The role of TRIP13 in DNA damage repair

by Bastian Föhr
Doctoral thesis
Date of Examination:2023-03-02
Date of issue:2023-08-10
Advisor:Dr. Alex Caspar Faesen
Referee:Dr. Alex Caspar Faesen
Referee:Prof. Dr. Henning Urlaub
Persistent Address: http://dx.doi.org/10.53846/goediss-10007

 

 

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Abstract

English

Double-strand breaks, the most toxic DNA lesions, can be repaired via homologous recombination or non-homologous end joining (NHEJ). Using the right pathway at the right time is crucial to maintain genome integrity. Shieldin (REV7-SHLD1-SHLD2- SHLD3) promotes DSB repair via NHEJ by inhibiting DNA end resection. TRIP13 and its cofactor p31 disassemble REV7-homolog MAD2 from its interaction partner. The interaction of REV7 and SHLD3 in cells is also TRIP13- and p31-dependent. Additionally, TRIP13 affects NHEJ efficiency in a REV7-dependent manner. Based on these observations, a model emerged, where TRIP13 and p31 control DSB repair pathway choice by disassembling Shieldin (specifically REV7-SHLD3). Here, I biochemically reconstituted the interactions between TRIP13, p31, and Shieldin. The aim was to provide a mechanical understanding of how TRIP13 and p31 regulate Shieldin. However, the proposed TRIP13-mediated disassembly of REV7- SHLD3 was not observed. Also, REV7 and its homolog MAD2 interacted differently with TRIP13 and p31, suggesting that the disassembly mechanism is not conserved. Yet, TRIP13, p31, and Shieldin formed multiple complexes with different stoichiometries, indicating a function of TRIP13-p31 in Shieldin regulation. To reveal the mechanical function of these complexes, I obtained electron density maps for TRIP13-Shieldin and TRIP13-p31-Shieldin via cryo-EM. They showed that even though dimeric REV7 binds TRIP13 on its own, p31 tethers TRIP13 and a REV7 dimer. REV7 is positioned above the pore of the TRIP13 hexamer, indicating REV7 as a substrate of TRIP13. Interestingly, p31 bound Shieldin at slow rates, suggesting that the p31-Shieldin interaction is dependent on structural changes. This slow p31- Shieldin binding was required for TRIP13 ATPase activity, indicating that it has a function in Shieldin regulation. While the nature of those rate-limiting structural changes remains unclear, I ruled out a topology change of one REV7 protomer. Together, my data suggest that TRIP13 and p31 regulate Shieldin via a novel mechanism. Based on my observations, I propose three models of how TRIP13 and p31 regulate Shieldin in DSB repair.
Keywords: Biochemistry; DNA repair; NHEJ; cryo-EM; HORMA domain proteins; Shieldin Complex
 
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