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53BP1 Inhibits Homologous

Recombination in Brca1-Deficient Cellsby Blocking Resection of DNA BreaksSamuel F. Bunting,1 Elsa Callen,1,8 Nancy Wong,1,8 Hua-Tang Chen,1 Federica Polato,1 Amanda Gunn,4 Anne Bothmer,5

Niklas Feldhahn,5 Oscar Fernandez-Capetillo,7 Liu Cao,2 Xiaoling Xu,3 Chu-Xia Deng,3 Toren Finkel,2

Michel Nussenzweig,5,6 Jeremy M. Stark,4 and Andre Nussenzweig1,*1Experimental Immunology Branch, National Cancer Institute2Translational Medicine Branch, National Heart, Lung, and Blood Institute3Genetics of Development and Disease Branch, National Institute for Diabetes and Digestive and Kidney Diseases

National Institutes of Health, Bethesda, MD 20892, USA4Department of Cancer Biology and Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of the

City of Hope, Duarte, CA 91010, USA5Laboratory of Molecular Immunology6Howard Hughes Medical Institute

Rockefeller University, New York, NY 10065, USA7Genomic Instability Group, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain8These authors contributed equally to this work

*Correspondence: andre_nussenzweig@nih.gov

DOI 10.1016/j.cell.2010.03.012

SUMMARY

Defective DNA repair by homologous recombination

(HR) is thought to be a major contributor to tumori-

genesis in individuals carrying Brca1 mutations.

Here, we show that DNA breaks in Brca1-deficient

cells are aberrantly joined into complex chromosomerearrangements by a process dependent on the

nonhomologous end-joining (NHEJ) factors 53BP1

and DNA ligase 4. Lossof53BP1 alleviates hypersen-

sitivity ofBrca1 mutant cells to PARP inhibition and

restores error-free repair by HR. Mechanistically,

53BP1 deletion promotes ATM-dependent process-

ing of broken DNA ends to produce recombinogenic

single-stranded DNA competent for HR. In contrast,

Lig4 deficiency does not rescue the HR defect in

Brca1 mutant cells but prevents the joining of chro-

matid breaks into chromosome rearrangements.

Our results illustrate that HR and NHEJ compete to

process DNA breaks that arise during DNA replica-tion and that shifting the balance between these

pathways can be exploited to selectively protect or

kill cells harboring Brca1 mutations.

INTRODUCTION

Mutations in the Brca1 gene predispose carriers to a high inci-

dence of breast and ovarian cancer (Venkitaraman, 2004).

In theabsenceof Brca1,Xrcc2, or other homologous recombina-

tion (HR) proteins, Rad51 foci formation and homology depen-

dent repair are impaired (Moynahan et al., 1999; Scully et al.,

1999). Since the HR pathway is required for repair of sponta-

neous double-stranded breaks (DSBs) that arise during DNA

replication, defects in HR result in an accumulation of chromatid

breaks (Andreassen et al., 2006; Sonoda et al., 1998). Cells that

cannot repair chromatid breaks by HR become more reliant on

other poorly defined alternative repair pathways. These path-

ways are not template based like HR and therefore have the

propensity to join together DSBs on different chromatids to pro-

duce complex chromosomal rearrangements, which promote

genomic instability and/or trigger loss in viability (Bryant et al.,

2005; Farmer et al., 2005; Sonoda et al., 1998). Genomic insta-

bility following loss-of-function ofBrca1 is hypothesized to be

a key factor leading to tumorigenesis in individuals with the

Brca1 mutation; nevertheless, additional mutations are required

to enable survival and outgrowth of tumor cells (Deng, 2006;

Venkitaraman, 2004).

HR-deficient cells exhibit an acute sensitivity to killing by

inhibitors of the single-stranded DNA (ssDNA) repair protein

poly(ADP-ribose) polymerase (PARP)(Bryantet al., 2005; Farmer

et al., 2005; Jackson and Bartek, 2009). Mechanistically, loss of

PARP activity prevents repair of ssDNA breaks, which are thenconverted into DSBs during DNA replication. These breaks are

normally repaired by Rad51-dependent HR using the sister chro-

matid as a template, hence PARP inhibition is particularly toxic in

Brca1- or Brca2-deficient cells, which are HR defective. The

ability of PARP inhibitors to selectively kill HR-deficient cells is

currently being used in clinical trials for treatment of breast and

ovarian cancers where Brca1 or Brca2 is mutated (Fong et al.,

2009; Jackson and Bartek, 2009). More recently, it has been

observed that Brca2-deficient tumors are capable of acquiring

reversion mutations that enable resistance to chemotherapeutic

agents (Edwards et al., 2008; Sakai et al., 2008). This observation

raises the possibility that additional secondary mutations could

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mediate resistance ofBrca-deficient tumors to the toxic effects

of PARP inhibitors.

