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Received 15 Apr 2014 | Accepted 7 Oct 2014 | Published 17 Nov 2014

Shailja Pathania1,2, Sangeeta Bade2, Morwenna Le Guillou3, Karly Burke1, Rachel Reed2, Christian Bowman-Colin1,2, Ying Su2, David T. Ting1,4, Kornelia Polyak1,2, Andrea L. Richardson2,5, Jean Feunteun3, Judy E. Garber1,2 & David M. Livingston1,2

BRCA1a breast and ovarian cancer suppressor genepromotes genome integrity. To study the functionality of BRCA1 in the heterozygous state, we established a collection of primary human BRCA1 / and BRCA1mut/ mammary epithelial cells and broblasts. Here we report that all BRCA1mut/ cells exhibited multiple normal BRCA1 functions, including the support of homologous recombination- type double-strand break repair (HR-DSBR), checkpoint functions, centrosome number control, spindle pole formation, Slug expression and satellite RNA suppression. In contrast, the same cells were defective in stalled replication fork repair and/or suppression of fork collapse, that is, replication stress. These defects were rescued by reconstituting BRCA1mut/ cells with wt BRCA1. In addition, we observed conditional

haploinsufciency for HR-DSBR in BRCA1mut/ cells in the face of replication stress. Given

the importance of replication stress in epithelial cancer development and of an HR defect in breast cancer pathogenesis, both defects are candidate contributors to tumorigenesis in BRCA1-decient mammary tissue.

DOI: 10.1038/ncomms6496 OPEN

BRCA1 haploinsufciency for replication stress suppression in primary cells

1 Harvard Medical School, Boston, Massachusetts 02115, USA. 2 Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA. 3 Stabilit Gntique et Oncogense, Universit Paris-Sud, CNRS-UMR8200, Gustave-Roussy, Villejuif 94805, France. 4 Department of Hematology/Oncology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA. 5 Brigham and Womens Hospital, Harvard Medical School, 75 Francis St, Boston, Massachusetts 02115, USA. Correspondence and requests for materials should be addressed to S.P. (email: mailto:[email protected]

Web End [email protected] ) or to D.M.L.(email: mailto:[email protected]

Web End [email protected] ).

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Germ line BRCA1 mutations increase greatly the risk of breast and ovarian cancer13. While all cells of males and females with germline BRCA1 mutations exhibit a

heterozygous BRCA1mut/ genotype, cancer develops primarily in females, often at young ages and almost exclusively affects the breast and ovaries. Why BRCA1 is largely a breast and ovarian cancer susceptibility gene, why males are largely protected from BRCA1 cancer and how an ostensibly normal mammary epithelial cell in a BRCA1 mutation carrier (BRCA1mut/ ) gives rise to breast cancer cells are largely unknown.

In addition, there is insufcient mechanistic insight into BRCA1 breast tumorigenesis on which to base rational preventive strategies. Their design will, in part, require a deeper appreciation of the biological properties of a heterozygous but ostensibly normal, mammary epithelium (BRCA1mut/ ).

The tumour-suppressing BRCA1 protein is an E3 ubiquitin ligase and a multi-functional scaffold that binds numerous partner proteins4,5. It plays a key role in genome integrity maintenance, which appears to be an essential component of its tumour-suppressing function2,5,6.

A BRCA1 loss of heterozygosity (LOH) event is a consistent characteristic of fully developed BRCA1-linked tumour cells. Two generic models describe the chain of events that precede it and the concomitant emergence of mammary tumour cells (human mammary epithelial cells (HMECs)). In one, HMECs, despite being heterozygous, are histologically and biologically normal before the emergence of LOH. They fail to exhibit a signicant defect in BRCA1 function. Here key events that transform a cell to malignancy follow the loss of all BRCA1 functions at the LOH event and are often preceded by acquisition of a p53 mutation that sustains cell viability in the face of emerging genome disorder7.

In the other model, BRCA1mut/ HMECs are haploinsufcient for the performance of one or more BRCA1 functions even before any signs of a neoplastic cell phenotype emerge. This model implies that, from the time that mammary epithelial development is complete or at some relatively early time thereafter, BRCA1mut/ HMECs cannot perform all BRCA1 genome integrity maintenance functions at normal amplitude.

These abnormalities may increase the likelihood that steps in the mammary tumorigenesis process begin long before they become clinically apparent.

In this regard, there is growing evidence of a defect in normal mammary epithelial progenitor differentiation in histologically normal, BRCA1 heterozygous mammary tissue811, implying that the second model is more likely valid than the rst. Thus, determining whether BRCA1 heterozygosity confers haploinsufciency on HMECs for any of the multiple, known, BRCA1 functions is a potentially valuable step in achieving a better understanding of BRCA1 mutation-driven cancer predisposition. In this regard, we have analysed a new collection of primary mammary BRCA1mut/ epithelial cells and skin broblasts obtained from BRCA1 mutation carriers for such functions.

ResultsPrimary cell genotyping and lineage determination. Established elements of BRCA1 function were analysed in freshly isolated, morphologically non-neoplastic, primary HMECs and skin broblasts derived from multiple BRCA1 / and BRCA1mut/ tumour-free women. Twenty-three primary BRCA1mut/ bro-blast cultures were derived from skin punch biopsies, and 15 primary BRCA1mut/ HMEC cultures were generated from individual prophylactic mastectomy samples (Table 1). HMECs were cultured in serum-free medium.

The properties of BRCA1mut/ HMECs were compared with BRCA1 / HMECs (N 7), freshly derived from reduction

mammoplasty tissue, and those of BRCA1mut/ skin broblasts with freshly isolated BRCA1 / broblasts (N 11; Table 1).

Mutations in BRCA1 mutant broblasts and HMECs were conrmed by homogenous Mass-Extend (hME) analysis12 and by direct BRCA1 gene sequencing (Supplementary Fig. 1ac).

Table 1 | Primary broblast and HMEC strains (BRCA1 / and BRCA1mut/ ) used in this study.

Number Study ID Procedure Age Mutation Fibroblasts

1 26 Skin Punch Biopsy 38 185delAG2 32 Skin Punch Biopsy 55 185delAG3 33 Skin Punch Biopsy 56 185delAG4 34 Skin Punch Biopsy 29 Y1463X5 39 Skin Punch Biopsy 50 S713X6 45 Skin Punch Biopsy 49 5083del197 46 Skin Punch Biopsy 26 1137delG8 47 Skin Punch Biopsy 48 185delAG9 48 Skin Punch Biopsy 32 4184del4 10 53 Skin Punch Biopsy 52 185delAG11 54 Skin Punch Biopsy 53 4154delA12 57 Skin Punch Biopsy 27 185delAG13 62 Skin Punch Biopsy 38 1294del40 14 65 Skin Punch Biopsy 36 3819del515 68 Skin Punch Biopsy 46 Q491X16 69 Skin Punch Biopsy 30 5385insC17 73 Skin Punch Biopsy 37 795delT18 76 Skin Punch Biopsy 43 2530delAG 19 78 Skin Punch Biopsy 42 W1815X20 80 Skin Punch Biopsy 49 185insA21 82 Skin Punch Biopsy 62 185delAG 22 83 Skin Punch Biopsy 45 IVS19 1G4A

23 1075 Skin Punch Biopsy 26 185delAG

1 WT Skin Punch Biopsy 56 N/A 2 1002 Skin Punch Biopsy 33 N/A 3 1004 Skin Punch Biopsy 58 N/A 4 1006 Skin Punch Biopsy 43 N/A 5 1007 Skin Punch Biopsy 50 N/A 6 1008 Skin Punch Biopsy 48 N/A 7 1009 Skin Punch Biopsy 69 N/A 8 1010 Skin Punch Biopsy 46 N/A 9 1011 Skin Punch Biopsy 58 N/A10 AR8F Reduction Mammo 45 N/A11 AR20L Reduction Mammo 31 N/A

Mammary Epithelial Cells1 79 Proph. Mx 41 E143X2 1046 Proph. Mx 28 3725C4T3 1048 Proph. Mx 50 185delAG4 CP10 Proph. Mx 43 1135insA5 CP16 Proph. Mx 45 4065-4068del6 CP17 Proph. Mx 28 2012insT7 AR1 Proph. Mx 46 R1443X8 AR9 Proph. Mx 34 1100delAT9 AR10 Proph. Mx 28 1081G-4A10 AR11 Proph. Mx 37 5385insC11 AR12 Proph. Mx 48 R1203X12 AR13 Proph. Mx 26 5385insC13 AR14 Proph. Mx 38 5385insC14 AR15 Proph. Mx 44 2530delAG15 AR16 Proph. Mx 41 2983insT

1 CP14 Reduc. Mammo. 31 N/A

2 CP22 Reduc. Mammo. 49 N/A

3 CP29 Reduc. Mammo. 28 N/A

4 CP32 Reduc. Mammo. 38 N/A

5 AR4 Reduc. Mammo. 25 N/A

6 AR7 Reduc. Mammo. 33 N/A

7 N202 Reduc. Mammo. 27 N/A

N/A, not applicable.

Twenty-three primary broblast strains were derived from skin punch biopsies and 15 primary, mammary epithelial cell (HMECs) strains from prophylactic mastectomies (Proph. Mx) performed on BRCA1 mutation carrying (BRCA1mut/ ) women. One primary broblast strain (1075) was derived from breast skin tissue obtained during prophylactic mastectomy. BRCA1 / control

HMECs (n 7) were derived from reduction mammoplasty tissue (Reduc. Mammo.), and control

broblasts (n 11) were derived from skin punch biopsies and reduction mammoplasties from

women lacking BRCA1 mutations.

