Repair of broken DNA is vital for genome stability and to prevent the development of cancer. Research shows how the tumour-suppressor protein BRCA1 promotes a DNA-repair pathway called homologous recombination. See Article p.360
DNA molecules can break on exposure to radiation or chemicals, and as a result of errors during DNA metabolism. When both strands of DNA are broken, this is known as a double-stranded DNA (dsDNA) break. Cells can repair such damage and restore the original DNA sequence — even if short pieces of DNA are lost from the broken ends — using a pathway called homologous recombination1 (often referred to simply as recombination). On page 360, Zhao et al.2 reveal that the complex formed between two tumour-suppressor proteins, BRCA1 and BARD1, directly promotes recombination. This helps to explain how BRCA1 and its partner prevent tumour development.
Defects in several recombination genes have been linked to a predisposition to cancer. More specifically, genes that encode the tumour-suppressor proteins BRCA1 and BRCA2 are frequently mutated in hereditary breast and ovarian cancers3. Working out their specific roles in recombination is therefore of great interest.
The first step in dsDNA-break repair by recombination involves the generation of single-stranded DNA (ssDNA) at the broken ends by nuclease enzymes (Fig. 1a). The resulting ssDNA overhangs are then bound by replication protein A (RPA, the protein that stabilizes and protects ssDNA in most DNA metabolic processes). The next step is the replacement of RPA by the protein RAD51 (Fig. 1b). This process is strictly regulated to ensure that RAD51 is loaded onto ssDNA and that recombination is activated only when needed, because unneccessary recombination threatens genome stability. Dysregulation of dsDNA-break repair can cause genome rearrangements, which are common in many cancers4.
In human cells, BRCA2 facilitates the exchange of RPA for RAD51 on ssDNA, which leads to the formation of a RAD51–ssDNA filament3,5. Once stably bound, RAD51 catalyses a signature step of recombination: the 'invasion' of the ends of the processed DNA into intact dsDNA that has a matching (homologous) sequence. The recombination machinery uses this template DNA molecule as a backup of genetic information to help it correctly repair the DNA break and replace any missing sequences1.
Findings from several laboratories have previously led researchers to propose that BRCA1 promotes recombination, but mechanistic insights have been lacking3. Specifically, it was suggested6 that BRCA1 functions upstream of recombination to regulate the balance between homologous recombination and non-homologous end-joining (an alternative dsDNA-break repair pathway that often causes mutations). BRCA1 was also proposed to stimulate the generation of ssDNA overhangs at broken ends, and to facilitate the loading of RAD51 onto ssDNA3. However, the mechanisms underlying these functions are unknown, and it has not been possible to conclude whether BRCA1 has a direct stimulatory function in recombination, or whether it acts indirectly, for example by counteracting recombination inhibitors.
The lack of insight into the mechanisms of BRCA1 action in recombination has largely been due to the fact that purified BRCA1 protein was not available for use in laboratory studies. One of Zhao and colleagues' key breakthroughs is their development of a method to purify high-quality, full-length BRCA1 in complex with its partner, BARD1, in quantities sufficient for experimentation. This allowed them to expand on previous observations7,8 to show that the BRCA1–BARD1 complex binds DNA with a preference for branched DNA structures, which form during recombination. The authors also found that RAD51 directly interacts with both subunits of the BRCA1–BARD1 complex.
Zhao et al. next performed a series of in vitro reactions to test whether BRCA1–BARD1 regulates the activities of RAD51. Although BRCA2 (in complex with its partner protein, DSS1) promotes the exchange of RPA for RAD51 on ssDNA5,9,10, the authors observed that BRCA1–BARD1 possesses no such activity.
Strikingly, they demonstrate that BRCA1–BARD1 instead promotes a reaction downstream of RAD51–ssDNA binding, namely the step in which the RAD51–ssDNA filament engages and invades the template dsDNA. They showed this using two approaches. First, they used biochemical techniques to monitor the invasion of circular dsDNA by short, RAD51-coated ssDNA. This revealed that BRCA1–BARD1 strongly stimulates formation of D-loops (the products of strand invasion). Second, they observed the stimulatory effect directly, using a single-molecule imaging technique called the DNA curtain assay.
Zhao and colleagues identified a site in the BARD1 subunit with which RAD51 interacts with high affinity. This allowed them to construct a mutant version of the BRCA1–BARD1 complex in which three amino-acid residues in BARD1 had been replaced by others, impairing the complex's association with RAD51 in vitro and in vivo. The authors observed that the mutant (known as BRCA1–BARD1AAE) impairs stimulation of recombination in the D-loop assay, or complex assembly in the DNA curtain assay.
When the researchers transiently replaced the wild-type BRCA1–BARD1 complex with BRCA1–BARD1AAE in cultured human cells, they observed defects in recombination assays. Furthermore, the cells could be killed by the compound mitomycin C and by an inhibitor of the PARP enzyme; cell sensitivities to mitomycin C and PARP inhibitors are hallmarks of impaired recombination. These experiments convincingly demonstrate that the mechanistic insights provided by the cell-free reactions with purified proteins in vitro are functionally relevant, and that the interplay of BRCA1–BARD1 with RAD51 is important for recombination in living cells.
How does the BRCA1–BARD1 complex function alongside other RAD51 regulators? Zhao et al. performed a D-loop assay using short ssDNA pre-bound with RPA. They found that D-loop formation was more robust when BRCA1–BARD1 and BRCA2–DSS1 were both present. This confirms the separate functions of the BRCA complexes: BRCA2 stimulates the exchange of RPA for RAD51 on ssDNA5,9,10, and BRCA1 subsequently promotes DNA invasion (Fig. 1c).
It will now be of interest to study the function of the PALB2 protein that brings the BRCA1 and BRCA2 complexes together3, and to delineate the interplay of these complexes with RAD51 'paralogue' proteins11,12, which probably also act downstream of RAD51–ssDNA filament formation in recombination11. The availability of the purified BRCA1–BARD1 complex will also help scientists to work out its functions in other steps of the recombination pathway, such as the initial processing of the broken DNA ends to generate overhanging ssDNA. Together, these experiments will reveal how key DNA-repair factors help to maintain genome stability and protect us from cancer.
Kowalczykowski, S. C. Cold Spring Harb. Perspect. Biol. 7, a016410 (2015).
Zhao, W. et al. Nature 550, 360–365 (2017).
Prakash, R., Zhang, Y., Feng, W. & Jasin, M. Cold Spring Harb. Perspect. Biol. 7, a016600 (2015).
Jeggo, P. A. & Löbrich, M. Biochem. J. 471, 1–11 (2015).
Jensen, R. B., Carreira, A. & Kowalczykowski, S. C. Nature 467, 678–683 (2010).
Bunting, S. F. et al. Cell 141, 243–254 (2010).
Paull, T. T., Cortez, D., Bowers, B., Elledge, S. J. & Gellert, M. Proc. Natl Acad. Sci. USA 98, 6086–6091 (2001).
Simons, A. M. et al. Cancer Res. 66, 2012–2018 (2006).
Thorslund, T. et al. Nature Struct. Mol. Biol. 17, 1263–1265 (2010).
Liu, J., Doty, T., Gibson, B. & Heyer, W.-D. Nature Struct. Mol. Biol. 17, 1260–1262 (2010).
Taylor, M. R. G. et al. Cell 162, 271–286 (2015).
Sigurdsson, S. et al. Genes Dev. 15, 3308–3318 (2001).
Related links in Nature Research
About this article
Nanoscopic analysis of 53BP1, BRCA1 and Rad51 reveals new insights in temporal progression of DNA-repair and pathway choice
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis (2019)