Please cite this article in press as: Periasamy et al., Targeting Phosphopeptide Recognition by the Human BRCA1 Tandem BRCT Domain to Interrupt
from BACH1 (Figure 4A). When this BACH1 sequence is phos- whether drug-like inhibitors of substrate recognition by tBRCT
phorylated by intracellular protein kinases, it is recognized by can elicit selective biological effects is uncertain. To test this
the BRCA1 tBRCT, apposing the Tag-BFP and Tag-GFP2 re- issue, we first determined the effects of MDC1 tBRCT overex-
porters to induce FRET. HEK293 cells were stably transfected pression on the formation of damage-induced MDC1 or BRCA1
with a construct encoding the biosensor under the control of a foci in cells (Figures 5C and 5D). As expected, MDC1 tBRCT
tetracycline-inducible promoter. Constructs encoding Tag-BFP suppresses both MDC1 and BRCA1 foci formation, since
or Tag-GFP2 alone were used as controls for spectral correction MDC1 recruitment to damage sites precedes and is required
to calculate FRET efficiency. For validation, we first exposed for BRCA1 accumulation (Huen et al., 2007; Kolas et al.,
cells stably expressing the biosensor to 16 Gy ionizing radiation 2007; Mailand et al., 2007). Interestingly, however, Bractoppin,
(
IR), which is reported to activate signals leading to BACH1 but not its inactive analog CCBT2047, selectively inhibits dam-
phosphorylation (Peng et al., 2006; Shiozaki et al., 2004). Indeed, age-induced BRCA1 foci formation, but has little effect on the
this suffices to induce FRET (Figure 4B). FRET is abrogated (Fig- radiation-induced accumulation of MDC1 at sites of DNA dam-
ure 4B) by the replacement of the Ser residue in the BACH1 age (Figures 5C, 5D, and S3E). Similarly, Bractoppin has little
substrate peptide that undergoes phosphorylation, with the effect on the radiation-induced recruitment of TOPBP1 (Fig-
non-phosphorylable residue, Ala (Ser990Ala biosensor), con- ure S3F), a protein containing multiple, structurally related
firming the specificity of pSer recognition by the BRCA1 tBRCT tBRCT domains, again speaking to the selectivity of its effects.
in the biosensor construct.
Thus, collectively, our observations provide multiple lines of
Overexpression of a construct encoding the BRCA1 tBRCT evidence that Bractoppin selectively inhibits the recognition of
domain––which is expected to mimic the cellular effects of Brac- phosphopeptide substrates by the human BRCA1 tBRCT, sup-
toppin by competitively inhibiting phosphopeptide substrate pressing the recruitment of BRCA1, but not other proteins con-
recognition––suffices to decrease FRET efficiency detected by taining structurally related tBRCT domains, to cellular DNA
the biosensor, as measured by sensitized emission (Figure 4C) damage sites.
or acceptor photobleaching (Figure 4D). Notably, Bractoppin–
but not its inactive iso-butyl substituted analog, CCBT2047–– Bractoppin Interrupts DNA Damage Signaling for G2
also inhibits FRET (Figures 4C, 4D, and S2).
Arrest
BRCA1 recruitment to sites of DNA damage initiates events lead-
ing to cell-cycle arrest at the G2 checkpoint (Yarden et al., 2002;
Yu and Chen, 2004). Overexpression of the BRCA1 tBRCT in-
Bractoppin Selectively Inhibits Cellular Substrate
Recognition by the BRCA1 but Not MDC1 tBRCT
Following exposure to IR, BRCA1 protein is recruited to cellular hibits damage-induced G2 arrest (Figures 6A and 6B), whereas
sites of DNA damage, where it assembles in microscopic foci, the single (M1775R) and the double (S1655A, K1702M) tBRCT
through the recognition of a phosphorylated motif in the adaptor mutants do not (Figures S4A–S4D). G2 arrest is also inhibited
protein ABRAXAS via the BRCA1 tBRCT (Wang et al., 2007; Wu in a dose-dependent manner by Bractopppin, but not by its inac-
et al., 2016). Again, overexpression of the BRCA1 tBRCT tive analog CCBT2047 (Figures 6A and 6B), suggesting that the
domain, but neither the tBRCT M1775R nor S1655A/K1702M compound interrupts signals that activate the G2 checkpoint.
mutant forms deficient in phosphopeptide substrate binding, Failure to engage the G2 checkpoint sensitizes cells to the cyto-
decreases BRCA1 foci formation following DNA damage as toxic effects of IR (Tenzer and Pruschy, 2003). Indeed, overex-
measured by high-content microscopy using a murine mono- pression of the BRCA1 tBRCT significantly enhances cytotox-
clonal antibody against BRCA1 (Figures S3A–S3C). Bractoppin, icity induced by exposure to 1 Gy IR, as does treatment with
but not its inactive analog, CCBT2047, also inhibits the formation Bractoppin in a dose-dependent manner (Figure 6C). Together,
of radiation-induced BRCA1 foci (Figures 5A and 5B). Similar re- these findings demonstrate that Bractoppin inhibits intracellular
sults were observed in a different cell line using an alternative signals essential for the response of human cells to DNA
monoclonal antibody directed against a distinct BRCA1 epitope damage.
(
Figure S3D).
The human protein MDC1 is also recruited to microscopic Bractoppin Discriminates BRCA1-Dependent Steps in
foci formed at sites of DNA damage through interactions medi- DNA Repair by Homologous Recombination
ated by its tBRCT domains (Lou et al., 2003; Stewart et al., IR-induced double-strand DNA breaks are repaired in dividing
2
003; Stucki et al., 2005). While the MDC1 tBRCT domain is cells by homologous DNA recombination (HR), a mechanism in
structurally related to that of BRCA1 (Campbell et al., 2010), which BRCA1, and the related tumor suppressor protein
the extent of substrate cross-recognition by these tBRCT BRCA2, have been implicated at several steps (reviewed in Ven-
domain family members remains unclear. In turn, given the po- kitaraman, 2014). HR is initiated by the resection of DNA ends to
tential for substrate cross-recognition in the cellular milieu, generate ssDNA tracts that are coated by the ssDNA-binding
(B) Percentage of cells positive for radiation-induced nuclear BRCA1 foci (mean ± SD; n = 15,000, 0 Gy, 20,000, 16 Gy; 10,500, BRCA1 tBRCT; 10,600, Brac-
toppin; 13,000, CCBT2047) enumerated by high-content imaging at low magnification (see the STAR Methods). Treatment conditions were as described in (A).
Statistical significance was determined using an unpaired two-tailed t test. ***p % 0.001. Similar results were observed in three independent repeats.
(
C) Percentage of cells positive for radiation-induced nuclear MDC1 foci (mean ± SD; n = 24,000, 0 Gy; 6,000, 16 Gy; 14,500, MDC1-tBRCT; 8,000, Bractoppin;
,600, CCBT2047) enumerated as above. Treatment conditions were as described in (A), except that the effect of Tet-induced MDC1 tBRCT expression was
tested. Statistical significance was determined using an unpaired two-tailed t test. ***p % 0.001; ns, not significant.
D) Cells treated as in (C) were stained for nuclear BRCA1 foci. Similar results were observed in three independent repeats. **p % 0.01; ***p % 0.001.
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Cell Chemical Biology 25, 1–14, June 21, 2018