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Together, this collection of BRCA1mut/ mutations spans nearly the entire BRCA1 genome (Fig. 1a).

To determine the lineage of cells that grew out of our primary tissue samples under the culturing conditions used, we carried out

ow cytometry (FACS)-based analysis of lineage markers (CD44, CD49f, CD24 and EpCAM). In this study, our primary BRCA1mut/ and BRCA1 / HMEC cultures were similarly enriched in early basal (CD44high, CD24low, CD49fhigh

EpCAMlow) relative to luminal progenitor cells (CD44low, CD49flow, CD24high, EpCAMhigh)9,13 (Supplementary Fig. 2a). For this analysis, MCF7 was used as a luminal cell line control and MCFDCIS.com as a basal cell line control.

Furthermore, western blot analysis of whole-cell (for HMECs) and nuclear extracts (for broblasts) revealed that full-length BRCA1 expression in BRCA1mut/ HMEC (Fig. 1b and

Supplementary Fig. 2c) and broblast strains (Fig. 1c and supplementary Fig. 2b) was lower than that detected in wt BRCA1 / lines. This was in keeping with the proven genetic heterozygosity in these cells. As BRCA1 is much more abundant in

S and G2 than in G1, we only analysed wt and heterozygous HMEC and broblast cultures that exhibited similar cell cycle proles and BUdR uptake (see for example, Supplementary Fig. 2c).

Non-DNA repair-driven BRCA1 genome integrity functions. BRCA1 exhibits two types of genome integrity maintenance functionsthose that are directed towards the repair of DNA damage and checkpoint control, and others that sustain genome integrity by contributing to homeostatic functions that are not necessarily driven by DNA damage.

In this context, we asked whether the lower expression of BRCA1 in BRCA1mut/ cell cultures was associated with a deciency in the latter BRCA1 functions. BRCA1 is required for the maintenance of centrosome number14, mitotic spindle pole formation1517, mammary development through the regulation of master genes like Slug11 and heterochromatin-based satellite RNA suppression18.

Each of these functions was compared in heterozygous (BRCA1mut/ ) and control (BRCA1 / ) cells. Spindle formation was analysed by staining mitotic cells with a TPX2 antibody.

No abnormal spindle formation was detected in BRCA1mut/ cells (Fig. 2a and Table 1). The effects of robust BRCA1 depletion on this function have been documented15.

Similarly, we found that none of the BRCA1mut/ and BRCA1 / cells contained greater than 2 centrosomes, implying that centrosome maintenance was normal in these different

BRCA1mut/ strains (Fig. 2b and Supplementary Table 1). Although we did not detect any evidence of centrosome amplication in multiple BRCA1 heterozygous cells, other work7 with BRCA1 heterozygous tissue has detected a small increase of centrosome amplication (B5%) in the epithelial cells of heterozygous mammary tissue compared with 2.5% in wt tissue.

De-repression of satellite RNA transcription is also a feature of BRCA1 mutant tumours18. Furthermore, Brca1 heterozygous cells do not show evidence of satellite de-repression18. To test whether this phenotype was present in heterozygous BRCA1 HMECs, two approaches were employed. Quantitative RT-PCR (q-RT-PCR) was performed for alpha satellite variants (SATIII, SATa and mcBox). Satellite RNA transcript levels were also estimated by RNA FISH directed at another satellite RNA, HSATII. Very low levels of satellite RNA were present in primary HMECs, making it difcult to detect any satellite RNA signal by these methods (Supplementary Fig. 3a and b).

To address the effect of BRCA1 heterozygosity on Slug expression11, we compared the Slug level in BRCA1 / and

BRCA1mut/ HMECs by western blot analysis. In these experiments, MCF7 (a luminal breast cancer line) was used as a negative control and MDA-MB-231 (a basal line) was used as a positive control. No reproducible difference in Slug expression was detected between the BRCA1 / and BRCA1mut/ strains

185delAG

1100delAT

1135insA

185delAG

Q491X

Mammary epithelial cells (MECs)

BRCA1+/+ BRCA1mut/+

BRCA1

185insA

RING

E143X

AR7

CP29

CP10

CP16

BRCA1

GAPDH

191

795delT

1081G->A

1137delG

1294del40

Fibroblasts

BRCA1+/+ BRCA1mut/+

47*

2012insT

S713X

EXON 11

BRCA1

Loading control

2530delAG

191

2983insT

3725C->T

3819del5

40654068del4

4154delA

4184del4

R1203X

R1443X

Y1463X

5083del19

5385insC

BRCTs

5385insC

W1815X

BRCA1 mutation in fibroblasts

BRCA1 mutation in MECs

Figure 1 | Distribution of BRCA1 mutations and BRCA1 protein in cells derived from BRCA1 mutation carriers. (a) Cells were derived from skin punch biopsies and prophylactic mastectomies performed on BRCA1 mutation carrying women. (b) Western blot analysis of total BRCA1 protein levels in BRCA1mut/ and BRCA1 / HMEC lines. Equivalent amounts of whole-cell lysate (prepared in NETN300) were electrophoresed, blotted and the blots probed with an anti-BRCA1 monoclonal Ab (SD118). GAPDH served as a loading control. (c) Western blot analysis of BRCA1 protein levels in the nuclear fraction of BRCA1mut/ and BRCA1 / broblast strains. Cells were pre-lysed in pre-extraction buffer (PEB, details in

Materials and Methods), and the pellet was re-suspended in NETN400 buffer to prepare a nuclear extract. The intense BRCA1 band in 47 (185delAG, marked by an asterisk) is likely the previously discovered truncated product of this mutant allele45. A non- specic band served as the loading control.

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TPX2 DAPI+TPX2 TPX2

DAPI+TPX2

CP29 (BRCA1+/+)

AR8F (BRCA1+/+)

46 (BRCA1mut/+)

47 (BRCA1mut/+)

DAPI DAPI

+ -tubulin + -tubulin

CP22 (BRCA1+/+)

1006 (BRCA1+/+)

(BRCA1mut/+)

AR15 (BRCA1mut/+)

CP17 (BRCA1mut/+)

CP17

(BRCA1mut/+)

Percentage of BrdU-positive cells

50 50 1.8

AR7 CP22 CP29 079 CP10 CP16

45 40 35 30 25 20 15 10

50 CP29

1.61.41.2 1

0.80.60.4

0.20 AR7 CP10 CP17 079 CP16

CP29

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Sample ID

BRCA1mut/+

BRCA1+/+

BRCA1mut/+

Percentage of H3

(pS28)-positive cells

0Gy

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DamUV (10 J m ) IR (10 Gy)

CP32

CP16 CP17 079

CP32

0 0 2 4

UV (J m2)

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Sample ID

-H2AX

Rad51

Merge

Mammary epithelial cells

% Cells with Rad51 in IR-

induced foci

70 60 50 40 30 20 10

BRCA1mut/+ (CP16)

CP14

0 CP22

CP29

CP32

N202

AR4

AR7

AR10

79 CP10

CP16

CP17

AR13

AR14

AR16

Sample ID

Rad51

-H2AX Merge

BRCA1+/+ (WT)

BRCA1mut/+ (46)

BRCA1+/+ (CP29)

% Cells with Rad51 in IR-induced foci

Fibroblasts

AR8F

1002

1006

1007

1008

1009

1010

1011

Sample ID

Figure 2 | Spindle pole formation, centrosome number, checkpoint activation and Rad51 recruitment to DSB. (a) Representative images of HMECs (left panel) and skin broblasts (right panel), from BRCA1mut/ and BRCA1 / were immunostained with an anti- TPX2 Ab to detect spindles; n 50 spindles

were analysed for each strain. A summary of all strains that were tested in this assay is listed in supplementary Table 1. (b) Centrosome number was determined by immunostaining HMECs (left panel) and broblasts (right panel) with Ab to g-tubulin; n 50 cells for each line were counted and cells with

centrosomes r2 were considered normal. A summary of the lines that were tested is presented in Supplementary Table 1. (c) S-phase checkpoint in response to UV- and IR-induced DNA damage in control and BRCA1mut/ strains. Three BRCA1 / (AR7, CP22 and CP29) and three BRCA1mut/ HMEC strains (79, CP10 and CP16) were irradiated with increasing doses of UV (left panel). For IR-induced S-phase checkpoint analysis (right panel), cells were exposed to IR (10 Gy, red). Non-irradiated cells (0 Gy, blue) served as controls. Error bars indicate the s.d. between the results of three, independent experiments. (d) G2/M checkpoint activation in response to UV- and IR- induced DNA damage in BRCA1mut/ and control cells. BRCA1/ and

BRCA1mut/ cells were irradiated with either UV (10 J m 2) or IR (10 Gy), allowed to recover for 2 h and then harvested for FACS analysis. The percentage of cells in mitosis was determined by staining cells with propidium iodine (PI) and phosphorylated histone H3 (S28) antibody. Mock-irradiated (-Dam) cells served as controls. (e) HMECs and (f) broblasts were exposed to IR (10 Gy) and allowed to recover for 4 h. Cells were xed and co-immunostained with Abs to g-H2AX and Rad51. Graphs depicting the fraction of cells with Rad51 foci that co-localized with g-H2AX foci for each line are plotted for both

HMECs and broblasts (right panels in e and f). Mean and s.d. of at least three experiments for each strain are shown. wt BRCA1/ (green) and BRCA1mut/ (red) lines.

that were tested (Supplementary Fig. 3c). Addition of serum11 had similar effect on Slug expression in BRCA1 / and

BRCA1mut/ strains.

