Biochemical characterization of human shieldin complex
by Vivek Susvirkar
Date of Examination:2022-05-30
Date of issue:2022-08-01
Advisor:Dr. Alex Faesen
Referee:Prof. Dr. Peter Rehling
Referee:Prof. Dr. Patrick Cramer
Referee:Dr. Sonja Lorenz
Referee:Prof. Dr. Matthias Dobbelstein
Referee:Dr. Alexander Stein
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EnglishShieldin is a newly identified DNA repair effector involved in repair of double strand breaks (DSBs) in G1 phase of cell cycle. Shieldin is a four-component complex consisting of proteins SHLD1, SHLD2, SHLD3 and HORMA REV7. Shieldin through its direct DNA binding activity inhibits homologous recombination (HR) and directs the repair pathway to non-homologous end joining (NHEJ). Molecular function of SHLD proteins and HORMA REV7 in context of shieldin recruitment and assembly at DSBs is not well understood. In this thesis, I reconstituted the individual components of shieldin complex to investigate the mechanism of shieldin recruitment and assembly using bacterial and insect cell expression systems. Using this approach, I was able to elucidate unusual stoichiometry of shieldin complex and provide functional relevance of HORMA REV7 dimerization. In presence of SHLD3 and SHLD2 N-terminal domain (NTD), I observe a constitutive dimer of REV7 in shieldin complex. HORMA REV7 exists in two topologically distinct states (open and close) which can be isolated using trapping mutants. The assembly of shieldin complex is surprisingly slow and depends on conversion of open REV7 (O-REV7) to close REV7 (C-REV7) upon binding to SHLD3. I observe a similar reaction kinetics between REV7 and REV3 subunits of the DNA Polymerase ζ. My results show that shieldin/Pol ζ assembly centered around REV7 is remarkably slow in-vitro and therefore would require catalysis in-vivo. In order to understand mechanism of shieldin recruitment, SHLD3 was purified and investigated due to it been the most upstream component of the shieldin complex in recruitment hierarchy. My results show SHLD3 harbours a DNA binding domain and forms DNA-protein complex independently as well as in complex with REV7-SHLD2. SHLD3 binds both single stranded DNA (ssDNA) and double stranded DNA (dsDNA) with similar affinities. It also shows ability to bind both telomeric and non-telomeric sequences. SHLD3 truncation studies show DNA binding activity lies in its conserved C-terminal domain (CTD). To understand molecular basis, I used predicted SHLD3 structure from Alphafold 2 and identified key residues involved in DNA binding. Mutagensis of the residues abolished DNA binding activity of SHLD3. In conclusion, this thesis provides valuable insights into assembly of shieldin complex mediated by REV7 topology switch and recruitment of shieldin complex at DSBs mediated by SHLD3. It also provides a tool to trap REV7 in either open or close topology for future functional and kinetic studies.
Keywords: REV7; SHLD3; Shieldin; NHEJ; DNA repair; HORMA domain