nascent mRNAs to be translated. Many eIFs have been identified in proteomic screens which aim to identify proteins that are subject to post--‐translational modification by SUMO. Despite this, many remain to be biochemically validated or functionally characterised. In this project, the DEAD-box helicases eIF4A1 and eIF4A2 were found to be SUMOylated on conserved lysine residues K216 and K226 respectively using an in vitro SUMOylation assay and mass spectrometry. Additionally, the SUMOylation of eIF4A2 was found to increase in response to arsenite-treatment (a treatment known to induce stress granule formation in mammalian cells). Furthermore, mutation of K226 in eIF4A2 affects the formation of stress granules. Further work will be required to determine the potential effect of SUMOylation of eIF4A2 on global translation.

Double‐strand breaks (DSBs) form some of the most deleterious and damaging lesions to DNA. The DNA damage mediator, 53BP1, governs the choice of repair method promoting error prone non-homologous end joining over homologous recombination. In this project, two novel phosphorylation sites within the N-terminus of 53BP1 were identified. Phosphorylation of these residues is critical for the localisation of TopBP1 to sites of damage. Pulse-labelling cells with traceable nucleotide analogues reveals that failure to phosphorylate these residues also results in a failure to sustain a G1/S-phase checkpoint when DSBs are present. These results indicate that 53BP1 integrates two elements of the stress response- choice of DSB repair pathway and cell-cycle regulation. Further work will elucidate the exact mechanism requiring both 53BP1 and TopBP1 to maintain the G1/S‐phase checkpoint.

53BP1 is a reader of the histone code, recognising both methylation and ubiquitination of histones through distinct domains. In this project, the BRCT2 domains at the C-terminus of 53BP1 were shown to recognise a third post-translational mark, phosphorylation. By analysing the dependence on phosphorylation of the localisation of the BRCT2 domains in isolation to laser-induced DNA damage alongside structural and biochemical data, it was shown that the BRCT2 domains interact directly with ΥH2AX in vitro and in vivo. Although not essential for localisation to DNA damage, abolition of this interaction results in a persistence of ΥH2AX foci at ‘late’ time points representing the heterochromatic fraction of breaks. Furthermore, it was shown that this defect is propagated by a failure to localise the activated checkpoint kinase, ATM. Further investigation will be required to determine the mechanism underlying the repair defect observed.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Biochemistry
Depositing User: Library Cataloguing
Date Deposited: 11 May 2016 14:39
Last Modified: 12 Jun 2018 10:51
URI: http://sro.sussex.ac.uk/id/eprint/60731

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