User talk:Hala Ouzon-Shubeita
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CH391L/S2013 Hala Ouzon Jan 30 2013
Purified human BRCA2 stimulates RAD51-mediated recombination 
DNA integrity is essential to life. However DNA is constantly subject to assaults from endogenous species, such as oxygen radicals, alkylating agents or replication errors. Or from exogenously generated agents like UV light and X-rays . DNA damage could manifest in the form of single-strand breaks, double- strands breaks, insertions, deletions or the formation of bulky adducts. Figure 1 below shows some common DNA damaging agents and some repair mechanisms .
Chromosomes Double-strand breaks (DSB) in particular are troublesome because a single unrepaired DSB could lead to chromosomal instability or cell death .There are two ways in which organisms can repair DSBs; Non Homologous Ends Joining (NHEJ) and Homologous Recombination (HR). HR is a very important repair pathway in mitosis and in chromosomal exchange during meiosis . Therefore HR is highly regulated. HR goes through three phases: presynapsis, synapsis and post-synapsis. In the presynapsis phase as a response to DSBs several sensor proteins detect the break and send signals to mediator proteins that activate effectors that repair the damage. Figure 2 below is a simplified representation of this process .
The focus of this study  is on the breast cancer susceptibility protein (BRCA2) and its goal in mediating the binding of the central protein in recombination (RAD51) to ssDNA, replacing replicating protein A (RPA) and stabilizing RAD51-ssDNA filament formation. Many factors affect the likelihood of developing breast cancer but individuals carrying mutation in BRCA1 or BRCA2 have 40-80% chance of developing breast cancer [ 5 ]. All the previous knowledge about this protein came from studying its fragments or exploring its orthologues. Because of the large size of BRCA2 it was very difficult to induce and purify a functional full length BRCA2.
Methods and Results: 
Dr. Kowalczykowski’s group was able to clone and purify the full length human BRCA2 (3,418 amino acid) . This was successfully done by adding two tandem repeats of the maltose binding protein tag (MBP) at the N terminus of the protein (increasing the size to 470KDa) and expressing the construct in human 293TD cells. Cloning was verified by Western blotting on the C-terminus region and the MBP tag. The two MBP tags did not affect the function of BRCA2 as tested by comparing cell survival between brca2 mutant cells and brca2 mutant cells transfected with 2XMBP-BRCA2.Furthermore to confirm that the purified BRCA2 protein was folded correctly and biochemically active, this group used pull-down assays to test its interactions with recombinant proteins that were previously shown to interact with BRAC2 fragments. BRAC2 interacts with the strand-exchange protein (RAD51) and its Meiotic counterpart DMC1. Despite a report that BRAC2 interacts with the ssDNA-binding factor, replication protein A (RPA), no significant interactions were detected.
Figure 3 shows the BRAC2 functional domains and some protein interactions .
As shown in figure 3 RAD51 binds the BRC repeats of BRCA2. Using known concentrations of BRCA2 and RAD51, this group was able to estimate that six RAD51 proteins bind to BRCA2 at once. Using mobility shift assays, it was shown that BRCA2 has preference binding ssDNA and tailed DNA over dsDNA and that this result does not change if BRCA2 was pre-incubated with RAD51. RAD51 has affinity for both dsDNA and ssDNA. Binding to dsDNA inhibits DNA strand exchange.
When incubated with ssDNA and dsDNA, RAD51 inhibits DNA strand exchange. However, this exchange is facilitated if RAD51 was pre-incubated with BRCA2 indicating that the latter either targets RAD51 to ssDNA or limits its binding to dsDNA or both. This effect was reduced if the mixture was supplemented with RPA, but the exchange was not fully suppressed. To confirm that BRCA2 limits binding of RAD51 to dsDNA, the authors incubated both proteins with ssDNA first then added dsDNA. The inhibitory effect of the excess RAD51 that binds the dsDNA was reduced in a BRCA2 concentration depended manner. BRCA2’s role in targeting RAD51 to ssDNA is accomplished by blocking the ATPase activity of RAD51. Addition of BRCA2 reduced the ATPase activity in a concentration dependent manner down to the levels measured in the absence of DNA. These findings confirm that BRCA2 plays two roles: it stabilizes RAD51 bound to the ssDNA by down-regulating its ATPase activity and limits its binding to dsDNA.
In vivo, there is competition between RAD51 binding to ssDNA and RPA binding. It was shown previously from studying fragments of BRCA2 that this protein also encourages RAD51 filament formation on RPA-coated ssDNA. To check whether full length BRCA2 also have similar effect this group used DNA strand exchange assay similar to that used to assist BRCA2’s role in mediating RAD51 binding to ssDNA but with ssDNA first complexed with RPA. And they found that increasing the concentration of BRCA2 increased DNA strand exchange as much as 20 fold and that its stimulatory effect was obvious even at 100-fold smaller concentration than RAD51.
In summary, this work investigated the biochemical functions of full-length BRCA2. The authors showed that it enhances the functions of RAD51 in recombinatorial DNA repair of breaks. Surprisingly, they found that the protein can bind to ssDNA and tailed DNA of either polarity. This suggests that BRCA2 could also have a role in the repair of DNA gaps. The ability to purify full length BRCA2 should facilitate studying its important role in increasing the risk of cancer in individuals with mutations in the protein.
1. Negritto, M.C., Repairing Double-Strand DNA Breaks. Nature Education, 2010. 3(9).
2. Krejci, L., et al., Homologous recombination and its regulation. Nucleic Acids Res, 2012. 40(13): p. 5795-818.
3. Roy, R., J. Chun, and S.N. Powell, BRCA1 and BRCA2: different roles in a common pathway of genome protection. Nat Rev Cancer, 2012. 12(1): p. 68-78.
4. Jensen, R.B., A. Carreira, and S.C. Kowalczykowski, Purified human BRCA2 stimulates RAD51-mediated recombination. Nature, 2010. 467(7316): p. 678-83.
5. James D.Fachenthal and Olufunmilayo I. Olopade, Breast cancer risk associated with BRCA1 and BRCA2 in diverse populations.Nature Reviews Cancer, 2007.7: p.937-948.