Abstract
MAD2L2 (REV7) plays an important role in DNA double-strand break repair. As a member of the shieldin complex, consisting of MAD2L2, SHLD1, SHLD2 and SHLD3, it controls DNA repair pathway choice by counteracting DNA end-resection. Here we investigated the requirements for shieldin complex assembly and activity. Besides a dimerization-surface, HORMA-domain protein MAD2L2 has the extraordinary ability to wrap its C-terminus around SHLD3, likely creating a very stable complex. We show that appropriate function of MAD2L2 within shieldin requires its dimerization, mediated by SHLD2 and accelerating MAD2L2-SHLD3 interaction. Dimerization-defective MAD2L2 impairs shieldin assembly and fails to promote NHEJ. Moreover, MAD2L2 dimerization, along with the presence of SHLD3, allows shieldin to interact with the TRIP13 ATPase, known to drive topological switches in HORMA-domain proteins. We find that appropriate levels of TRIP13 are important for proper shieldin (dis)assembly and activity in DNA repair. Together our data provide important insights in the dependencies for shieldin activity.
Highlights
MAD2L2 (REV7) plays an important role in DNA double-strand break repair
To address whether MAD2L2 dimerization affects shieldin complex formation we focused on the two shieldin proteins that directly interact with MAD2L2: SHLD3 and SHLD2
We co-transfected 293T cells with epitope-tagged SHLD2, SHLD3, and wild-type MAD2L2 or the different MAD2L2-mutants and immunoprecipitated MAD2L2 in order to assess its interaction with SHLD2 and SHLD3
Summary
MAD2L2 forms a dimer mediated by SHLD2 and important for shieldin assembly. To understand to what extent MAD2L2’s ability to dimerize affects its function in shieldin we introduced dimer-breaking mutations in a flag-tagged RNAi-resistant (RR) MAD2L2 cDNA expression construct. Three single amino acid changes in the dimerization domain of MAD2L2 completely disrupted its ability to interact with SHLD2, while only slightly affecting its interaction with SHLD3 (Fig. 1c). Size-exclusion chromatography (SEC) on purified shieldin proteins indicated that SHLD21-95 induces the dimerization of MAD2L2 in vitro, suggesting that a dimer of MAD2L2 is present in a full-length shieldin complex interacting with single molecules of SHLD3 and SHLD2 (Fig. 1d and Supplementary Fig. 1b, c). MAD2 conversion and Absorbance (280nm) % of maximal binding a MAD2L2 1 13 K44
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