DNA damage checkpoints. BRCA1 plays an important role in regulating both the S phase and G2 checkpoints after DNA damage19,20. The efciency of post-damage checkpoint activation

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6496 ARTICLE

was also tested in BRCA1 heterozygous cells. We failed to detect any signicant difference in the ability of BRCA1 / and mut/ lines to mount either an S phase (Fig. 2c, left and right panel) or a

G2 checkpoint response (Fig. 2d) following IR or UV-induced DNA damage.

BRCA1 DNA repair functions double-strand break repair. BRCA1 plays an essential role in homologous recombination-type double-strand break repair (HR-DSBR)21,22. Defective HR-DSBR is a well-known property of BRCA1 and related, inherited breast cancers; molecular epidemiology results suggest that it is a risk factor for these cancers2325.

BRCA1 is attracted to discrete sites of DSB-containing damage, where it directs a complex HR repair response5,26. Long-standing results show that in BRCA1 / ES cells27, HR function is normal until both copies of BRCA1 are inactivated (BRCA1 / ).

By contrast, others have reported that targeting one copy of BRCA1 with a mutation (for example, 185delAG) in an established, spontaneously immortal line of human HMECs resulted in a subtle HR defect28. Thus, a detailed analysis of multiple, primary human BRCA1mut/ and BRCA1 / HMECs and broblasts was undertaken to search for evidence of BRCA1 haploinsufciency for HR-DSBR in this setting.

Two, well-validated assays were set up to measure HR-DSBR, by testing the recruitment of Rad51 (an indicator of a key step in HR)29 to sites of DSBs and by measuring the sensitivity to PARP inhibitors (PI). The rst assay clearly showed that BRCA1mut/ cells were competent in recruiting Rad51 to sites of DSBs (Figs 2e,f). Moreover, like HR-DSBR-competent cells, they were also insensitive to olaparib (a PI). This assay, described below, relies on the observation that sensitivity to a PI is dependent on the existence of an HR defect30. Indeed, BRCA1 tumour lines (which lack functional BRCA1 and reveal a defect in HR) are more sensitive to these agents than BRCA1 / cells31,32.

To study the effect of PARP inhibitors in our collection of BRCA1mut/ and BRCA1 / cells, a FACS-based cell survival assay of co-cultured cells was employed. Cells were colour-coded

and tested in pairs, where one cell strain emitted a uorescent signal (for example, strain A, GFP ) and the other (strain B) did

not. Strains A and B were mixed, co-plated and then exposed to a DNA damaging agent of choice. After 7 days of recovery, they were harvested and the relative abundance of each cell population was analysed by FACS (Fig. 3a). The ratio of green/non-green or non-green/green cells reected the relative survival of the two strains.

When BRCA1mut/ and BRCA1 / cells were compared for their sensitivity to olaparib, BRCA1mut/ cells were not found to be demonstrably sensitive (Fig. 3b,c). As a positive control, U20S cell line, made HR-DSBR incompetent by depleting BRCA1 (ShBRCA1 treated), was used along with control (ShLuc treated) cells. Once BRCA1 depleted, these cells proved to be highly sensitive to olaparib, while control cells were not (Fig. 3d). In addition, BRCA1mut/ HMEC viability was reduced by olaparib, again only after BRCA1 depletion (siBRCA1, Fig. 3e).

Thus, despite the linkage of HR to BRCA1 breast cancer suppression and in keeping with results obtained in mouse ES cells27, these results, too, suggest that BRCA1mut/ cells are not defective for HR-dependent DSBR function.

Stalled replication fork repair. BRCA1 also protects the genome from DNA damage resulting at stalled replication forks3336. It is rapidly attracted to these damage sites where it joins other proteins that are required for stalled fork repair (SFR). For example, BRCA1 is required for the generation of

phospho-RPA32-coated single-stranded DNA (ssDNA), a pre-repair step needed for recruitment of ATRIP/ATR to activate the intra-S and G2/M checkpoints that support SFR35,3739.

In the absence of BRCA1, a stalled fork is more likely to be bypassed by translesional synthesis35, or, it may collapse into DSB, a hallmark of replication stress and an established force in support of epithelial cancer development40,41. In the mammary epithelium, which undergoes normal periods of extreme proliferation (for example, during pubertal development and/or pregnancy), an accumulation of stalled forks, when not resolved, is likely to result in signicant replication stress.

Thus, we asked whether BRCA1mut/ cells are haploinsufcient in their ability to support SFR. Employing validated assays, we found that, by comparison with control cells, BRCA1mut/ broblasts and HMECs were defective in their SFR responses to replication-stalling agents like hydroxyurea (HU) and UV-C (ultraviolet radiation). We have shown previously that, in cells that were heavily depleted of BRCA1, recruitment of phospho-RPA32 (pRPA32) to chromatin was defective in response to UV35. This defect was also evident after treatment with HU (Supplementary Fig. 4a). When BRCA1mut/ cells were tested for their ability to recruit pRPA32 to chromatin after UV and/or HU treatment, a defect was detected in BRCA1mut/ cells (Fig. 4ac, and Supplementary Fig. 4ad).

To test whether these abnormal RPA binding observations in BRCA1mut/ cells are specically linked to BRCA1 haploinsufciency, we asked whether ectopic wt BRCA1 expression in

BRCA1mut/ cells corrects them. Infection by a lentiviral BRCA1 coding vector led to wt BRCA1 (HA-tagged) expression in primary BRCA1mut/ cells (Fig. 4f,g; Supplementary Fig. 5a).

This protein was recruited to DSBs and stalled forks in HMECs and broblasts like endogenous wt BRCA1 (Fig. 4f,g). Its expression suppressed the apparent, post-UV haploinsufcient defect in pRPA32 chromatin recruitment (Fig. 4h,i, respectively). Thus, this defect is a valid representation of BRCA1 haploinsufciency.

To test the generality of SFR haploinsufciency, we isolated MECs from Brca1 / and Brca1 / mice. These cells were used to study the generation of phospho-RPA32-coated ssDNA after UV- and HU-induced stalled fork formation. In keeping with results obtained with BRCA1 heterozygous human cells, we observed reduced phospho-RPA32 coating of ssDNA in Brca1 / mouse cells compared with WT Brca1 / cells (Fig. 4d). This underscores the generality of the nding that cells with one mutated allele for BRCA1 are haploinsufcient for pRPA32 loading on chromatin.pRPA32 loading on chromatin is dependent on the generation of ssDNA. Its generation after replication arrest is BRCA1-dependent35. To detect the generation of ssDNA, a BrdU immunoassay42 performed under non-denaturing conditions (HCl) was used. Here, using the same assay, we found that strain 39 (BRCA1mut/ ) generated less ssDNA (Supplementary

Fig. 4e,f) compared with strain 1002 (BRCA1 / ). This supports the hypothesis that BRCA1mut/ cells generate less ssDNA, which in turn results in less pRPA32 chromatin loading.

Of note, 1075 (a human 185delAG/ strain) failed to exhibit a

defect in ssDNA generation. This suggests that the post UV generation of ssDNA was not affected in these cells and explains why we did not observe a defect in pRPA32 loading in them. Possibly, steps downstream of ssDNA generation and pRPA32 loading are defective in 185delAG strains (see for example, below).

Finally, to test whether the inefcient loading of RPA at stalled forks in BRCA1mut/ cells is a reection of innately reduced RPA activation after DNA damage, we assayed for RPA recruitment to

DNA in response to UV laser-induced DSBs. As shown in Fig. 4e,

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GFP + ht ht DNA damage

Co-culture BRCA1mut/+ and BRCA1+/+ cells

8 Days later

FACS

Mammary epithelial cells

1.5

1.0

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Mut/WT

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0.6

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PARP inhibitor (M)

0.6

Fibroblasts (average)

Mut_54/WT_1004

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Mut_34/WT_1011

Mut_65/WT_1008

Mut_68/WT_1004

Mut_45/WT_1008

Mut_34/WT_1008

Mut_47/WT_1011

Mut_82/WT_1011

Mut_48/WT_1010

Mut_39/WT_1008

Mut_47/WT_1010

Mut_46/WT_WT

Mut_65/WT_AR8F

Mut_65/WT_1011

Mut_65/WT_WT

Mut_62/WT_1006

Mut_46/Mut_46

Mut_39/Mut_47

Mut_39/Mut_39

WT_1010/WT_1008

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WT_1006/WT_1011

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0.5

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0.2 0.4 PARP inhibitor (M)

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ShLuc/ShLuc

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PARP inhibitor (M)

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PARP inhibitor (M)

0.6

Figure 3 | FACS-based cell survival assay shows that HR-DSBR is not defective in BRCA1mut/ cells. (a) FACS-based cell survival assay was used to determine the sensitivity of cells to various DNA damage inducing agents. BRCA1mut/ and BRCA1 / colour-coded cells were co-plated and exposed to

DNA damaging agents. Cell survival data are plotted as a ratio of GFP positive to GFP negative cells. Ratio between WT/WT(Green), Mutant/Mutant (Blue) and Mutant/WT (Red) is plotted in the graphs below. (b) Combinations of BRCA1mut/ and BRCA1 / HMECs were exposed to different concentrations of a PARP inhibitor, and the ratio of each of these combinations was plotted (left). The average ratio of WT/WT, Mut/Mut and Mut/WT was also calculated and plotted (right). (c) (Left) Combinations of BRCA1mut/ and BRCA1 / broblasts were exposed to different concentrations of a

PARP inhibitor, and the survival ratio of each of these combinations was plotted (left). An average ratio of WT/WT, Mut/Mut and Mut/WT was also calculated and plotted (right). (d) U20S cells (containing or lacking a GFP reporter) were infected with ShLuc (control) or ShBRCA1 coding lentiviral vectors. Green ratio of number of ShLuc-treated cells to ShLuc-treated cells, that is (ShLuc/ShLuc), Blue ratio of number of ShBRCA1-treated cells to

ShBRCA1 treated-cells, that is, ShBRCA1/ShBRCA1 and Red ratio of number of ShBRCA1-treated cells to ShLuc-treated cells, that is, ShBRCA1/ShLuc.

