Intrinsic Disorder and Phosphorylation in BRCA2 Facilitate Tight Regulation of Multiple Conserved Binding Events.

Manon Julien,Sandrine M Caputo,Rania Ghouil,Aura Carreira,Ambre Petitalot,Sophie Zinn-Justin

doi:10.3390/biom11071060

Abstract

The maintenance of genome integrity in the cell is an essential process for the accurate transmission of the genetic material. BRCA2 participates in this process at several levels, including DNA repair by homologous recombination, protection of stalled replication forks, and cell division. These activities are regulated and coordinated via cell-cycle dependent modifications. Pathogenic variants in BRCA2 cause genome instability and are associated with breast and/or ovarian cancers. BRCA2 is a very large protein of 3418 amino acids. Most well-characterized variants causing a strong predisposition to cancer are mutated in the C-terminal 700 residues DNA binding domain of BRCA2. The rest of the BRCA2 protein is predicted to be disordered. Interactions involving intrinsically disordered regions (IDRs) remain difficult to identify both using bioinformatics tools and performing experimental assays. However, the lack of well-structured binding sites provides unique functional opportunities for BRCA2 to bind to a large set of partners in a tightly regulated manner. We here summarize the predictive and experimental arguments that support the presence of disorder in BRCA2. We describe how BRCA2 IDRs mediate self-assembly and binding to partners during DNA double-strand break repair, mitosis, and meiosis. We highlight how phosphorylation by DNA repair and cell-cycle kinases regulate these interactions. We finally discuss the impact of cancer-associated variants on the function of BRCA2 IDRs and more generally on genome stability and cancer risk.

Highlights

Introduction iationsThe BReast CAncer protein 2 (BRCA2) is a ubiquitous protein essential for embryonic development: BRCA2 knockout mice show early embryonic lethality and hypersensitivity to irradiation [1]
These protein regions lack a stable secondary structure. They interact with their partners through interfaces that never reach the size of the largest interfaces of ordered complexes but are characterized by a large surface per residue: intrinsically disordered regions (IDRs) use a larger portion of their surface for interaction with their partner, sometimes 50% of the whole, as opposed to only 5–15% for most ordered proteins [27,28]
We recently showed that S206C and T207A lead to chromosomal instability including aneuploidy as observed in BRCA2 mutated tumors, which we proposed could have an impact on cancer [57]

Summary

BRCA2 Exhibits a Set of Conserved Motifs Predicted to Be Disordered

Analysis of BRCA2 protein sequence using disorder prediction tools provided a general view of the structural organization of this protein. BRCA2 interacts with both RAD51 and the meiosis-specific recombinase DMC1 It binds DMC1 via the RAD51-binding BRC repeats [33,45] and a DMC1-specific site located in a region predicted to be disordered between Ser2386 and. Ser3291, in the BRCA2 C-terminal region binding to RAD51-ssDNA filaments, is strictly conserved (Figure 1E). It undergoes CDK (for Cyclin Dependent Kinase)-dependent phosphorylation at the G2 -M phase transition and dephosphorylation upon DNA damage [50]. At blocked replication forks, BRCA2 disordered regions, including its C-terminus, are essential for binding to RAD51 filaments and stimulating the initiation of DNA synthesis by Polη in vitro. Phosphorylation of Ser3291 promotes RAD51 filament disassembly, which in turn promotes entry into mitosis [52]

Nuclear Magnetic Resonance Analyses Support the Presence of Additional

15 N-labeled BRCA2

Both Purified

A Conserved and Disordered BRCA2 Region Contains a Cryptic Repeat That

Findings

11. Conclusions