Mice homozygous for the exon 11 deletion (D11) isoform of

Brca1 (Brca1D11/D11) die in utero (Xu et al., 2001). Embryonic

cell death is associated with extensive apoptosis and activation

of the ATM-Chk2-p53 arm of the DNA damage response (Cao

et al., 2006). Indeed, embryonic lethality can be rescued by

complete or heterozygous loss ofp53 (Xu et al., 2001) or deletion

of ATM or Chk2 (Cao et al., 2006). Deletion of 53BP1 also

rescues the viabilityofBrca1D11/D11 mice(Cao etal.,2009). How-

ever, in contrast to rescue by loss ofp53, Brca1D11/D1153BP1/

mice exhibit a low incidence of tumor formation and near-normal

life span (Cao et al., 2009). Nevertheless, Brca1D11/D1153BP1/

cells showed elevated levels of DSBs, intact ATM-Chk2-p53

signaling, and ionizing radiation (IR)-induced apoptosis (Cao

et al., 2009).Theonly known functionsof 53BP1 arein transducing a subset

of ATM-dependent cell-cycle checkpoints (DiTullio et al., 2002;

Fernandez-Capetillo et al., 2002; Lee et al., 2010; Wang et al.,

2002) and facilitating the joining of distal DSBs formed at

dysfunctional telomeres and during lymphocyte antigen receptor

recombination (Difilippantonio et al., 2008; Dimitrovaet al., 2008;

Manis et al., 2004; Reina-San-Martin et al., 2007; Ward et al.,

2004). None of these activities would appear to account for the

survival of Brca1D11/D11 mice in the absence of 53BP1. Thus,

the underlying mechanism by which loss of53BP1 rescues cell

death and prevents tumorigenesis inBrca1 mutant mice remains

unclear.

Here, we show that the presence of 53BP1 limits the capacity

ofBrca1-deficient cells to accurately repair DSBs. Importantly,

the high levels of genomic instability and cell death induced in

Brca1-deficient cells by treatment with an inhibitor of PARP are

not present in Brca1/53BP1 double-deficient cells. Genomic

stability is rescued because the HR pathway is largely restored

in cells lacking Brca1 and 53BP1. In contrast, Brca1-deficient

repair is not normalized by deletion of the nonhomologous

end-joining (NHEJ) factor DNA ligase 4 (Lig4), although deletion

of Lig4 does prevent accumulation of chromosomal fusions.

Our results indicate a role for 53BP1 and Brca1 in regulating

the choice between NHEJ and HR pathways, which has implica-

tions for anticancer therapies using PARP inhibitors.

RESULTS

53BP1 Promotes Genomic Instability and Mammary

Tumorigenesis in Brca1 Mutant Mice

Brca1D11/D11 mice rescued by loss of one or both copies of p53

(Brca1D11/D11p53+/or Brca1D11/D11p53/) develop multiple

types of tumors (Cao et al., 2006; Xu et al., 1999), whereas

rescue by deletion of53BP1 (Brca1D11/D1153BP1/) results in

near normal life span with significantly reduced tumorigenesis.

