The BRCT Domains of the BRCA1 and BARD1 Tumor Suppressors Differentially Regulate Homology-Directed Repair and Stalled Fork Protection

Abstract

The BRCA1 tumor suppressor preserves genome integrity through both homology-directed repair (HDR) and stalled fork protection (SFP). In vivo, BRCA1 exists as a heterodimer with the BARD1 tumor suppressor, and both proteins harbor a phosphate-binding BRCT domain. Here, we compare mice with mutations that ablate BRCT phospho-recognition by Bard1 (Bard1^S563F and Bard1^K607A) or Brca1 (Brca1^S1598F). Brca1^S1598F abrogates both HDR and SFP, suggesting that both pathways are likely impaired in most BRCA1 mutant tumors. Although not affecting HDR, the Bard1 mutations ablate poly(ADP-ribose)-dependent recruitment of BRCA1/BARD1 to stalled replication forks, resulting in fork degradation and chromosome instability. Nonetheless, Bard1^S563F/S563F and Bard1^K607A/K607A mice, unlike Brca1^{S1598F/S1598F} mice, are not tumor prone, indicating that HDR alone is sufficient to suppress tumor formation in the absence of SFP. Nevertheless, because SFP, unlike HDR, is also impaired in heterozygous Brca1/Bard1 mutant cells, SFP and HDR may contribute to distinct stages of tumorigenesis in BRCA1/BARD1 mutation carriers.

Keywords: BARD1; BRCA1; BRCT domain; DNA break repair; familial breast cancer; genome instability; poly(ADP-ribose); stalled replication forks; tumor suppression.

Figure 1.. Bard1^SF/SF cells are hypersensitive to DNA damaging agents and display genotoxin-induced chromosomal instability.

A) Colony survival analysis of MMC-treated isogenic Bard1^+/+ and Bard1^SF/SF MEFs, along with Brca1^+/+ and Brca1^SF/SF MEFs. Survival is quantified as percentage of colonies on MMC-treated relative to untreated plates. Each condition was tested in triplicate, and error bars represent standard error of the mean. B) Colony survival analysis of olaarib-treated Bard1^+/+ and Bard1^SF/SF MEFs, along with Brca1^+/+ and Brca1^SF/SF MEFs. C) Bard1^+/+ and Bard1^SF/SF primary MEFs were cultured with or without 40 ng/mL MMC for 16 hours and structural chromosome abnormalities were quantified by T-FISH. The mean number of aberrations per cell is denoted by a horizontal red line, and the error bars represent standard error of the mean. P values were calculated by unpaired Student’s T-Test (n.s. = no significance, ** = p<0.01, *** = p<0.001). D) T-FISH analysis of Bard1^+/+ and Bard1^KA/KA primary MEFs.

Figure 2.. Bard1^SF/SF and Bard1^KA/KA cells are competent for Rad51 focus formation and homology-directed repair (HDR).

A) Brca1 focus formation in isogenic Bard1^+/+ and Bard1^SF/SF MEFs and Brca1^+/+ and Brca1^SF/SF MEFs was measured 1 hour after exposure to 10 Gy. Each bar graph is an average of three independent experiments, and the error bars represent standard error of the mean. Statistical analyses were conducted using unpaired Student’s T-Test (** = p<0.01, **** = p<0.0001). B) Bard1 focus formation evaluated as in panel A C) Rad51 focus formation evaluated as in panel A. D) HDR efficiency was measured in Brca1^+/+ and Brca1^SF/SF embryonic stem (ES) cell subclones containing an integrated DR-GFP reporter. Cells were transfected with an empty (EV) or I-SceI-expressing (I-SceI) vector and the percentage of GFP-positive cells quantified by flow cytometry. Each ES cell subclone was analyzed in triplicate transfections. Error bars represent standard error of the mean. E) HDR efficiency was measured in independent subclones of isogenic Bard1^+/+ and Bard1^SF/SF ES cells (left) and Bard1^+/+ and Bard1^KA/KA ES cells (right) containing an integrated DR-GFP reporter.

Figure 3.. Bard1^SF/SF, Bard1^KA/KA, and Brca1^SF/SF cells have a defect in stalled fork protection.

A) Schematic of the DNA fiber assay. MEFs were exposed to sequential 20-minute pulses of IdU and CldU and then harvested immediately (–HU control) or after a 90-minute incubation with 2 mM hydroxyurea (HU). As indicated, cells were exposed to 50 µM mirin to inhibit Mre11 nuclease activity. B) DNA fiber analysis of isogenic immortalized Bard1^+/+ and Bard1^SF/SF MEFs. For each condition, the CldU/IdU ratios of at least 150 individual DNA fibers are presented as a dot plot, and the median CldU/IdU ratio is denoted by a horizontal red line. Statistical analyses were conducted using the Mann-Whitney rank sum test (**** p<0.0001). C) DNA fiber analysis of immortalized Bard1^+/+ and Bard1^KA/KA MEFs. D) DNA fiber analysis of primary Bard1^+/+, Bard1^SF/SF, and Bard1^KA/KA MEFs. E) DNA fiber analysis of immortalized Brca1^+/+ and Brca1^SF/SF MEFs.

Figure 4.. The recruitment of Brca1/Bard1 heterodimers to PCNA replication factories is defective in HU-treated Bard1^SF/SF and Bard1^KA/KA cells.

