BRCA1 Phosphorylation and Isomerization Enhances Replication Fork Protection

At some stalled replication forks, the regressed arm of nascent DNA is structurally similar to single-ended DNA with a double-strand break. RAD51 protects these ends with help from several other proteins, including BRCA1 and BRCA2. Daza-Martin and colleagues found that the BRCA1–BARD1 complex is required for fork protection independently of the canonical PALB2–BRCA2–RAD51 pathway. BARD1 and BRCA1 have serine residues (Ser148 and Ser114, respectively) that are normally phosphorylated and predicted to be close to the BARD1–RAD51 interface; substituting these serine residues with alanine residues caused defects in fork protection. In BRCA1, substitution with aspartic acid residues (which chemically resemble phosphorylated serine residues) promoted fork protection, suggesting BRCA1 phosphorylation is important for fork protection, whereas this was not the case with BARD1. BRCA1′s Ser114 residue is part of a Ser–Pro motif that forms the minimal consensus sequence for PIN1, a phosphorylation-targeted peptidyl–prolyl isomerase that catalyzes the cis–trans isomerization of the proline–peptidyl bond in the phopsho-Ser–Pro motif. PIN1 coimmunoprecipitated with BARD1 and BRCA1, implying it may interact with the complex. A proximity ligation assay suggested that BRCA1(pS114) localizes to stalled replication forks. Depletion or inhibition of PIN1 led to a decrease in fork protection; however, the requirement for PIN1 was mostly overcome by mutation of Pro115 to favor a trans-peptidyl bond in this region. In addition, BRCA1–BARD1 heterodimers containing BRCA1^P115A showed increased trypsin accessibility of BARD1 and increased ability to bind RAD51. The results suggest that a conformational change in BRCA1 caused by PIN1-dependent isomerization regulates BRCA1–BARD1 activity. PIN1 increased interactions between the BRCA1–BARD1 complex and RAD51, recruiting RAD51 to stalled replication forks and increasing protection of the nascent DNA strand. Analysis of BRCA1 variants from patients with cancer also uncovered variants associated with degradation of nascent strands without defects in homologous recombination. The mechanistic insights provided by this work will aid in further investigation of the possible role of fork protection in cancer.

Daza-Martin M, Starowicz K, Jamshad M, Tye S, Ronson GE, MacKay HL, et al. Isomerization of BRCA1–BARD1 promotes replication fork protection. Nature 2019 Jul 3 [Epub ahead of print].

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