LC-MS/MS identification of phosphosites at the N-terminal region of MUS81 by in vitro CHK2 kinase assay

ABSTRACT: MUS81 is a structure-specific endonuclease that processes DNA intermediates during mitosis and in S-phase following replication stress. It plays a crucial role in cleaving deprotected reversed forks in BRCA2-deficient cells. However, how MUS81 is regulated during replication stress in human cells remains unknown. In this study, we combined biochemical and single-cell assays to investigate whether CHK2 regulates the formation of MUS81-dependent DSBs at replication forks, and to determine whether CHK2 and MUS81 functionally interact during sustained replication arrest or in the absence of BRCA2. We demonstrate that human CHK2 and MUS81 physically interact through the FHA domain, and that the I157T mutation significantly impairs this interaction, whereas the R117A mutation does not. We further show that CHK2 contributes to the regulation of MUS81-dependent DSB formation at replication forks during prolonged arrest or destabilization due to BRCA2 deficiency. In addition, we identify specific MUS81 residues phosphorylated by CHK2 that are functionally required for DSB formation at reversed replication forks in BRCA2-deficient cells. These findings uncover a critical regulatory mechanism governing the MUS81 complex at demised replication forks and indicate that CHK2 activity, and its interaction with MUS81, controls the cleavage of reversed forks in the absence of BRCA2.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Sara Rinalducci  

LAB HEAD: Sara Rinalducci

PROVIDER: PXD064334 | Pride | 2026-09-14

REPOSITORIES: Pride