Abstract 3669: “DNA binding region” of BRCA1 affects genetic stability through modulating the intra-S-phase checkpoint

Abstract

Abstract Introduction. BRCA1, a tumor suppressor gene in breast or ovarian cancer, is a large multifunctional protein implicated in DNA double-strand break repair, centrosome duplication, transcription regulation, DNA damage response, and cell cycle control, all of which are important for maintaining genomic stability, BRCA1 contains 3 domains; a N-terminal RING domain, C-terminal BRCT domain and a central region (CR). RING and BRCT domains are well addressed, but the function of the CR remains unclear. The CR is reported to have DNA binding region (DBR). Our aim of this study is to identify DBR and to clarify the biological significance of BRCA1 binding to DNA. Materials and Methods. 1) Identification of DBR of BRCA1. First, electrophoretic mobility shift assay (EMSA) was performed to identify an essential region responsible for BRCA1 binding to DNA using purified tagged full length BRCA1 protein or the fragments and DNA oligos, and to investigate features of the DNA binding using various structures or lengths of DNA. Next, atomic force microscopy (AFM) was performed to see how BRCA1 binds to DNA. 2) Analysis of the biological significance of BRCA1 binding to DNA using mouse ES cells which are deficient in the DBR of BRCA1(ΔDBR) by TALEN-mediated Gene Targeting. 1. MTT survival assay under PARP1 inhibitor or Hydroxyurea (HU) to see the ability to homologous recombination (HR) repair or the sensitivity to replication stress, respectively. 2. HR assay in vitro. 3. EdU incorporation assay in the presence of HU to see cell cycle under replication stress. 4. Western blot to evaluate the phosphorylation status of CHK1 which regulates intra-S-phase checkpoint. 5. Karyotyping analysis to check for the genetic instability. Results. 1) We identified an essential DBR (421-701 amino acids) within the CR of human BRCA1 by EMSA or AFM, which preferably binds to splayed-arm DNA (mimic of replication fork) in a sequence-independent manner and brings DNA strands together. These in vitro data suggest that binding of BRCA1 to DNA may affect protection of the DNA replication fork. 2) 1. The ΔDBR cells exhibited decreased survival as compared to wild type (WT) cells treated with a PARP1 inhibitor or HU in MTT assay. 2. There was not statistical difference with a HR assay between WT cells and ΔDBR cells. 3. ΔDBR cells continued to incorporate more EdU in the presence of HU in EdU incorporation assay. 4. Phosphorylation of CHK1 was moderately decreased in ΔDBR cells in Western blot. 5. The ΔDBR cells exhibited an increase in abnormal chromosome structures in Karyotyping analysis. Conclusion. In this study, we demonstrate that the DBR of BRCA1 modulates genetic stability through the intra-S-phase checkpoint that is activated by replication stress. Thus, our finding refines a functional domain of BRCA1, DBR, deficiency of which results in moderately impaired DNA replication upon replicative stress, leading to accumulative DNA mutations and cancer development. Citation Format: Takaaki Masuda, Koshi Mimori, Chuxia Deng. “DNA binding region” of BRCA1 affects genetic stability through modulating the intra-S-phase checkpoint. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3669.