What question did this study set out to answer?

To clarify the role of BRCA1 in the DNA damage response by comparing it with BRCA2 using isogenic cell lines.

April 26, 2026Mutagenesis0 citationsOpen Access

Differential Roles of BRCA1 and BRCA2 in the DNA Damage Response Revealed by isogenic V79 Mutant Cell lines

PCPiyawan ChailapakulColorado State University JMJunko MaëdaColorado State University TMTomisato MiuraHirosaki University

Key Points

To clarify the role of BRCA1 in the DNA damage response by comparing it with BRCA2 using isogenic cell lines.
Created a novel BRCA1 mutant cell line (B1-21) through CRISPR/Cas9 from V79 cells
Analyzed DDR phenotypes including RAD51 foci formation and homologous recombination repair
Assessed sensitivity to DNA-damaging agents like camptothecin and etoposide
B1-21 cells displayed impaired RAD51 foci formation and increased sensitivity to camptothecin, while V-C8 showed higher sensitivity to etoposide and cisplatin
B1-21 exhibited chromosomal instability with 19-20 chromosomes compared to stable karyotypes in V79 and V-C8
Checkpoint failures were confirmed as both BRCA mutant cells entered mitosis after gamma irradiation unlike wild-type cells.

Abstract

This study aimed to establish a novel BRCA1 mutant cell line from Chinese hamster V79 cells and clarify the role of BRCA1 in the DNA damage response (DDR) by comparison with a BRCA2 mutant line, V-C8. Using CRISPR/Cas9 editing, we generated a hypomorphic BRCA1 mutant, designated B1-21, carrying a 27-bp in-frame deletion in exon 4. This mutation deletes nine amino acids within the RING domain. B1-21, V79, and V-C8 cells were analyzed for DDR phenotypes. Both mutants showed impaired RAD51 foci formation, defective homologous recombination repair, and increased sensitivity to DNA-damaging agents. B1-21 cells were particularly sensitive to camptothecin and the PARP inhibitor NU1025, whereas V-C8 cells showed higher sensitivity to etoposide, cisplatin, mitomycin C, and bleomycin. Although γH2AX and FANCD2 focus responses were similar between V79 and B1-21, RAD51 recruitment was only partially reduced in B1-21 and completely absent in V-C8. B1-21 also displayed chromosomal instability (19-20 chromosomes), while V79 and V-C8 maintained a stable karyotype. After gamma irradiation, V-C8 cells accumulated substantially more chromatid-type aberrations and retained unrepaired chromatin longer than B1-21. Neither mutant showed normal RAD51 foci formation, radiation-induced sister chromatid exchange or an effective G2/M checkpoint arrest, unlike wild-type cells. Mitotic index measurements further confirmed checkpoint failure: V79 cells suppressed mitotic entry after irradiation, while B1-21 and V-C8 continued to enter mitosis, with V-C8 showing the most complete checkpoint breakdown. These findings indicate that partial disruption of the BRCA1 RING domain results in a hypomorphic phenotype with impaired homologous recombination, defective checkpoint control, and enhanced genotoxic sensitivity. The isogenic BRCA1 and BRCA2 mutant V79 lines offer a valuable model for dissecting DDR pathway differences and developing mutation-specific therapeutic strategies targeting BRCA-mutant cancers.

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