To further investigate the requirement for 53BP1 in tumorigen-

esis in Brca1-deficient animals, we followed the onset of devel-

opment of mammary tumors in cohorts of female mice with dele-

tion mutations in Brca1 exon 11 (Figure 1A). As expected, mice

with breast-specific deletion of Brca1 exon 11 succumbed to

A

D

Brca111/11

53BP1-/-

0

20

40

60

80

100

Percentlivecells

(re

lativetountreated)

Brca111/11

p53+/-

WT

Brca111/11

53BP1-/-

Brca111/11

p53+/-

WT

Brca111/11

53BP1-/-

Brca111/11

p53+/-WT

PI

Caspase

C

Untreated+ PARPi

Brca111/11

p53+/-

Brca111/11

53BP1-/-

10.7% 1.0%

WT

0%

B

untreated + PARPi

Brca1/ ;MMTV-Cre

Brca111/1153BP1-/-

CFSE

49.69 49.06

45.03 12.88

55.64 46.78

(n = 22)

(n = 27)

Figure 1. Deletion of 53BP1 Reduces

Mammary Tumorigenesis, Radial Chromo-

some Formation, and Cellular Proliferation

Defects in Brca1D11/ D11 Cells

(A) Breast cancer incidence in mice deficient in

Brca1. The red line indicates tumor incidence inmice with deletion of Brca1 exon 11 targeted to

the mammary cells with a Cre transgene under

control of the MMTV LTR promoter (Brca1f/f;

MMTV-Cre). The blue line shows mammary tumor

incidencein Brca1D11/D11

53BP1/ mice, in which

Brca1 exon 11 and 53BP1 are deleted in all cells.

(B) Radial chromosome structures characteristic

of metaphases from Brca1D11/ D11p53

+/ B cells.

The percentage of metaphases containing radial

chromosomes in Brca1D11/ D11

p53+/ (n = 300),

Brca1D11/ D1153BP1/ (n = 300) and WT (n =

100) cells is indicated. Note that equivalent

results were seen with Brca1D11/ D11p53+/ and

Brca1D11/ D11p53

/cells.

(C) Flow cytometry analysis of B cells to measure

cell survival. The labeled populations in the dot

plots are viable cells, identified by their ability to

exclude PI and their lack of caspase 3 activation.

The chart shows the frequency of live cells treated

with PARP inhibitor normalized to the untreated

population.

(D) Proliferation of B cells pulsed with CFSE and

cultured with and without PARP inhibitor. CFSE

signal diminishes with increasing cell division, so

that cells that do not divide have the highest

CFSE signal.

See also Figure S1.

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tumors of the mammary tissue (Brodie et al., 2001; Xu et al.,

1999), with 12 out of 27 animals affected by 18 months of age

(Figure 1A). Mice that were doubly deficient for Brca1 exon 11

and 53BP1, by contrast, developed almost no breast tumors,

with just one animal affected at the age of 22 months (Fig-

ure 1A; data not shown).

Brca1 is thought to suppress malignancy by promoting HR

(Moynahan et al., 1999; Scully et al., 1999; Venkitaraman,2004). In light of the dramatic reduction in the frequency of

mammary tumors in Brca1D11/D1153BP1/ animals, we hypoth-

esized that loss of 53BP1 might specifically affect the ability

of Brca1-deficient cells to repair replication-associated DNA

damage. To test this, we examined chromosomal aberrations in

metaphase spreads from WT, Brca1D11/D11p53+/ and

Brca1D11/D1153BP1/ B cells that were stimulated to divide ex

vivo for 3 days. Asymmetric radial chromosome structures, a

type of chromatid exchange characteristic of HR deficiency,

were found in 10.7% of Brca1D11/D11p53+/B cells (n= 300)(Ven-

kitaraman, 2004) (Figure 1B). Strikingly, thesechromosome aber-

rations were present in just 1.0% ofBrca1D11/D1153BP1/ cells,

1 0 31 3 27

28

102

8 1022

0

50

100

150

0 10 nM 1 M

WT

53BP1-/-

Brca111/11p53-/-

Brca111/11

53BP1-/-

0 0 01 0 04

6876

40

16

0

20

40

60

80

0 10 nM 1 M

WT

53BP1-/-

Brca111/11

p53-/-

Brca111/11

53BP1-/-

0 0 00 0 111

94

320

1 6 8

0

100

200

300

400

0 10 nM 1 M

WT

53BP1-/-

Brca111/11

p53-/-

Brca111/11

53BP1-/-

Kap1-S824 Kap1-S824

Tubulin Tubulin

PARPi - + - + - + IR - + - + - +

semosomor

hcl

aid

aR

se

sa

hpatem

001

rep

sk

aer

bemos

omor

hC

sesa

hpatem

001

rep

A

Brca111/11

p53-/-Brca111/11

53BP1-/-

WT Brca111/11

p53-/-Brca111/11

53BP1-/-

WT

sk

aer

bdit

amor

hC

sesa

hpatem

001

rep

[PARPi]