Averages of the results of individual experiments are plotted. (e) BRCA1mut/ (CP10 and CP16) were transduced with shRNA directed at GAPDH (siGAPDH) or BRCA1 (siBRCA1). Three days post transfection, combinations of siGAPDH or siBRCA1-transduced BRCA1mut/ HMECs (CP10 and CP16)

were co-plated with AR7 (a BRCA1 / HMEC) and exposed to various doses of a PARP inhibitor. Averages of the results generated by these combinations were plotted. Error bars were calculated as the standard error propagation (SEP) in the ratios of each of the combinations in three independent experiments.

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RPA was equivalently recruited to these stripes in BRCA1mut/ and / cells. This rules out the possibility of an innate defect

in RPA activation after DNA damage.

An inability to form pRPA32-coated ssDNA after DNA damage may result in relevant checkpoint defects43. Although we detected an incomplete reduction in pRPA32-coated chromatin after UV-induced DNA damage in BRCA1mut/

HMECs, there was no obvious S or G2 checkpoint defect. Thus, incomplete formation of pRPA32-coated ssDNA, in the conditions tested, was, nonetheless, sufcient to initiate a proper checkpoint response.

Given that inefcient loading of pRPA32 on ssDNA is associated with an SFR defect, we asked whether BRCA1mut/ strains also experience an abnormally high frequency of collapsed forks compared with their WT counterparts (BRCA1 / ). Fork collapse can be captured by staining the cells with antibody to 53BP1 and/or g- H2AX, which is routinely recruited to these damaged structures35,44.

BRCA1mut/ cells, stained 18 h post UV with p-S1778 53BP1 and g-H2AX Abs, revealed an increase in fork collapse by comparison with wt controls (Fig. 5a,b). This again implies that the efciency of SFR is compromised in BRCA1mut/ cells, leading to higher fork collapse and incomplete resolution/repair of these structures. Thus, BRCA1 is haploinsufcient for the suppression of replication stress in primary HMECs and broblasts.

Of note, 185delAG-bearing strains (that is, 26, 47, 53, 57, AR19L) exhibited near normal loading of pRPA32 onto chromatin (marked with asterisk in Fig. 4a and Supplementary Fig. 4b), but more abundant 53BP1 foci by comparison with control cells (Fig. 5b). Others have shown that the 185delAG allele expresses a modestly truncated BRCA1 protein, translation of which is initiated immediately downstream of the mutation near the 50 end of the gene45. Thus, one might hypothesize that 185delAG is a hypomorph, capable of supporting some but not all BRCA1 SFR support functions.

To better understand the fate of collapsed forks in BRCA1mut/ cells, we carried out DNA bre analysis. We wished to assess two phenotypes: (1) the stability of nascent replication tracts after fork stalling and (2) the efciency of replication restart. Cells were pulse labelled with IdU for 20 min followed by treatment with or without 5 mM HU for 3 h. After washing off HU, cells were incubated in the presence of CldU for 30 min (Fig. 5c). This protocol allows the analysis of the fate of nascent replication tracts (synthesized before HU addition) during replication stalling, as well as replication fork restart after a replication block is eliminated (Fig. 5c).

DNA bre assays have been used previously with Brca1 / mouse ES cells to show that BRCA1 is required to suppress degradation of nascent strands after replication stalling induced by HU treatment36. In that study, replication restart was not affected by the absence of BRCA1.

In keeping with these results, we nd that, in the presence of HU, BRCA1mut/ cells exhibited increased degradation of the nascent strand (shorter green tracts) at stalled forks compared with the BRCA1 / cells (Fig. 5d,e; Supplementary

Fig. 5c). As shown in Fig. 5e, the distribution of nascent DNA tract lengths (green tracts, Fig. 5d) for BRCA1 /

MECs (CP32 and CP29) was not different between HU-treated and -untreated samples (red and grey curves, respectively). However, the red curves shifted towards shorter lengths (increased degradation) after treatment with HU in BRCA1mut/ cells (CP10 and CP17). By contrast, no signicant difference in the ability of BRCA1mut/ cells to restart replication was detected after replication stress had abated (Fig. 5ce;

Supplementary Fig. 5c).

These results further support our conclusion that the stability of stalled forks is compromised in BRCA1 heterozygous (BRCA1mut/ ) cells.

To assess further the conclusion that inefcient SFR in BRCA1mut/ cells results in increased DNA breaks, we employed comet assays. In UV-treated cells there was a greater increase in DNA breaks in BRCA1mut/ when compared with BRCA1 / cells (Supplementary Fig. 5d,e). This result reafrms the nding that, faced with replication stalling, BRCA1 heterozygous primary cells exhibited signs of replication stress, unlike BRCA1 / cells.

Roles of BRCA1-associated proteins in SFR in BRCA1mut/ cells. A stalled fork serves as a scaffold to recruit and concentrate proteins that play critical role/s in stabilizing, processing, repairing and restarting a stalled fork. This is essential to prevent the risk of its collapse into a DSB, a prime contributor to genomic instability. We tested a subset of the proteins that are known to be recruited to a stalled fork, with an eye towards those that interact and/or function together with BRCA1 to carry out SFR.

Specically, we asked whether recruitment of Rad51, a BRCA1 partner in HR-based DSBR46,47 and a protein known to play an HR-independent repair role at stalled forks48, is affected in BRCA1mut/ cells. We found that Rad51 recruitment to UV-induced stalled forks was reduced in BRCA1mut/ compared with

BRCA1 / cells (Supplementary Fig. 6b). This was not surprising, given that Rad51 is recruited to RPA-coated ssDNA, the generation of which is compromised in BRCA1mut/ cells.

We also found that the same BRCA1mut/ strains that revealed efcient Rad51 recruitment to DSBs (Fig. 2e,f) were defective in recruiting Rad51 to stalled forks. This implies that the role of Rad51 at a stalled fork is different from that at a DSB and further conrms the observations made by other groups who found that Rad51 helps restart stalled forks in an HR-independent manner36,48,49. In addition, Scully et al.50 detected signicant differences between the mechanism of repair at a non replication fork-associated DSB and at a stalled fork-induced break.

We next assayed the efciency of CtIP recruitment to UV-induced stalled forks. CtIP is an established BRCA1 partner51 and plays an important role in replication restart after stalled fork formation52,53. We found previously that BRCA1 is required for the recruitment of CtIP to UV-induced stalled forks35. In light of this evidence, we asked whether CtIP recruitment is compromised in BRCA1mut/ cells. Just as for Rad51,

BRCA1mut/ cells exhibited reduced CtIP recruitment to sites of UV-induced fork stalling (Supplementary Fig. 6a). It is unclear whether this defect in CtIP recruitment to stalled forks is a direct result of a reduced BRCA1 protein level or reduced pRPA32-coated ssDNA. Nonetheless, these data further conrm that BRCA1mut/ cells are defective in SFR.

Finally, we addressed the fate of Mre11 at stalled forks in BRCA1mut/ and BRCA1 / cells. Mre11 is a BRCA1-associated nuclease that has been implicated in helping restart collapsed and stalled replication forks via resection and initiation of repair at these sites49,54,55. Given that BRCA1mut/ cells exhibit reduced ssDNA generation, defective pRPA32 loading on chromatin and collapsed forks, we asked whether Mre11 recruitment mirrors this phenotype. The data revealed increased Mre11 recruitment to the sites of UV-induced stalled forks in BRCA1mut/ cells compared with BRCA1 / cells (Supplementary Fig. 6c). Given that Mre11 is a nuclease that is recruited to DSBs, it seems reasonable to propose that increased fork collapse in BRCA1mut/ cells results in DSBs, that, in turn, recruit Mre11. Thus, in lieu of possibilities discussed above,

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increased Mre11 recruitment to UV-induced stalled forks in BRCA1mut/ cells may be yet another indicator of the reduced stability of stalled forks in BRCA1mut/ cells.

Cell sensitivity to different DNA damage inducing agents. In an effort to validate the observation that primary BRCA1mut/ cells are defective for SFR and suppression of replication stress,

BRCA1+/+

BRCA1mut/+

BRCA1+/+

BRCA1mut/+

BRCA1+/+

BRCA1mut/+

1010

+ + +

WT WT

+ + + +

1002

45 46 47*

+ +

Loading control

UV UV

Loading control

UV 97

Lamin B1

pRPA32

BRCA1+/+ BRCA1+/+ BRCA1+/+

BRCA1mut/+ BRCA1mut/+ BRCA1mut/+

BRCA1+/+

BRCA1mut/+

N202

CP22

CP17

CP16 CP37 CP32 CP10 CP16 AR13

1002 53* 65 69

+ +

+ + + + + +

Loading control

pRPA32

-H2AX Merge

RPA

L/1 (Brca1+/ )

V/1 (Brca1+/+ )

L/10 (Brca1+/ )

V/1 (Brca1+/+)

3478/30 (Brca1+/)

UV + +

+ +

BRCA1+/+

(1008)

Loading control

BRCA1mut/+

(78)

pRPA32

48 (BRCA1mut/+), fibroblast

CP17 (BRCA1mut/+), MEC

BRCA1 Merge

BRCA1

Merge

IR-induced

DSBs

IR-induced

DSBs

UV induced

stalled forks

UV-induced

stalled forks

(BRCA1+/+) 1004

(BRCA1mut/+) 69

(BRCA1mut/+) 65

(BRCA1mut/+) 48

(BRCA1+/+) (BRCA1mut/+)

CP17

eGFP

+ +

eGFP

CP22 CP16

+ + + + + +

UV + + + +

Loading control

pRPA32 pRPA32

191

97 51

39 39

Loading control

pRPA32

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the relative sensitivity of these primary cells to stalled fork-inducing agents, like UV and cisplatin56, was tested in differentially coloured, co-cultured cells. In multiple comparisons of primary BRCA1mut/ and BRCA1 / broblasts and HMECs, the heterozygotes were signicantly more sensitive than the wt cells to UV (Fig. 5e,f) and cisplatin (Fig. 5g,h).

