Plasticity of BRCA2 Function in Homologous Recombination: Genetic Interactions of the PALB2 and DNA Binding Domains

Abstract

The breast cancer suppressor BRCA2 is essential for the maintenance of genomic integrity in mammalian cells through its role in DNA repair by homologous recombination (HR). Human BRCA2 is 3,418 amino acids and is comprised of multiple domains that interact with the RAD51 recombinase and other proteins as well as with DNA. To gain insight into the cellular function of BRCA2 in HR, we created fusions consisting of various BRCA2 domains and also introduced mutations into these domains to disrupt specific protein and DNA interactions. We find that a BRCA2 fusion peptide deleted for the DNA binding domain and active in HR is completely dependent on interaction with the PALB2 tumor suppressor for activity. Conversely, a BRCA2 fusion peptide deleted for the PALB2 binding domain is dependent on an intact DNA binding domain, providing a role for this conserved domain in vivo; mutagenesis suggests that both single-stranded and double-stranded DNA binding activities in the DNA binding domain are required for its activity. Given that PALB2 itself binds DNA, these results suggest alternative mechanisms to deliver RAD51 to DNA. In addition, the BRCA2 C terminus contains both RAD51-dependent and -independent activities which are essential to HR in some contexts. Finally, binding the small peptide DSS1 is essential for activity when its binding domain is present, but not when it is absent. Our results reveal functional redundancy within the BRCA2 protein and emphasize the plasticity of this large protein built for optimal HR function in mammalian cells. The occurrence of disease-causing mutations throughout BRCA2 suggests sub-optimal HR from a variety of domain modulations.

Resistance to therapy in BRCA2 mutant cells due to loss of the nucleosome remodeling factor CHD4

“…Consistent with the conclusion that HR is not restored, CHD4 depletion did not enhance break-induced HR in the BRCA2 mutant VC-8 hamster cell line that includes an integrated SCE-1 break-inducible DR-GFP reporter, (Pierce et al 1999), but GFP-positive cells were recovered, as expected, by introduction of a functional BRCA2 ( Fig. 3D; Siaud et al 2011).…”

Dual DNA-binding domains shape the interaction of Brh2 with DNA

“…There is no evidence as yet to suggest that there is a second cryptic DNA-binding domain in the N-terminal region of BRCA2 as in the case with Brh2 and the NBD. But a similar kind of dualistic behavior as noted with Brh2 seems apparent from the studies with the synthetic BRCA2 fusions [29]. HR is functional in the absence of the DBD so long as there is PALB2 interaction, and conversely, HR is functional when PALB2 is removed so long as the DBD remains.…”

“…A similar situation prevails in the mammalian system where it was found using synthetic BRCA2 constructs prepared by fusing different domains that abolishing the DBD had little impact on HR so long as the ability to associate with PALB2 was maintained [29]. PALB2 is a partner of BRCA2 that physically interacts with the N-terminal region of BRCA2 and that has its own intrinsic DNA binding domain [30].…”

Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography

“…Prior work using BRC repeat regions shows that they also (less efficiently) specifically promote the formation of ssDNA-RAD51 complexes by blocking the ATPase activity of RAD51, which promotes disassembly [1171][1172][1173]. Genetic analysis in V79 hamster cells suggests functional redundancy within this complex HRR protein [1174].…”