Suppression of the DNA repair defects of BRCA2-deficient cells with heterologous protein fusions

Abstract

The BRCA2 tumor suppressor plays an important role in the repair of DNA damage by homologous recombination, also termed homology-directed repair (HDR). Human BRCA2 is 3,418 aa and is composed of several domains. The central part of the protein contains multiple copies of a motif that binds the Rad51 recombinase (the BRC repeat), and the C terminus contains domains that have structural similarity to domains in the ssDNA-binding protein replication protein A (RPA). To gain insight into the role of BRCA2 in the repair of DNA damage, we fused a single (BRC3, BRC4) or multiple BRC motifs to the large RPA subunit. Expression of any of these protein fusions in Brca2 mutant cells substantially improved HDR while suppressing mutagenic repair. A fusion containing a Rad52 ssDNA-binding domain also was active in HDR. Mutations that reduced ssDNA or Rad51 binding impaired the ability of the fusion proteins to function in HDR. The high level of spontaneous chromosomal aberrations in Brca2 mutant cells was largely suppressed by the BRC-RPA fusion proteins, supporting the notion that the primary role of BRCA2 in maintaining genomic integrity is in HDR, specifically to deliver Rad51 to ssDNA. The fusion proteins also restored Rad51 focus formation and cellular survival in response to DNA damaging agents. Because as little as 2% of BRCA2 fused to RPA is sufficient to suppress cellular defects found in Brca2-mutant mammalian cells, these results provide insight into the recently discovered diversity of BRCA2 domain structures in different organisms.double-strand break ͉ mammalian cells ͉ Rad51 ͉ homologous recombination ͉ BRC repeat

The roles of BRCA1 and BRCA2 and associated proteins in the maintenance of genomic stability

“…However, in these experiments individual BRC repeats were utilized, which may not be representative of their function in the context of full-length BRCA2. This is supported by experiments investigating the importance of the BRC repeats, along with the C-terminal DBD (DNA/DSS1-binding domain), in the repair of DNA damage by BRCA2 using a fusion protein (Saeki et al, 2006). The DBD of BRCA2 has five domains, including three oligonucleotide/oligosaccharide-binding (OB) fold domains (OB1, OB2 and OB3) (Yang et al, 2002).…”

Modification of the DNA Damage Response by Therapeutic CDK4/6 Inhibition

“…DSB Repair Assays-Homologous recombination activity was quantified using a synthetic repair substrate (pDR-GFP) as previously described (16). Circular DR-GFP was digested using ISce1 enzyme.…”

BRCA2: a universal recombinase regulator

“…Remarkably, BRCA2-deficient cells can also be, at least partially, rescued by artificial BRCA2 fusions containing just BRC3 or BRC4 fused to the ssDNA-binding domain of RPA (Saeki et al, 2006). These fusions also restored the ability to form RAD51 foci after DNA damage, supporting the notion that the primary role of BRCA2 is to target RAD51 to ssDNA generated at a DSB, and that both the BRC motifs and the DNA-binding region of BRCA2 are indispensable for this RAD51-targeting activity.…”