HR-DSBR in BRCA1mut/ cells undergoing replication stress. The discordance between multiple intact and one defective

BRCA1-associated functions in numerous, primary heterozygous cell strains suggests that BRCA1mut/ cells can preferentially direct their limited stores of intact BRCA1 protein to checkpoint activation, HR-DSBR, centrosome, SLUG control and spindle pole function, and less effectively to SFR. Alternatively, less BRCA1 protein is required for the former functions than the latter one. In either case, we asked whether, when these cells encounter sufcient replication stress, BRCA1 becomes preferentially dedicated to SFR and, in doing so, the pool of BRCA1 available for otherwise intact functions is reduced. If it falls sufciently, do BRCA1mut/ cells now become multiply haploinsufcient, that is, for other known BRCA1 functions that were formerly intact in these cells.

To address this possibility, we pre-exposed cells to increasing doses of UV and then assayed them for other BRCA1 functions (other than SFR). To assay for HR, the UV-treated cells were irradiated with IR and analysed for recruitment of Rad51 to DSBs (Fig. 6a). To assay for spindle formation and centrosome maintenance, we allowed the cells to recover for one and/or two full cycles of cell division and then analysed the cells for spindles as well as centrosomes.

As shown in Fig. 2e,f, multiple BRCA1mut/ and BRCA1 / cell strains recruited Rad51 to IR-induced DSBs with equal efciency in the absence of UV pre-treatment. However, the ability of BRCA1mut/ cells to recruit Rad51 to DSBs became increasingly defective after exposure to increasing doses of UV (Fig. 6a,b). No such effect was detected in BRCA1 / cells. We asked whether changes in BRCA1 protein levels in the UV pre-treated heterozygotes could account for reduced Rad51 recruitment, but no obvious alterations were observed (Supplementary Fig. 4e). This result, along with the observation that Rad51 protein levels in BRCA1mut/ and BRCA1 / were also similar (Fig. 6c), suggests that a defect in Rad51 recruitment to DSBs, in

UV- pretreated BRCA1mut/ cells, is a result of a defect in the ability of a limited pool of BRCA1 protein to respond to DSBs by driving the HR-DSBR process.

To assess further the apparent emergence of conditional haploinsufciency for HR-DSBR in the presence of replication

stress, we used the FACS-based assay described earlier to determine the survival efciency of BRCA1mut/ cells in the presence of olaparib. The question here was whether pre-exposure of cells to stalled fork-inducing damage (for example, UV) compromises the ability of these cells to carry out DSBR. If so, the BRCA1mut/ cells should become olaparib-sensitive.

Evidence presented in Fig. 6d showed this to be the case. Exposure of BRCA1mut/ cells to increasing doses of UV before adding olaparib rendered them acutely sensitive to a relatively low concentration of olaparib (Fig. 6d).

Centrosome number and spindle formation in the same cell strains were not altered under these conditions (data not shown). This implies that, at the very least, there is conditional haploinsufciency57 for HR-DSBR in BRCA1mut/ cells facing sufcient replication stress.

DiscussionMultiple primary broblast and HMECs derived from nontumour tissue of BRCA1 mutation carriers reveal, for the rst time, the existence of BRCA1 haploinsufciency for one of its established, genome integrity maintenance functions, that is, its ability to support SFR and to prevent replication stress. By contrast, no such defect was detected among several other such functions. Conceivably, haploinsufciency for some of these apparently unaffected BRCA1 functions occurs but takes considerably longer to develop during the life of a BRCA1mut/ individual than did the defect in SFR. In addition, the quantity of

BRCA1 needed to sustain at least some of its other functions may be signicantly less than that required for this activity.

Furthermore, in keeping with a model rst proposed by Bartek et al.57, the data presented here also reveal a possible hierarchy of DNA repair functions in BRCA1mut/ cells, wherein a defect in

SFR, if not resolved, can trigger an otherwise undetectable defect in HR-DSBR following enhanced replication stalling. In effect, representative primary BRCA1mut/ HMECs and broblasts exhibited a state of innate haploinsufciency for SFR and conditional haploinsufciency for HR-DSBR.

Thus, in keeping with the model of Bartek et al.57, we hypothesize that, when the amplitude of replication stalling rises above a threshold level in cells that are already deprived of a full complement of intact BRCA1, the available BRCA1 pool is dedicated rst to preventing and repairing collapsed forks. This leaves even less BRCA1 available to form complexes that are required for the execution of HR at DSB that are not associated with fork collapse. The latter effect can be hypothesized to give rise to the de novo development of an HR defect. This prediction was borne out experimentally.

Figure 4 | BRCA1mut/ cells derived from human and mouse tissue are defective in the generation of phospho-RPA32-coated ssDNA. (a) Phospho-RPA32 (pRPA32) loading on chromatin is BRCA1 dependent. After UV-induced DNA damage, BRCA1mut/ broblasts exhibited reduced pRPA32 loading on ssDNA, compared with BRCA1 / lines. Cells were irradiated with 30 J m 2 of UV and harvested 3 h post damage. Chromatin extracts were prepared, and the relevant western blot was probed with an antibody to phosphorylated RPA32 (S4/S8). The replication status for each line was tested on the day of the experiment by BrdU uptake measurement, and only those lines that exhibited similar replication proles were analysed. A subset of lines tested is shown here. Western blots for other WT and BRCA1 mutant lines are shown in Supplementary Fig. 4b. (b) BRCA1mut/ broblasts reveal reduced pRPA32 loading on ssDNA compared with BRCA1 / lines, after HU exposure (10 mM for 3 h). An asterisk marks strains with the 185delAG mutation.

(c) BRCA1mut/ HMECs reveal reduced pRPA32 loading on ssDNA, compared with BRCA1/ HMECs after UV irradiation. (d) Mammary epithelial cells derived from Brca1 / (L/10 and 3478/30) and/or Brca1 / (V/1) mice were analysed for pRPA32 levels on chromatin after UV- and HU-induced damage. (e) BRCA1mut/ cells efciently recruit RPA32 to DSBs. RPA32 loading at laser-induced DSBs was equivalently efcient in BRCA1mut/ and

BRCA1 / lines. Cells were co-stained with anti- g-H2AX to reect the existence of DSBs. (f) BRCA1mut/ skin broblasts (48) and (g) mammary epithelial cells (CP17), each infected with a lentiviral vector expressing HA- tagged BRCA1, were either irradiated with 10 Gy IR (upper panel) or 30 J m 2 of UV (lower panel). Cells were co-immunostained with Abs to BRCA1 and HA. (h,i) Phospho-RPA32 recruitment to ssDNA was analysed with a subset of primary BRCA1mut/ and BRCA1/ broblasts (h) and HMECs (i), infected with a lentiviral vector expressing either full-length WT BRCA1 (HA-tagged)

or eGFP (control). Western blots were immunostained with Ab to phospho-RPA32.

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53BP1

-H2AX DAPI

53BP1 -H2AX DAPI

BRCA1mut/+ (CP16)

BRCA1mut/+ (57)BRCA1+/+ (1002)BRCA1+/+ (CP32)

P =2.0 106

P =6.4 106

Mammary epithelial cells

% Of cells with >10

53BP1 foci/cell

CP14

0 CP22

CP29

CP32

N202

AR4

AR7 79

1046

1048

CP10

CP16

CP17

AR9

AR11

AR12

AR13

AR14

AR15

AR16

Sample ID

% Of cells with >10

53BP1 foci/cell

1011 26 32 33 34 39 45 46 47 48 54 57 62 65 68 69 73 76 78 80 82 83

Skin fibroblasts

0 WT

1002

1004

1006

AR8F

1007

1008

1009

1010

Wash off IdU and grow cells with +/ 5 mM HU for 3 h

50 45 40 35 30 25 20 15 10

5 0

Wash off HU add CldU (30 min)

BRCA1+/+(CP29) BRCA1mut/+(CP10)

CP10 (BRCA1mut/+)

Add IdU (20 min)

10 m 10 m

5 mM HU for 3 h 5 mM HU for 3 h

CP32 (BRCA1+/+) CP29 (BRCA1+/+)

CP17 (BRCA1mut/+)

50 45 40 35 30 25 20 15 10

5 0

50 45 40 35 30 25 20 15 10

5 0

50 P = 0.0734 P = 0.396 P < 0.000001 P < 0.000001

HU + HU

Frequency of tract length (%)

Survival ratio

45 40 35 30 25 20 15 10

5 0

0 2 4 6 8 10 12 14 16 18 20 22 24 26 Tract length (m)

MECs

1.5

1.0

0.5

0.0

Frequency of tract length (%)

0 2 4 6 8 10 12 14 16 18 20 22 24 26 Tract length (m)

MECs (average) Fibroblasts Fibroblasts (average)

MECs MECs (average) Fibroblasts Fibroblasts (average)

P=1.71009

P=4.21006

Survival ratio

1.5

1.0

0.5

0.0

Survival ratio

1.5

1.0

0.5

0.0

Survival ratio

1.5

1.0

0.5

0.0

P=0.000125 P =7.7410 06

0 5 10

UV (J m2)

8 10

15 20 0 2 4 6 UV (J m2)

8 10 0 2 4 6 UV (J m2)

Survival ratio

1.5

1.0

0.5

0.0

0 5 10

UV (J m2)

15 20

Survival ratio

1.5

1.0

0.5

0.0

Survival ratio

1.5

1.0

0.5

0.0

Survival ratio

1.5

1.0

0.5

0.0

0.0 0.5 1.0 Cisplatin (M)

1.5 2.0 0.0 0.5 1.0 Cisplatin (M)

1.5 2.0

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Why replication stress did not affect any other BRCA1 genome integrity maintenance function, other than HR-DSBR, is unknown. One possible explanation is that BRCA1 appears to execute each of these unaffected functions as a member of a large, multi-subunit complex(es)4,15,21,58,59. Conceivably, these complexes are sufciently stable, well-compartmentalized and function efciently enough before the onset of replication stalling that they are not disadvantaged by such an event.

