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Twist in the Tail: Regulation of BRCA1 for Replication Fork Protection

In this webinar, Professor Jo Morris, Ph.D., Birmingham University, UK, discusses the roles of BRCA1 in replication fork protection and homologous recombination, and implications in cancer predisposition. Replication fork protection is a process by which recently replicated DNA is stabilized and protected from nuclease mediated degradation (e.g., DNase 2, MRE11). While BRCA1, a gene frequently studied for its association to early onset ovarian and breast cancer, is known to play a role in homologous recombination, its role in replication fork protection is less understood.

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Roles of BRCA1 in homologous recombination include

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1. Counteracting of 53BP1-RIF2-Shieldin and the fill-in that is promoted by CST-Polα.

2. Promoting BRCA2, PALB2 and RAD51 loading onto 3’ exposed single stranded DNA ends.

3. Stabilizing the synapse in the D loop.

Many of the proteins important for homologous recombination also play roles in replication fork protection, which is mediated by RAD51. Dr. Morris’s research aims at elucidating how BRCA1, along with a number of other proteins important for homologous recombination, is involved in this process.

To achieve this goal, Dr. Morris has evaluated the requirement of various BRCA1 domains for replication fork protection.

Dissecting the role of BRCA1 domains in replication fork protection

• BRCA1 RING domain

  1.BRCA1 RING domain- found in the N-terminus, is the ring coordinating around two zinc ions and known to interact with BARD1. Together, BRCA1 and BARD1 form an E3 ubiquitin ligase. Mutation of BARD1 at R99, interrupts the ability of BRCA1/BARD1 to interact with the ubiquitin loaded onto E2 and reduces the rate of ubiquitin transfer to lysines of substrate proteins. Interestingly, the R99E mutation specifically affects BARD1’s ligase activity but does not affect BRCA1:BARD1 heterodimer formation, stability or BRCA1 localization to IR foci. However, the BARD1-R99E mutation affects the placement of 53BP1 relative to BARD1 and BRCA1 within DNA repair foci. Surprisingly, when tested in vitro by incubating cells with nucleotide analogs followed by treatment with hydroxyurea to induce fork stalling, the BARD1 R99E mutation does not affect replication fork protection.

  
• BRCA1 Coiled-Coil domain

  2.BRCA1 Coiled-Coil domain- important for interaction with PALB2, BRCA2 and for loading RAD51, which is critical for the protection of replication forks. However, introduction of a mutation (M1411T) in the coiled-coil domain of BRCA1 does not affect replication fork protection. This is a surprising finding considering that both BRCA1 and BRCA2 are important for replication fork protection and their interaction should be disrupted by the introduced mutation (M1411T-BRCA1). However, this finding was further confirmed by deleting the N terminus of PALB2, which removes the coiled coil domain required for interaction with BRCA1.

  
• BRCA1 phosphorylation site, pS114-BRCA1

  3.BRCA1 phosphorylation site, pS114-BRCA1- phosphosite located in the N terminus of BRCA1 after the Ring region. By using an antibody generated by GenScript against a BRCA1 peptide containing the phosphorylated S114 residue, Dr. Morrison was able to demonstrate the localization of phosphorylated BRCA1 to replication foci through a proximity ligation assay. Additionally, Dr. Morrison demonstrated that phosphorylation of BRCA1 at S114 is required for replication fork protection, as cells expressing the mutant form of BRCA1 S114A showed decreased ability to protect replication forks from degradation.

  

  

  GenScript’s antibody to pS114 BRCA1 allows detection of wildtype BRCA1 in cell extracts but does not detect the BRCA1 S114A mutant form.

  

  In the Nature publication "Isomerization of BRCA1-BARD1 promotes replication fork protection", Dr. Jo Morris relied on monoclonal and polyclonal antibodies against BRCA1(pS114) developed by GenScript. These antibodies are available upon request to Dr. Jo Morris and subject to completion of a standard MTA.

  Monoclonal Polyclonal

  Additionally, a site in BARD1 required for RAD51 interaction and important for BRCA1’s role in synapse stability is also required for replication fork protection.

  

  The phosphorylated S114 site followed by a Proline residue in BRCA1 is a potential site for interaction with Pin1, which is enriched at nascent DNA that is treated with hydroxyurea. Depletion of Pin1 resulted in short forks, while depletion of both Pin1 and BRCA1 did not have an additive effect, suggesting that Pin1 and BRCA1 act via the same pathway in replication fork protection.

  Pin1 is a phospho-specific peptidyl-prolyl-isomerase, which means that Pin1 can catalyze conformational changes in proline to achieve a cis or trans orientation. Dr. Morris went on to show that the interaction between Pin1 and BRCA1 S114 is critical for BARD1-RAD51 binding. In fact, replacing proline with an alanine, which effectively mimics a trans conformation, increased the interaction between BARD1-RAD51, relative to wildtype control. Importantly, a BRCA1 double mutant (S114A and P115A) was able to rescue the defect in replication fork protection seen with the BRCA1 S114A mutant, demonstrating that the BRCA1 trans form at position 115 is able to rescue the need for the phosphorylation at S114.