B

[PARPi]

[PARPi]

C

snoitarr

ebA

sesa

hpatem

001

rep

0

8

0

54

18 14

0

6

00

10

20

30

40

50

60

Radial

chromosomes

Chromosome

breaks

Chroma dbreaks

WT

Brca111/11

p53-/-

Brca111/11

53BP1-/-

+ CPT

Figure2. Deletion of53BP1 Reverses Sensi-

tivity of Brca1D11/ D11 Cells to PARPi and

Camptothecin

(A) Analysis of genomic instability in metaphases

from B cells treated with 0, 10 nM, and 1 mM

PARP inhibitor. Charts show the number of radialchromosomes, chromatid breaks, and chromo-

some breaks per 100 metaphases (n = 50 meta-

phases analyzed in each case). Note that genomic

instability in Brca1D11/ D11

cells is independent of

p53 status, and equivalent results were seen in

Brca1D11/ D11p53+/ and Brca1D11/ D11p53/

cells.

(B) Western blot showing Kap1 phosphorylation in

B cells from the indicated genotypes treated with

PARP inhibitor or 5Gy ionizing radiation.

(C) Analysis of genomic instability in metaphases

from B cells treated with 4 nM camptothecin

(CPT). B cells were cultured overnight with CPT

prior to fixation and preparation of metaphase

slides.

equivalent to a 10-fold reduction relative

to Brca1D11/D11p53+/ (n = 300). Radial

chromosomes were undetectable in WT

and53BP1/cells(n = 100cells analyzed

for each genotype; Figures 1B and 2A).

To exclude the possibility that p53

heterozygosity provides a survival advan-

tage to genomically unstable Brca1-defi-

cient cells by allowing aberrant chromo-

somes to persist (Callen et al., 2007;

Difilippantonio et al., 2008), we quantified

the incidence of radial chromosomes in

Brca1D11/D11conditional B cells. By infect-

ing these cells with a virus expressing Cre

recombinase, we were able to specifically

delete Brca1 exon 11 in p53-sufficient

cells.We found that theseBrca1f/f cells still showeda significant

increase (8-fold) in the number of radial chromosomes relative

to Cre-infected Brca1f/f 53BP1/cells (Figure S1A available

online). Radial chromosome formation in Brca1f/f cells is there-

fore independent ofp53 status but dependent on 53BP1.

To investigate further the effect of 53BP1 in regulating repair

of DNA breaks occurring during replication, we challenged

Brca1 mutant cells with a chemical inhibitor of PARP (PARPi;KU58948) (Figures 1C and 1D). This drug has been shown to

cause cell death in Brca1-deficient cells, associated with high

levels of genomic instability (Farmer et al., 2005). In the presence

of PARPi, Brca1D11/D11p53+/ cells showed much lower levels of

survival relative to either WT or Brca1D11/D1153BP1/ cells (Fig-

ure 1C). Equivalent PARPi-induced cell death was seen with

Brca1D11/D11p53/ cells, which were nonetheless resistant to

IR-induced apoptosis (Figure S1B). Thus, PARPi-mediated

apoptosis in Brca1D11/D11 cells, in contrast to IR-induced apo-

ptosis, occurs independently ofp53 status.

The ability of PARPi to inhibit cell proliferation in culture

was measured by pulsing of the cells with the fluorescent dye

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CFSE (carboxyfluorescein succinimidyl ester). In this assay,

cellular proliferation is revealed by stepwise loss of CFSE during

the culture period. PARPi inhibited growth in Brca1D11/D11p53+/

cells (Figure 1D), as well as in Brca1D11/D11p53/ B cells (see

below). Brca1D11/D11

53BP1/

cells were distinct in that theywere practically insensitive to PARPi-induced apoptosis and

divided almost normally whether or not PARPi was present

(Figures 1C and 1D). Consistent with these findings in B cells,

Brca1D11/D1153BP1/ mouse embryonic fibroblasts (MEFs)

were also insensitive to cell killing with PARPi, whereas

Brca1D11/D11 MEFs were hypersensitive to the drug (Fig-

ure S1C). We conclude that 53BP1 is required for the toxic effect

of PARP inhibition on Brca1D11/D11 cells.

