Recombination Mediator and Rad51 Targeting Activities of a Human BRCA2 Polypeptide

Abstract

BRCA2 likely exerts its tumor suppressor function by enhancing the efficiency of the homology-directed repair of injured chromosomes. To help define the DNA repair role of BRCA2, we expressed and purified a polypeptide, BRC3/4-DBD, that harbors its BRC3 and BRC4 repeats and DNA binding domain. BRC3/4-DBD interacted with hRad51 and bound DNA with a distinct preference for single-stranded (ss) DNA. Importantly we demonstrated by biochemical means and electron microscopy that BRC3/4-DBD nucleates hRad51 onto ssDNA and acts as a recombination mediator in enabling hRad51 to utilize replication protein A-coated ssDNA as recombination substrate. These functions of BRC3/4-DBD required both the BRC repeats and the BRCA2 DNA binding domain. The results thus clarify the role of BRCA2 in Rad51-dependent DNA recombination and repair, and the experimental strategies described herein should be valuable for systematically deciphering this BRCA2 function. Homologous recombination (HR)3 helps eliminate DNA breaks, cross-links, and other deleterious lesions from chromosomes. A failure in HR leads to cancer formation (1, 2), which aptly underscores the importance of delineating the mechanism of the HR machinery. HR is best understood in the context of DNA double strand break repair wherein the break is processed nucleolytically to yield ssDNA, which serves as the nucleation site for the Rad51 recombinase. Polymerization of Rad51 onto the ssDNA results in a helical protein filament called the presynaptic filament. The presynaptic filament harbors a binding site for duplex DNA and provides the catalytic center for the search of homology in the duplex and the formation of a joint between the ssDNA and duplex substrates. Assembly of the presynaptic filament is slow, rendering it prone to interference by the single strand binding factor RPA. In addition, the timely delivery of Rad51 to the single-stranded recombination substrate is complicated by a high affinity of this recombinase for duplex DNA. In Saccharomyces cerevisiae, several HR factors, termed recombination mediators, act to promote the assembly of the Rad51 presynaptic filament (3, 4).Mutations in BRCA2 account for a significant portion of familial breast and ovarian cancers (5) and can lead to the cancer-prone syndrome Fanconi anemia (2). BRCA2 mutant cells exhibit hypersensitivity to genotoxic agents and HR deficiency (1, 6, 7). BRCA2 harbors eight copies of a Rad51 binding motif called the BRC repeat (8 -10). In addition, the carboxyl terminus of BRCA2 contains a distinct Rad51 binding domain, and phosphorylation of this domain by cyclin-dependent kinases appears to modulate the interaction with Rad51 (11). Crystallographic and biochemical characterization of mouse Brca2 has revealed a DNA binding domain that comprises three oligonucleotide/oligosaccharide binding (OB) folds (12).Several lines of evidence suggest that BRCA2 functions as a recombination mediator. First, as noted above, BRCA2 interacts with Rad51 (10) and possesses ssDNA binding activity (12), characteristics...

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

“…Although the ability of the BRC-RPA proteins to complement Brca2 mutants is quite surprising, our results provide insight into the recently discovered plasticity of BRCA2 structures in different organisms (18,19,36) and biochemistry on BRCA2 fragments (37). For example, our smallest fusion, BRC3RPA, may be considered analogous to BRCA2 orthologs, which contain a single BRC repeat [U. maydis Brh2 (18) and C. elegans BRC-2 (19)] or have truncated DNA-binding regions (e.g., C. elegans BRC-2, which does not have a tower domain).…”

Minding the gap: The underground functions of BRCA1 and BRCA2 at stalled replication forks

“…Consistent with this, isolated BRC fragments of BRCA2 inhibit Rad51-mediated strand exchange in vitro and interfere with HR in vivo [135,139]. In contrast, certain combined domains of BRCA2 promote Rad51-mediated strand exchange in vitro, suggesting that there may be intramolecular collaboration between distinct domains of BRCA2 [140,161]. The U. maydis BRCA2 homolog, Brh2, possesses only one BRC domain and has been purified with DSS1 in vitro [20,25,141].…”

Hed1 regulates Rad51-mediated recombination via a novel mechanism

“…We next used an oligonucleotide-based homologous DNA pairing assay (Fig. 2B;San Filippo et al 2006) to ask whether Hed1 has any effect on the recombinase function of Rad51. In this assay, ssDNA (150-mer) is incubated with Rad51 in the presence of ATP to assemble the presynaptic filament, which is then mixed with a radiolabeled duplex (40-mer), in which the radiolabeled strand is complementary to the middle portion of the ssDNA.…”

“…The assay was performed as described previously (San Filippo et al 2006). Briefly, Rad51 (3.45 µM) or RecA (3.9 µM) was incubated with 150-mer ssDNA oligonucleotide (6 µM nucleotides) in 9 µL of Buffer D containing 2.5 mM ATP and 30 mM KCl for 5 min at 37°C.…”