Insights into the function of DNA repair factors MRN and ATM

Rotheneder, Matthias (2022): Insights into the function of DNA repair factors MRN and ATM. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

DNA double strand breaks (DSB) are a particularly deleterious threat to genomic integrity throughout all domains of life. DSBs can cause chromosomal aberrations, tumorigenesis and cell death if left unre-paired and are caused by either endogenous or exogenous sources. Cells rely on efficient detection, repair and response upon occurrence of DSBs. In eukaryotes, DSBs are mostly repaired by either end joining pathways or homologous recombination (HR). HR, in contrast to the end joining pathways, en-ables error-free DSB repair in presence of a template sister chromatid. The Mre11-Rad50-Nbs1 (MRN) complex recognizes and tethers DNA ends, even if they are obstructed by proteins to initiate HR. In order to respond to DSBs, the MRN complex recruits and activates the signaling kinase Ataxia-telangiectasia mutated (ATM), that belongs to the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family. Activated ATM in turn initiates the cellular DNA damage response (DDR). Mre11 and Rad50 are highly conserved and form a topology-specific, ATP-dependent nuclease complex that pro-cesses DNA ends but leaves genomic DNA intact. The eukaryote specific Nbs1 subunit finetunes MRN’s endonuclease activity by providing interaction with proteins (e.g. CtIP). Apart from its nucleo-lytic activity, MRN has a scaffolding function that promotes DNA end tethering, repair foci formation and possibly signal amplification. Although the complex has been studied for more than two decades, a model that integrates both MRN’s enzymatic and scaffolding functions has not yet been established. In the first part of the thesis, such a model was elaborated by combining both structural and biochemical data from this and previ-ous studies. A cryo-electron microscopy (cryo-EM) structure of the Chaetomium thermophilum (Ct)MRN catalytic head domain in its ATPγS-bound state not only clarifies its atomic architecture but also reveals how a core part of Nbs1 stabilizes and possibly locks the Mre11 dimer. In this structure significant parts of the Rad50 coiled-coils were resolved in a rod configuration, stabilized by several interaction points. A previously uncharacterized C-terminal Mre11 domain, denoted bridge could fur-ther stabilize the rod configuration. The rod configuration and the bridge domain restrict access to the Rad50 DNA binding site. Biochemical analysis revealed the Rad50 DNA binding site is extremely specific for DNA ends. However, an additional, eukaryote-specific DNA binding site at the C-terminus of Mre11 enables binding to internal DNA. The Rad50 coiled-coil domains are linked at the apex via a zinc hook dimerization motif to form a large proteinaceous ring/rod. Cryo-EM data and crystal structures ex-plained how two MRN complexes can tether DNA ends via dimerization of these apical domains. In vivo assays indicate that mutation of the apex tethering element negatively impacts DSB repair. Mutations in DDR pathways allow cancer cells to cope with increased replication and genotoxic stress. For this reason, proteins involved in DDR were described to be promising targets in cancer therapy. Due to its central role in DSB induced DDR, ATM is an auspicious target for drug development. Howev-er, lack of ATM high-resolution structures, as well as atomic details of small molecule inhibitor binding modalities hampered the application of structure-based drug design. In the second part of the thesis, the binding modalities of two ATP-competitive ATM-inhibitors were described. This project was a col-laborative work with Merck KGaA, that provided a novel inhibitor (M4076) with improved pharmacoki-netics. Comparison of the inhibitor-bound kinase active sites with the likewise resolved ATPγS-bound active site explains the high affinities that were determined in biochemical assays. Superposition and sequence alignment of the ATM kinase active site with other PIKK active sites enables to rationalize the molecular reasons for selectivity. In biochemical assays, IC50 values of the inhibitors for ATM, PIKKs and CHK2 showed high selectivity towards ATM. The binding of the inhibitors stabilized the N-terminal solenoid domain of ATM, this enabled the generation of a high-resolution structure of the entire ATM protein. The quality of the map allowed the identification of two zinc binding sites that possibly stabi-lize loops and the generation of a near-complete ATM structure. Taken together, the structural data provides the framework for structure-based ATM inhibitor design and allows mapping of cancer muta-tion as well as functionally important protein interaction sites.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 5. Oktober 2022
• 1. Bericht­erstatter:in: Hopfner, Karl-Peter