Investigation of the CK2-mediated MUS81 phosphorylation and analysis of its relevance for chemosensitivity in BRCA2-deficient human cells [Tesi di dottorato]

The to be carefully regulated to avoid unscheduled targeting structure-specific endonuclease MUS81/EME1 plays important roles in the resolution of recombination intermediates, however, its function needs intermediates during DNA replication, which may result in genome instability. Little is known about the regulation of the human MUS81 complex. Hence, we undertook a proteomic analysis to identify regulatory phosphorylation events of MUS81. Our analysis identified several hits and among them we functionally characterized the residue targeted by the pleiotropic kinase CK2. Using biochemical and cell biological approaches, we demonstrated that accurate MUS81 phosphorylation on S87 by CK2 is not required to prevent DNA damage in S-phase but for resolution of branched DNA intermediates in mitosis, which is crucial to facilitate proper DNA segregation under replication stress but contributes modestly to chromosome integrity. However, constitutive S87 MUS81 phosphorylation triggers DNA damage and largely undermines genome integrity in normal cells. Next, using our S87-MUS81 phosphorylation mutant as a tool to define which function of MUS81, the S-phase-related one or that performed in M-phase, was essential under pathological conditions. As a prototype of pathological condition, we used BRCA2-deficient cells that needs MUS81 to recover from replication stress. Using cell biology approaches to evaluate survival, DNA damage and resolution of mitotic interlinked intermediates, we show that the S87 MUS81 phosphorylation is involved to ensure viability of BRCA2-deficient cells mostly because it is the M-phase function of MUS81 to be essential. Altogether, our data described a novel regulatory mechanism required to control MUS81 complex function in M-phase in human cells and involved in the viability of BRCA2-deficient cells. As CK2 inhibitors are under evaluation as anti-cancer drugs, our data may be useful to evaluate their use in tumors with signs of BRCAness.

diritti: info:eu-repo/semantics/openAccess
In relazione con info:eu-repo/semantics/altIdentifier/hdl/11573/1231785
Tutor esterno: P. Pichierri
Valutatori esterni: D. Barilà, F. Rosselli

Tesi di dottorato. | Lingua: Inglese. | Paese: | BID: TD20018559