Structural and Functional Characterization of Two Phosphorylation Site Clusters in the DNA Damage Response Adaptor Protein MDC1

DNA double strand breaks (DSBs) are amongst the most genotoxic and dangerous lesions and can therefore severely threaten the integrity of our genome. To avoid that, DSBs are signaled and repaired by the DNA damage response (DDR) pathways that initiate a series of chromatin remodeling events and recruit many DDR factors to the site of lesion. This will ultimately lead to the repair of DSBs via one of the two main pathways involved: the non-homologous end joining (NHEJ), which is an error-prone mechanism and is mainly active in G1 and early-S phases of the cell cycle, and the homologous recombination (HR), which is a more faithful process that requires the presence of the sister chromatids as template for the repair and takes place from mid-S to G2 phases. The first chromatin modification that takes place when a DSB occurs is phosphorylation of histone variant H2AX (yH2AX). The main factor that recognizes and binds to yH2AX is MDC1, a large nuclear adaptor protein involved in the DDR. MDC1 is composed of several domains and regions that are implicated in direct protein-protein interactions, allowing for the recruitment of downstream DDR factors. Most of these interactions are dependent on post-translational modifications, mainly phosphorylation. In my PhD thesis I characterized two phosphorylation site clusters in MDC1 with regards to their role in phosphorylation-dependent protein-protein interactions. The first phosphorylation site cluster is referred to as SDTD repeat region and was shown to interact with the DNA damage response factor MRE11/RAD50/NBS1 (MRN) complex via its NBS1 subunit. The second phosphorylation site cluster is referred to as TQXF cluster and was shown to interact with the ubiquitin ligase RNF8. In my thesis project, I contributed to the discovery of new phosphorylation-dependent interaction partners of the MDC1 TQXF cluster. Pellino proteins are FHA domain-containing E3 ubiquitin ligases known to be involved in signaling events in the Toll and interleukin-1 (IL-1) pathways, responsible for ...