Given the strong contributory history of inadequate SFR to epithelial cancer development and the fact that it is, thus far, the only apparent haploinsufcient BRCA1 DNA repair abnormality, we speculate that SFR haploinsufciency serves as an early and persistent contributor to the long process that gives rise to BRCA1 breast cancer (Fig. 6e). The fact that, when exposed to sufcient levels of replication stress, conditional BRCA1 HR-DSBR haploinsufciency emerged in BRCA1 heterozygous cells suggests that this defect might join SFR haploinsufciency as a BRCA1 breast cancer co-contributor in mammary epithelial progenitor cells that are experiencing sufcient ongoing replication stress. This would bet its widely accepted role as a major BRCA1 breast cancer risk factor.

Assuming that other BRCA1 functions remain to be discovered, it is conceivable that one or more of them, too, is haploinsufcient in the cells we have analysed. Thus, the current picture, while new, may be incomplete.

BRCA1 haploinsufciency for SFR was also apparent in Brca1 / mouse MECs. Given that Brca1 / mice are not

tumour prone60, this suggests that haploinsufciency for SFR in mMECs is not sufcient to drive tumorigenesis, especially given the short life span of mice. Furthermore, despite the tissue specicity (breast and ovary) of BRCA1 mutant cancer, a haploinsufcient phenotype was not limited to mammary epithelial cells. Similar defects were also displayed by BRCA1mut/ broblasts, thereby supporting the notion that multiple factors combine to generate the tissue specicity of

BRCA1-mutant cancer.

Indeed, while drafting this manuscript, a new haploinsufcient role for BRCA1 was reported61. The authors showed that transcription of the CYP1A gene, which encodes an estrogenmetabolizing enzyme, is upregulated in BRCA1 heterozygous cells61. In addition, Savage et al.61 showed that these oestrogen metabolites result in increased DNA damage in BRCA1 heterozygous cells. In light of the existence of defective SFR in BRCA1 heterozygous cells, it is reasonable to predict that such a defect would be a key avenue through which haploinsufciency for oestrogen metabolite detoxication could result in DNA damage.

Even if SFR haploinsufciency was not the only DNA repair defect in BRCA1 heterozygous HMECs, the high potential for it to give rise to chronic replication stress may well be clinically signicant. This is because chronic replication stress is an established and common force in human epithelial cancer formation40,41,62,63.

Moreover, others have observed defects in differentiation in populations of primary BRCA1mut/ HMECs811. As yet undened BRCA1 functional abnormalities underlie this set of phenotypes and could, when deciphered, enlarge the results described here. The extent to which SFR and, possibly, conditional HR-DSBR haploinsufciency contribute to them is unknown but worthy of investigation. DNA damage is known to perturb the differentiation of certain cell types64.

Recently, Winqvist et al.65 reported the existence of haploinsufciency for replication stress responsiveness in EBV-immortalized B lymphocytes and primary T cells derived from PALB2 heterozygotes. Their ability to perform HR was not analysed. These data obtained from cells with a single PALB2 mutant genotype represent an example of haploinsufciency for a known BRCA1- and BRCA2- interacting protein that is also a breast cancer suppressor. Thus, those results and evidence reported here imply that haploinsufciency in replication stress suppression is a feature of ostensibly normal mammary epithelial cells of two, different sets of mutation carriers.

In this regard, evidence of BRCA1 haploinsufciency was sought by Buchholz et al.66 in BRCA1 heterozygous broblasts and lymphocytes, and by Konishi et al.28 in a human HMEC line where a BRCA1 mutation (185delAG) was introduced into one allele by gene targeting. Buchholz et al.66 observed increased sensitivity of BRCA1 heterozygous broblasts to ionizing radiation (IR) and increased chromatid breaks in lymphocytes after IR. Given the pleiotropic effect of IR on DNA (for example, strand breaks, fork stalling, base damage, DNA-adducts6769), one cannot rule out that the sensitivity to IR is a result of contribution of multiple forms of DNA damage and not just a response to DSB formation.

Similarly, in Konishi et al.28 it was suggested that the targeted heterozygous clones were defective in HR-DSBR. Although increased sensitivity of these clones to IR and a reduced HRDSBR signal in HR reporter-containing cells were detected, they, like we, failed to observe any sensitivity of their BRCA1mut/ cells to PARP inhibition, raising a question regarding the existence of an HR defect. Given that the test cells were reported to be slow to proliferate, this could have contributed towards apparent HR deciency.

The possibility that persistent replication stress is a tumour-promoting force in BRCA1mut/ mammary epithelial cells offers,

Figure 5 | The stalled fork repair pathway is defective in BRCA1mut/ cells. Heterozygous BRCA1mut/ cells reveal increased DNA break formation, after stalled fork-inducing DNA damage, show reduced replication fork stability, and are more sensitive than WT BRCA1/ cells to stalled fork-inducing agents.

After exposure to a stalled fork-inducing agent (UV and/or HU), BRCA1mut/ cells were prone to increased fork collapse compared with BRCA1/ cells. (a) Skin broblasts, and (b) HMECs derived from BRCA1 mutation carriers (BRCA1mut/ ) and wild type BRCA1 counterparts (BRCA1/ ), were irradiated with low dose UV (5 J m 2) and allowed to recover for 18 h. Cells were immunostained with Ab to 53BP1 and g- H2AX (a marker for collapsed replication forks). The right (R) panel depicts the percentage of cells with Z10 53BP1 foci per cell in HMECs and broblasts. Mean and s.d. of at least three experiments for each strain are shown (green: wt BRCA1 /; red: BRCA1mut/ ). (c) Schematic representation of DNA bre experiment.

(d) Representative tracts from DNA bre experiments with HMECs (BRCA1 / , CP29; BRCA1mut/ , CP10) treated with 5 mM HU for 3 h. Green and red tracts correspond to IdU and CldU incorporation, respectively. Red scale bar represents 10 mm length. (e) Distribution curves of IdU tract lengthsin the presence and absence of HU (5 mM for 3 h) for both BRCA1 / (rst two plots, CP32 and CP29) and BRCA1mut/ (last two plots, CP10 and CP17)

cells. Red and Grey curves represent the presence and absence of HU in the culture medium, respectively. At least 200 tracts were scored for each distribution curve. (f,g) (Left panels) Combinations of BRCA1mut/ and BRCA1 / HMECs (f) and broblasts (g) were irradiated with different doses of UV. (Right) Average of data plotted on left. (h) Combinations of BRCA1mut/ and BRCA1 / HMECs and (i) broblasts were incubated with increasing concentrations of cisplatin for 15 h. Cells were allowed to recover for 6 days and then harvested for FACS analysis. Panels on the right show the averages of data plotted on the left.

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a hypothetical, mechanism-based route to BRCA1 breast cancer prevention. If specic subsets of BRCA1mut/ HMECs normally advance beyond the manifestation of an SFR defect to develop

additional BRCA1 functional deciencies accompanied by a much higher risk of tumorigenicity, their selective elimination might suppress subsequent BRCA1 breast cancer development.