A) Representative images of late S phase PCNA⁺ cells. Upon immunoflourescent co-staining with antibodies specific for PCNA (red) and Bard1 (green), three distinct populations of PCNA⁺ cells were identified: 1) cells displaying ≥5 PCNA foci but no Bard1 foci (PCNA ⁺Bard1^–, top); 2) cells displaying both ≥5 PCNA foci and ≥5 Bard1 foci that were spatially non-overlapping (PCNA⁺Brca1⁺ non-co-localizing, middle); and 3) cells displaying both ≥5 PCNA foci and ≥5 Bard1 foci in which more than half of the Bard1 foci co-localize with PCNA foci (PCNA⁺Brca1⁺ co-localizing, bottom). In panels B and C, the percent co-localization is the number of PCNA⁺Bard1⁺ co-localizing cells divided by the total number of PCNA⁺ late S phase cells (PCNA⁺Bard1^– + PCNA⁺Bard1⁺ co-localizing + PCNA⁺Bard1⁺ non -co-localizing). B) The percentage of late S phase cells with co-localizing PCNA and Bard1 foci in isogenic Bard1^+/+ and Bard1^SF/SF MEFs cultured for 90 minutes in the presence or absence of 2mM hydroxyurea (HU) and/or 100 nM olaparib (PARPi). At least 200 late S phase PCNA⁺ cells were examined for each condition. The histogram presents the average of three independent experiments and the error bars represent standard error of the mean. Statistical analyses were performed using one-way ANOVA (**** p<0.0001). C) The percentage of late S phase cells with co-localizing PCNA and Bard1 foci in Bard1^+/+ and Bard1^KA/KA MEFs cultured in the presence or absence of HU and/or PARPi. D) The percentage of late S phase cells with co-localizing PCNA and Brca1 foci in Bard1^+/+ and Bard1^SF/SF MEFs cultured in the presence or absence of HU and/or PARPi. E) The percentage of late S phase cells with co-localizing PCNA and Brca1 foci in Bard1^+/+ and Bard1^KA/KA MEFs cultured in the presence or absence of HU and/or PARPi.

Figure 5.. The assembly of Brca1/Bard1 heterodimers onto stalled replication forks is defective in Bard1^KA/KA and Bard1^SF/SF cells.

A) For iPOND analysis (Sirbu et al., 2012), cell lysates and iPOND-purified fractions were prepared from untreated cell cultures and from parallel cultures pulsed-labeled for 10 minutes with IdU. The IdU-labeled cultures were harvested immediately or after a subsequent 90-chase with 2mM hydroxyurea (HU) and/or 100 nM olaparib (PARPi). B) Immunoblot analysis of protein abundance in the input cell lysates (left) and the corresponding iPOND-purified fractions (right) from Bard1^+/+ and Bard1^SF/SF cells. C) Immunoblot analysis of protein abundance in the input cell lysates (left) and the corresponding iPOND-purified fractions (right) from Bard1^+/+ and Bard1^KA/KA cells.

Figure 6.. Although Bard1^SF/SF and Bard1^KA/KA cells accumulate DNA damage during replication stress, Bard1^SF/SF and Bard1^KA/KA mice are not tumor prone.

A) The alkaline comet assay was used to assess HU-induced DNA damage in isogenic Bard1^+/+ and Bard1^SF/SF MEFs, as well as isogenic Brca1^+/+ and Brca1^SF/SF MEFs. For each condition, the individual tail moments of at least 75 cells are presented as a dot plot; the mean tail moment is denoted by a horizontal red line and the standard error of the mean is indicated by error bars. Statistical analyses were conducted using one-way ANOVA (Bard1 clones) or unpaired Student’s t test (Brca1 clones) (**** p<0.0001). B) Alkaline comet assay to assess HU-induced DNA damage in Bard1^+/+ and Bard1^KA/KA MEFs, as well as Brca1^+/+ and Brca1^SF/SF MEFs. C) Kaplan-Meier tumor-free survival curves of the Bard1^+/+ (n = 28), Bard1^SF/SF (n = 39), and Bard1^KA/KA (n=34) cohorts. Using the log-rank (Mantel-Cox) test, no statistical significance (defined as p<0.05) was observed between the Bard1^SF/SF and Bard1^+/+ curves (p = 0.9854) or the Bard1^KA/KA and Bard1^+/+ curves (p = 0.8387). The Kaplan-Meier survival curve of Brca1^SF/SF mice (n=72) from (Shakya et al., 2011) is superimposed for comparison.

Figure 7.. Heterozygous Bard1^SF/+, Bard1^KA/+, and Brca1^SF/+ cells are haploinsufficient for stalled fork protection and accumulate DNA damage upon replication stress.

A) DNA fiber analysis (as described in Figure 3) of Brca1^+/+, Brca1 ^SF/SF, and Brca1^SF/+ MEFs. B) DNA fiber analysis of Bard1^+/+, Bard1^KA/KA, and Bard1^KA/+ MEFs. C) Alkaline comet assay (as described in Figure 6) to assess HU-induced DNA damage in isogenic panels of wild type, homozygous-mutant, and heterozygous-mutant MEFs harboring the Brca1^SF, Bard1^SF, and Bard1^KA alleles. Statistical analyses were conducted using one-way ANOVA (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001). D) DNA fiber analysis of Bard1^+/+, Bard1^Q552X/+, Bard1^co-rec/+, Bard1^SF/+, and Bard1^KA/+ MEFs.