PARPi is predictedto increase thefrequency of S phase-asso-

ciated chromosomal aberrations (Bryant et al., 2005; Farmer

et al., 2005; Jackson and Bartek, 2009). To determine whether

loss of53BP1 affects S phase-specific genomic instability, we

monitored the level of chromosome breaks and radial structures

in Brca1-deficient cells treated with andwithout PARPi. WhereasWT and 53BP1/ cells showed very low levels of genomic

instability in the presence or absence of PARPi, exposure of

Brca1D11/D11p53/ to PARPi caused a substantial increase in

the level of genomic instability (Figure 2A). For example, treat-

ment of Brca1D11/D11p53/ cells with 1 mM PARPi increased

the frequency of radial chromosomes from 0.1 per cell to an

average of 3.2 per cell and increased the number of DSBs (chro-

matid and chromosome) from 0.1 to 1.8 per cell. Thus, PARPi

treatment of Brca1D11/D11p53/ cells leads to a greater than

20-fold increase in aberrations (Figure 2A). In contrast, the total

frequency of chromosomal aberrations in PARPi-treated

Brca1D11/D1153BP1/ cellswas just 0.5per cell,which is 10-fold

lower than PARPi-treated Brca1D11/D11p53/ cells. Neverthe-

less, PARPi-induced genomic instability in Brca1/53BP1double-

mutant mice was somewhat elevated relative to the WT.

Our findings were further substantiated by the observation

that PARPi induced a significant amount of DNA damage

signaling in Brca1D11/D11p53/ cells. Brca1D11/D11p53/ cells

preincubated with PARPi for 24 hr showed induction of Kap1

phosphorylation (Figure 2B), while phosphorylated Kap-1 was

undetectable in PARPi-treated WT and Brca1D11/D1153BP1/

cells (Figure 2B). This was not due to an intrinsic defect in

ATM signaling, because similar levels of Kap1 phosphorylation

were detected in cells from all genotypes treated with IR

(Figure 2B).

To determine the effects of other chemotherapeutic agents

that induce replication damage, we challenged WT, Brca1-,and Brca1/53BP1-deficient B cells with camptothecin (CPT).

CPT and its derivatives are topoisomerase I poisons that induce

DSBs during replication and are widely used as anticancer drugs

(Jackson and Bartek, 2009). Brca1-deficient cells are hypersen-

sitive to CPT (Nakamura et al., 2010). Consistent with this,

after treatment of Brca1D11/D11p53+/ B cells with 4 nM CPT,

radial chromosomes formed at a frequency of 0.54 per cell,

whereas these aberrations were undetectable in CPT-treated

Brca1D11/D1153BP1/ B cells (Figure 2C). Similarly, loss of

53BP1 suppressed CPT-induced chromosome and chromatid

breaks in Brca1-deficient cells (Figure 2C). Thus, loss of53BP1

greatly alleviates replication-associated aberrations and toxicity

that clinically relevant chemotherapeutic agents confer on

Brca1-deficient cells.

Loss of 53BP1 Increases HR in Brca1 Mutant Cells

Replication-associated breaks are primarily repaired by HR. Wetherefore hypothesized that loss of 53BP1 might restore HR,

a DNA repair pathway that is significantly compromised in the

absence ofBrca1 (Moynahan et al., 1999). To test this, we first

examined Rad51 foci formation, a marker of HR, which is

impaired in Brca1 mutant cells (Bhattacharyya et al., 2000;

Scully et al., 1997). WT, Brca1D11/D11p53+/, and Brca1D11/D11

53BP1/ B cells were treated with IR, fixed, and stained with

a Rad51 antibody (Figure 3A). Whereas Rad51 foci formed in

just 6.8% of irradiated Brca1D11/D11p53+/ cells, more than

30% ofBrca1D11/D1153BP1/ cells exhibited Rad51 foci, similar

to that observed in WT cells (Figure 3A). Differences in the fre-

quency of Rad51 foci formation was not due to alterations in

cell-cycle distribution, as the percentage of cycling cells was

equivalent in all genotypes tested (Figure S2).As a second measure of HR, we monitored sister chromatid

exchanges (SCEs), which are dependent on Rad51 activity

(Sonoda et al., 1999). While the basal level of SCEs was the

same, treatment with PARPi increased the frequency of SCEs

in Brca1D11/D1153BP1/ but not in Brca1D11/D11p53+/ cells

(Figure 3B). PARPi treatment of Brca1D11/D11p53+/ therefore

leads to an increase in aberrant radial chromosome formation,

which is a marker of HR deficiency (Figure 2A), whereas PARPi

treatment ofBrca1D11/D1153BP1/ cells promotes sister chro-

matid exchange, which is a marker of HR proficiency (Figure 3B).

