Deficiency of human BRCA2 leads to impaired homologous recombination but maintains normal nonhomologous end joining

Abstract

Carriers of BRCA2 germline mutations are at high risk to develop early-onset breast cancer. The underlying mechanisms of how BRCA2 inactivation predisposes to malignant transformation have not been established. Here, we provide direct functional evidence that human BRCA2 promotes homologous recombination (HR), which comprises one major pathway of DNA double-strand break repair. We found that up-regulated HR after transfection of wild-type (wt) BRCA2 into a human tumor line with mutant BRCA2 was linked to increased radioresistance. In addition, BRCA2-mediated enhancement of HR depended on the interaction with Rad51. In contrast to the tumor suppressor BRCA1, which is involved in multiple DNA repair pathways, BRCA2 status had no impact on the other principal double-strand break repair pathway, nonhomologous end joining. Thus, there exists a specific regulation of HR by BRCA2, which may function to maintain genomic integrity and suppress tumor development in proliferating cells.

Figure 1

Expression of BRCA2 in Capan-1 cell. Nuclear extracts from untransfected Capan-1 cells and Capan-1 cells transfected (transiently or stable integration) with a pcDNA3-based expression vector coding for HA-tagged wt BRCA2 or an empty control vector were immunoprecipitated with anti-HA antibody, followed by Western blotting with anti-BRCA2 antibody. The nuclear extracts from MCF7 were used as control for endogenous expression of wt BRCA2.

Figure 2

Human BRCA2 promotes HR. (a) Schematic map of the chromosomal homologous-targeting substrates. Donation of wt xgprt sequence from the episomal pΔ3 to the chromosomally integrated pΔ2-puro through either gene conversion or crossover events restores xgprt function and confers resistance to XHATM. (b) Reconstitution of wt BRCA2 function in BRCA2-deficient Capan-1 cells enhances HR frequencies. Capan-1 cells with stable pΔ2-puro integration (Capan/pΔ2-puro) were transfected with donor plasmid pΔ3 and pcDNA3 based HA-BRCA2 expression plasmid (19) or control plasmid. The plating efficiency of cells was used to calculate the HR frequencies, i.e., HR frequency equals the number of XHATM-resistant colonies per total number of viable cells seeded. Logarithmic means of HR frequencies +/− SEM are based on four independent experiments.

Figure 3

The function of BRCA2 on HR enhancement depends on its interaction with Rad51. HR frequencies were assessed with the same assay as described in Fig. 2b. Donor plasmid pΔ3 and BRCA2-HA expression vector or control vector were cotransfected with wt BRC4 or mutant BRC4-M5 repeats into Capan/pΔ2-puro cells. The wt BRC4, but not mutant BRC4-M5 repeat, dominant-negatively inhibited the enhancement effect of BRCA2 on HR. Logarithmic means +/− SEM are based on four independent experiments.

Figure 4

Restoration of wt BRCA2 enhances clonogenic survival of Capan-1 cells after exposure to ionizing radiation, but not to UV light. (a) Surviving fractions after irradiation were determined for untransfected Capan-1 cells and in derivatives with or without constitutive wt BRCA2 expression. (b) Clonogenic survival following exposure to UV-C was assessed as for x-ray exposure. Error bars are SEM.

Figure 5

BRCA2 is not involved in NHEJ. (a) Random integration of plasmid pΔ2-puro with or without BRCA2 expression vector was assessed via selection by puromycin in Capan-1 cells. Integration frequencies were calculated from the number of puroR colonies per number of viable cells seeded. Logarithmic means +/− SEM are based on four independent experiments. (b) Human BRCA2 deficiency does not compromise rejoining of IR-induced DSBs. Induction of DSB (Left) and DSB-rejoining kinetics (Right) by pulse-field gel electrophoresis in Capan-1 cells with or without constitutive wt BRCA2 expression. (Top Left and Right) Typical gels. (Bottom Left and Right) Quantification of the results obtained in three independent experiments. Plotted is the fraction of activity released (FAR) as a function of radiation dose or as a function of time after exposure to 40-Gy x-rays.