Rad51

-H2AX Merge

BRCA1+/+ (CP29)

BRCA1mut/+ (CP16)

% Cells with Rad51 foci colocalized

with -H2AX foci

AR7

0J + 10Gy

0 J m2

5 J m2

10 J m2

15 J m2

15J + 10Gy 0J + 10Gy 15J + 10Gy

0 CP29

CP22 CP32

79 CP10 CP16 CP17

BRCA1+/+ BRCA1mut/+

Patient ID

(BRCA1+/+)

(BRCA1mut/+)

(BRCA1+/+)

(BRCA1mut/+)

CP22

CP29

CP17

AR7

CP29

CP10

CP16

Rad51

GAPDH

0 J m2

3 J m2

6 J m2

9 J m2

BRCA1+/+/BRCA1+/+ BRCA1mut/+/BRCA1mut/+ BRCA1mut/+/BRCA1+/+

WT_CP29/WT_CP29 WT_CP32/WT_CP32 Mut_CP16/Mut_CP16 Mut_079R/Mut_079R Mut_CP16/WT_CP32 Mut_79R/WT_CP32 Mut_CP16/WT_CP32

survival raio

1.40

1.20

1.00

0.80

0.60

0.40

* *

* * * *

* *

0.20

0.00

UV+PI(0.2 M)

UV UV

UV+PI(0.2 M))

UV+PI(0.2 M)

UV UV+PI(0.2 M)

UV+PI(0.2 M)

UV UV+PI(0.2 M)

Patient sample ID and experimental set up

Ostensibly normal

Breast cancer

BRCA1 LoH

BRCA1+/ Defectivestalled fork repairand ? MEC differentiation

Replication stress genomic instability

p53 +/+

SFR haplo

HR cond_haplo

Others + or ?

p53 mut

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Immnoblotting and antibodies. Whole-cell extracts were prepared by lysing cells in NETN300 lysis buffer (300 mM NaCl, 20 mM TrisHCl buffer pH7.8, 0.5% NP-40, 1 mM EDTA) for 1 h at 4 C. Nuclear extracts were prepared by pre-extracting the cytoplasmic protein fraction by incubating the cells in pre-extraction buffer, that is, PEB (0.5% Triton -X-100, 20 mM HEPES pH 7.0, 100 mM NaCl, 3 mM MgCl2 and 300 mM Sucrose). Incubation was carried out at 4 C for 20 min. Cells were pelleted, washed once in PEB, and lysed in NETN 400 lysis buffer(400 mM NaCl, 20 mM Tris-HCl buffer pH7.8, 0.5% NP-40, 1 mM EDTA) for45 min at 4 C. All lysis buffers were supplemented with 1 protease inhibitor

(Roche) and Halt Phosphatase inhibitor (Thermo Scientic). Chromatin extracts were prepared as described previously43. Immunoprecipitation for HA- tagged BRCA1 was carried out by incubating whole-cell extracts with an HA antibody (Covance) for 2 h, followed by 1 h incubation with Protein A beads (GE healthcare) at 4C. The beads were washed in NETN 150 buffer (150 mM NaCl, 20 mM TrisHCl buffer pH7.8, 0.5% NP-40, 1 mM EDTA). Antibodies used forwestern blotting were phospho-RPA32 (Bethyl Labs, A300-245A; 1:2,000), BRCA1 (SD118; 1:1,000), GAPDH (Santa Cruz, SC-25778; 1:4,000), pS53BP1-S25 (Novus Biologicals, NB100-1803; 1:5,000), Rad51 (Santa Cruz, SC-8349; 1:600), Slug (Cell Signaling, C19G7; 1:3,000), Vinculin (Santa Cruz, SC-55465; 1:1,000), BRCA1 (MS110; 1:1,000) and HA (Covance, MMS-101P; 1:4,000). Uncropped western blots for Figs 1,4,6, and Supplementary Figs 25 are shown in Supplementary Fig. 7.

Immunouorescence and antibodies. Cells on coverslips were xed with 4% paraformaldehyde/2% Sucrose for 15 min, and triton extracted (0.5% Triton X-100 in PBS) for 4 min. Cells were blocked with 5%BSA/PBST and then incubated with respective antibodies for 30 min at 37 C followed by incubation with secondary antibodies (FITC or Rhodamine) for 30 min at 37 C. Primary antibodies usedin immunouorescence studies were BRCA1 (Upstate; 1:500), phospho-53BP1(S1778) (Cell Signaling, 2675S; 1:200), RPA (Cal Biochem, NA13; 1:100), 53BP1 (Bethyl Labs, A300-272A; 1:2,000), Rad51(Santa Cruz, SC-8349; 1:150), Mre11 (Genetex, GTX70212 1:200), CtIP (generous gift from Dr. Richard Baer), HA (Covance, MMS-101P; 1:500) and g-H2AX (Millipore, 05-636; 1:5,000).

For TPX2 (Bethyl Labs, A300-429A; 1:400) and g-tubulin (Sigma- Aldrich, T6557; 1:1,000) staining, the cells were pre-xed with acetone:methanol (3:7) at

20 C for 10 min, followed by triton extraction (0.2% triton-X-100 in20 mM HEPES, pH 7.4, 50 mM NaCl, 3 mM MgCl2, 300 mM Sucrose) at room temperature. Primary and secondary antibody staining was carried out as described above.

Cell treatments. For analysis of phospho-RPA32 loading on chromatin, cells were treated with stalled fork inducing agents like HU (Sigma) and/or UV. Cells were incubated in HU (10 mM)-containing medium for 4 h before harvesting for further analysis. For UV treatment, cells were irradiated with 30 J m 2 UV with a 254 nm

UV-C lamp (UVP Inc., Upland, CA) and harvested 4 h post UV. UV-irradiation through a micropore membrane was performed as described previously43. For colour- coded FACS-based cell survival assays, the Parp inhibitor, olaparib (Selleck), was added at nal concentrations of 0.2, 0.4 and 0.6 mM for 6 days.

cisplatin (Novaplus) was added at nal concentrations of 0.5, 1.0 and 1.5 mM for 24 h. Medium was replaced, and the cells were allowed to grow for ve more days.The doses of UV used were 5, 10 and 15 J m 2, and cells were allowed to recover for 6 days before they were harvested for FACS analysis. Laser-induced DNA breaks were generated as described in Greenberg et al.4

DNA bre assay. DNA bres were prepared and analysed as described previously48,49 with a few modications. In brief, cells were labelled with 25 mM IdU for 20 min, washed two times and incubated in presence of 5 mM HU for 3 h. This

Figure 6 | Evidence of conditional haploinsufciency for DSBR in BRCA1mut/ HMECs after pre-exposure to a stalled fork- inducing agent.(a) Recruitment of Rad51 to IR-induced DSBs is reduced in heterozygous BRCA1mut/, and not in WT BRCA1 / HMECs, when pre-exposed to stalled fork-inducing damage. HMECs derived from a BRCA1 mutation carrier (CP16, BRCA1mut/) and a wt counterpart (CP29, BRCA1 /) were irradiated with different doses of UV (5, 10 or 15 J m 2) and allowed to recover for 1 h. Cells were then irradiated with IR (10 Gy) and xed 4 h post IR. Fixed cells were coimmunostained with Abs to g-H2AX and Rad51. Additional wt and heterozygous strains were also assayed (in panel b). (b) Additional BRCA1/ and BRCA1mut/ strains were analysed as described in (a). A graph depicting the fraction of cells in each additional HMEC strain that contains Rad51 foci after exposure to increasing doses of UV followed by 10 Gy dose of IR was plotted. The mean results and s.d. of data from at least three experiments are shown for each line. (c) Rad51 expression in BRCA1mut/ and BRCA1 / HMEC lines. Whole-cell extracts from various BRCA1mut/ and BRCA1/ strains were analysed by western blot. GAPDH was used as a loading control in these blots. (d) Combinations of BRCA1mut/ and BRCA1 / HMECs (green: BRCA1 //BRCA1 /, blue: BRCA1mut// BRCA1mut/ and red: BRCA1mut//BRCA1 /) were irradiated with different doses of UV(0, 3, 6 and 9 J m 2), allowed to recover for 1 h, and then treated with either 0.2 mM PARP inhibitor (PI olaparib; UV PI) or DMSO as control (UV).

Cells were grown for ve more days before harvesting for FACS analysis. Data are plotted for the three, different cell combinations, and the error bars were calculated as the standard error propagation (SEP) in the ratios of each of the combinations in three, independent experiments. Data marked with an asterisk (*) reveal statistically signicant differences (P-value o0.05) between UV and UV PI sets. (e) One Possible Model of BRCA1 mutation- driven

tumorigenesis. This model speculates that certain abnormal developments might occur during the extended period between full mammary development and the appearance of a BRCA1 breast cancer.

Methods

Isolation and culture of human MECs and broblasts from tissue biopsies.

Tissue samples were briey washed in PBS and then minced and digested overnight at 37 C in medium containing 1 mg ml 1 of collagenase type III (Roche). For digestion,

MEGM medium (Lonza) was used for breast tissue, and Dulbeccos modied Eagles Medium (DMEM) with 5% fetal bovine serum (FBS) for skin tissue. The digested tissue was pelleted and broblasts were cultured in DMEM supplemented with 15% FBS (Gibco), 1% Pen/Strep (Gibco) and 1% Glutamine (Gibco), and HMECs were grown in MEGM medium supplemented with 1% Pen/Strep.

Isolation and culture of mouse MECs from mouse mammary tissue. Primary mouse MEC cultures were generated from the 4th and 5th pairs of mammary fat pads using a sterile technique. The tissue was digested overnight at 37 C in serum-free Leibovitz-15 medium containing 3 mg ml 1 of collagenase A (Sigma). Digestion was stopped by adding 1 volume of 10% serum containing DMEM. The pelleted cells

and organoids were plated and cultivated in DMEM/F12 50:50 medium supplemented with 10% FCS, 50 units ml 1 penicillin, 50 mg ml 1 streptomycin (Life

Technologies), 5 mg ml 1 recombinant human insulin (Sigma), 5 ng ml 1 recombinant human EGF (Sigma) and 5 ng ml 1 cholera toxin (Calbiochem).

