Mutation of the BRCA1 SQ-cluster results in aberrant mitosis, reduced homologous recombination, and a compensatory increase in non-homologous end joining

Abstract

Mutations in the breast cancer susceptibility 1 (BRCA1) gene are catalysts for breast and ovarian cancers. Most mutations are associated with the BRCA1 N- and C-terminal domains linked to DNA double-strand break (DSB) repair. However, little is known about the role of the intervening serine-glutamine (SQ) - cluster in the DNA damage response beyond its importance in regulating cell cycle checkpoints. We show that serine-to-alanine alterations at critical residues within the SQ-cluster known to be phosphorylated by ATM and ATR result in reduced homologous recombination repair (HRR) and aberrant mitosis. While a S1387A BRCA1 mutant - previously shown to abrogate S-phase arrest in response to radiation - resulted in only a modest decrease in HRR, S1387A together with an additional alteration, S1423A (BRCA12P), reduced HRR to vector control levels and similar to a quadruple mutant also including S1457A and S1524A (BRCA14P). These effects appeared to be independent of PALB2. Furthermore, we found that BRCA14P promoted a prolonged and struggling HRR late in the cell cycle and shifted DSB repair from HRR to non-homologous end joining which, in the face of irreparable chromosomal damage, resulted in mitotic catastrophe. Altogether, SQ-cluster phosphorylation is critical for allowing adequate time for completing normal HRR prior to mitosis and preventing cells from entering G1 prematurely resulting in gross chromosomal aberrations.

Orally Bioavailable and Blood–Brain Barrier-Penetrating ATM Inhibitor (AZ32) Radiosensitizes Intracranial Gliomas in Mice

“…Lentiviruses were generated in HEK293T cells (23). Lentivirus expressing H2B-mCherry has been described (23).…”

“…Lentiviruses were generated in HEK293T cells (23). Lentivirus expressing H2B-mCherry has been described (23). The virus expressing the fusion EGFP-Centrin2 was constructed from pEGFP-Centrin 2 (provided by E. Nigg; Addgene plasmid #41147) and pWPXLd (23).…”

Novel BRCA1 splice-site mutation in ovarian cancer patients of Slavic origin

“…for ATR [27]. Mutations of serine residues may affect localization of BRCA1 to sites of DNA damage and DNA damage response function [28]. Mutation detected in this study, c. in BRCA1 gene might be of clinical significance for hereditary ovarian cancer.…”

A Multiplexed Mass Spectrometry-Based Assay for Robust Quantification of Phosphosignaling in Response to DNA Damage

“…The ATR kinase is autophosphorylated after DNA damage at multiple sites, including Thr1989 (55). The activity of the DDR can be seen by an increase in the levels of several phosphosites [e.g., phospho BRCA1 pS1524 (56), phospho CHK1 pS317 (57), phospho NBN pS343 (58)]. The pharmacologic inhibition of the ATR kinase was profiled using the ATR kinase inhibitor AZD-6738 in conjunction with the 4NQO treatment.…”