Mouse model and genotyping. The mouse Brca1 null allele used in this study was generated by crossing mice harbouring the BRCA1F5-13 conditional allele (kindly provided by Dr Jos Jonkers group)60 with Meox2-Cre deleter mice purchased from Jackson Labs (Bar Harbor, MEStock #003755). Mouse genotyping was performed on genomic DNA extracted from mouse-tail snips using standard procedures60. Genotyping for Cre-generated Brca1 null allele was carried out with primers GenoB1-A (50-AGGTACCAGTTATGAGTTAGTCGTGTGCCTGAGTCA-30) and

GenoB1-D (50-GGCTACCTATAACTACTCTCTAACAACGAAGTGCAA-30), which yielded a 654-bp fragment. The wt brca1 allele was genotyped using primers GenoB1-A and GenoB1-B (50-GCTGAGATTAAAGTGCAGGCCACCACACTCA

GTGAT-30), which yielded a PCR product of 495 bp for the wt allele and 624 bp for the oxed allele. PCR amplication conditions used were as described previously60. Primers Meox2Cre1 (50-CCTGAAAGCAGTTCTCTGGGACCACCTTCTTTTGG

CTTC-30) and Meox2Cre2 (50-CTTCTTCTTGGGTCCTCCCAGATCCTCCTCAG AAATCAGC-30) were used to verify the presence of Meox2 Cre allele. Amplied fragment was 423 bp.

Transfection, infection and selection. For siRNA experiments, cells were grown in six-well plates and transfected with 100 pmoles of siRNA with RNAiMAX (Invitrogen) according to the manufacturers protocol. Where relevant, experiments were initiated 48 h after transfection. All siRNA oligonucleotides were purchased from Thermo Scientic. siRNA oligonucleotides used were siBRCA1 (On Target Plus BRCA1, catalog number CTM-41735) and siGAPDH (On Target Plus GAPDH, catalog number D-001830-01-20). For shRNA experiments, shRNA encoding lentiviruses were generated using 293FT-packaging cells in the presence of lipofectamine (Invitrogen). Cells infected with puroR-encoding lentiviruses were selected transiently using 2.5 mg ml 1 puromycin (Santa Cruz). ShBRCA1 and shLuc were acquired from The RNAi Consortium (TRC). The target sequence for shBRCA1 was 50-AGAATCCTAGAGATACTGAA-30. For BRCA1 reconstitution experiments, lentiviral packaging plasmids, VSVG and PSPAX, were used to package BRCA1 and/or eGFP plasmids in 293FT cells using lipofectamine (Invitrogen). Cells were infected with the lentivirus and selected using 6 mg ml 1 of

Blasticidin (Invitrogen). For colour-coding experiments, hTERT and GFPhTERT containing retroviruses were prepared by packaging the plasmids pMIG-hTERT and pBABE-hygro-hTERT with the retrovirus packaging plasmids, pMD-MLV and pMD-G, in 293FT cells. hTERT-infected cells were selected with hygromycin B (Roche) (50 mg ml 1).

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was followed by incubation with 250 mM CldU for 30 min. Labelled cells were harvested, mixed 1:5 with unlabelled cells, lysed and spread on slides toobtain single-DNA tracts. After xation, denaturation and blocking, the DNA tracts were stained with rat anti-CldU (Abcam, ab6326), followed by staining with a secondary antibody Alexa uor 555 goat anti-rat overnight at 4 C. DNA tracts were then stained with mouse anti- IdU (BD Biosciences, 555627) followed by a secondary antibody Alexa- 488 goat anti mouse. ImageJ software wasused for determining the tract lengths based on scale bar generated during microscopy.

Detection of ssDNA (BrdU ssDNA Assay). BrdU ssDNA assay was performed as described previously42. In brief, cells on coverslips were cultured with 30 mM BrdU for 20 h, and then released in BrdU-free medium for 16 h. Cells were stained as described previously42.

Sequencing and hME. Cells lines were sequenced to conrm their mutations via direct sequencing or by the hME sequencing method. Genomic DNA was prepared from Blood and a DNeasy kit (Qiagen), and a mutation locus-specic PCR reaction was carried out to amplify the region of interest. For direct sequencing, the amplied PCR products were puried using a Qiagen PCR purication kit and were sent for sequencing. For hME analysis, a locus-specic primer extension reaction of the PCR amplied region was carried out in the presence of a mixture of di-deoxy and deoxy NTPs. Allele-specic extension products were analysed by mass spectrometry to determine the specic sequence. More details of the protocol are available at the following link: http://cancer-seqbase.uchicago.edu/documents/AssayDesign3.1Guide.pdf

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Comet assay and analysis. For detection of DNA breaks, alkaline comet assays were performed using the Single-Cell Gel Electrophoresis Assay kit (Trevigen) according to the manufacturers instructions. The quantication of tail DNA was carried out using CellProler software.

Flow cytometry, checkpoints and colour-coding-based cell survival FACS assay. For cell cycle analysis, cells were pulse-labelled with 10 mM BrdU for 30 min (for HMECs) and 1.5 h (for broblasts) in respective culture media. Single-cell suspensions were xed in 70% ice-cold ethanol. Cells were incubated with an anti-BrdU FITC conjugate antibody (Becton Dickinson, 1:10 dilution made in Blocking solution from Thermo Scientic) at room temperature in the dark for 45 min. Finally, the cells were resuspended in propidium iodide and RNAse staining buffer (Becton and Dickinson) and analysed using a Becton Dickinson FACS (Mountain View, CA).

For checkpoint assays, cells were irradiated with UV and/or IR and allowed to recover for 2 h. For S-phase checkpoint analysis, cells were incubated with BrdU, as described above, before harvesting and xing for FACS analysis. For G2 checkpoint, xed cells were incubated with an Alexa Fluor anti-phospho-histone H3 (Ser10) antibody diluted in 2% BSA/PBS at room temperature in the dark for 2 h. Cells were washed and resuspended in propidium iodide and RNAse-containing staining buffer.

For colour-coded FACS-based assays, GFP-positive and -negative cells were mixed in equal numbers (8,000 cells per strain) and plated in 6 cm2 plates. After drug and/or UV treatment, cells were allowed to recover for 6 days before being harvested for FACS analysis.

Satellite RNA q-RT-PCR. Cells grown in 6 cm2 plates were collected, RNA was prepared using an RNeasy Plus Mini Kit (Qiagen), followed by cDNA preparation. q-RT-PCR was carried out with primers for SatA, SatIII, mcbox and b-Actin. More details and primer sequences are described in Zhu et al.18

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Acknowledgements

We express our deep gratitude to the women who donated their tissues for these studies. We are extremely grateful to Ildi Hajdu for teaching us the DNA bre assay and analysis. We also thank Stoil Dimitrov and Jorge Ernesto Gonzlez for help and advice in setting up the comet assay and the CellProler pipeline for comet data analysis, and Manish Neupane for pMIG-hTERT and pBABE-hygro-hTERT plasmids. We thank Aaron R. Thorner and Paul Van Hummelen from the DFCI Center for Cancer Genome Discovery (CCGD) for help with genotyping the BRCA1 mutations. We also thank multiple members of the Livingston laboratory for valuable discussions. This work was supported in part by grants to D.M.L from the National Institutes of Health (PO1CA80111-15), the Susan G. Komen Foundation for the Cure (SAC110022), the Breast Cancer Research Foundation, and a generous gift from Richard and Ann Solomon. S.P. was also supported by a grant from Susan G. Komen for the Cure (CCR13264590). This work was also supported in part by La Ligue Franaise contre le Cancer and The National Cancer Institute (contract BRACAPS) to J.F.

Author contributions

D.M.L., S.P., J.E.G. and J.F. conceptualized the study. S.P. and D.M.L. designed the experiments and wrote the manuscript. S.P. performed the experiments, analysed the data and oversaw the experiments carried out by S.B., R.R. and K.B. Tissue from BRCA1 mutation carriers and non-mutation carriers was collected under the guidance of J.E.G. A.L.R. helped provide breast tissue, and K.P. provided some of the MEC strains for the MEC collection. M.G. helped derive some of the MEC strains and carried out western blot analysis for SLUG. M.G. and Y.S. assisted in determining the lineage of theMEC strains by FACS analysis. C.B.-C. provided the mice to carry out breedings for Brca1 / mouse MEC-based experiments and also assisted in statistical analysis of the cell sensitivity assays. D.T.T. helped with satellite RNA FISH experiments. All authors read and contributed to editing the manuscript.

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BRCA1--a breast and ovarian cancer suppressor gene--promotes genome integrity. To study the functionality of BRCA1 in the heterozygous state, we established a collection of primary human BRCA1^+/+ and BRCA1^mut/+ mammary epithelial cells and fibroblasts. Here we report that all BRCA1^mut/+ cells exhibited multiple normal BRCA1 functions, including the support of homologous recombination- type double-strand break repair (HR-DSBR), checkpoint functions, centrosome number control, spindle pole formation, Slug expression and satellite RNA suppression. In contrast, the same cells were defective in stalled replication fork repair and/or suppression of fork collapse, that is, replication stress. These defects were rescued by reconstituting BRCA1^mut/+ cells with wt BRCA1. In addition, we observed 'conditional' haploinsufficiency for HR-DSBR in BRCA1^mut/+ cells in the face of replication stress. Given the importance of replication stress in epithelial cancer development and of an HR defect in breast cancer pathogenesis, both defects are candidate contributors to tumorigenesis in BRCA1-deficient mammary tissue.

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Title

BRCA1 haploinsufficiency for replication stress suppression in primary cells

Author

Pathania, Shailja; Bade, Sangeeta; Le Guillou, Morwenna; Burke, Karly; Reed, Rachel; Bowman-colin, Christian; Su, Ying; Ting, David T; Polyak, Kornelia; Richardson, Andrea L; Feunteun, Jean; Garber, Judy E; Livingston, David M

Pages

5496

Publication year

2014

Publication date

Nov 2014

Publisher

Nature Publishing Group

e-ISSN

20411723

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms6496

ProQuest document ID

1625357820

BRCA1 haploinsufficiency for replication stress suppression in primary cells

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