As a third measure of HR, we used the DR-GFPhyg reporter

system, which is designed to measure error-free HR of a site-

specific break formed by the rare-cutting endonuclease I-SceI

(Nakanishi et al., 2005) In this reporter, error-free HR repair of

the I-SceI-induced DSB leads to restoration of a GFP+ gene

that uses iGFP as the template (Figure 3C). To analyze repair

of chromosomal breaks, we integrated DR-GFPhyg into WT,

Brca1D11/D11, and Brca1D11/D11 53BP1/ immortalized MEF cell

lines. From these experiments, we found that HR in Brca1D11/D11

cells wasreduced relative to theWT (3-fold,p < 0.0001), whereas

HR in Brca1D11/D11 53BP1/ cells was increased relative to the

WT (5-fold, p < 0.0028) (Figure 3C). Thus, in Brca1D11/D11 MEFs,

loss of 53BP1 causes a 16-fold increase in HR repair of a site-

specific chromosomal break. In conclusion, loss of 53BP1

increases HR in Brca1 mutant cells, as evidenced by Rad51

foci, sister chromatid exchange, and recombination reporter

assays.

53BP1 Does Not Affect the Activity of Xrcc2 in HR

To examine whether loss of53BP1 could rescue the defects in

cells deficient in other HR components, we examined chromo-

somal aberrations in cells from mice deficient for Xrcc2, a

Rad51 paralog that, like Brca1, is required for Rad51 foci forma-

tion (Liuet al., 1998; Takata etal.,2001). Metaphasespreads were

prepared from conditional Xrcc2f/f B cells, in which Xrcc2 was

deleted with a B cell-specific CD19-Cre transgene. As expected,

Xrcc2-deficient cells stimulated to proliferate in vitro exhibited

chromosome and chromatid breaks and radial chromosomes,

which was increased by treatment with PARPi (Figure 3D).

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Surprisingly, whereas53BP1deletion promoted genome stability

in Brca1-deficient cells (Figure 2A and Figure S1), this effect was

not seen in Xrcc2 knockout cells (Figure 3D). Rather, thefrequency of breaks and asymmetric radial chromosomes was

similar in Xrcc2f/f53BP1/ and Xrcc2f/f cells. Thus, despite

the fact that both Brca1 and Xrcc2 are required for Rad51 foci

formation during HR, loss of53BP1 reverses the HR defect in

Brca1- but not in Xrcc2-deficient cells. We hypothesize that

Brca1 affects an early stage of HR (Stark et al., 2004), whereas

Xrcc2 acts at a downstream step that is not affected by the pres-

ence of 53BP1 (Nagarajuet al., 2009) (see the Discussion, below).

Lig4 Is Required for RadialFusions inBrca1Mutant Cells

As measured by reporter substrates, the frequency of HR is

enhanced in the absence of the NHEJ proteins Ku70, XRCC4,

A

C

0

10

20

30

untreated

PARPi

Exchangesper100metaphas

es

0

10

20

30

40

%

CellswithRad51foci

Radialchromos

omes

per100metaphases

0

20

40

60

80

100

untr ea te d PAR Pi

Xrcc2/

Xrcc2/ 53BP1-/-

0

20

40

60

80

100

untreated PARPi

Xrcc2/

Xrcc2/ 53BP1-/-

Chromatidbreaks

per100me

taphases

0

10

20

untr ea ted PARPi

Xrcc2/

Xrcc2/ 53BP1-/-

Chromosome

breaks

per100metaphases

W T Brca1 11/11

5 3 B P 1 - / -

Brca1 11/11

p 5 3 + / -

W T Brca111/11p53+/-

Brca111/1153BP1-/-

Brca111/11p53+/-

Brca 1 11/11

53BP1-/-

B

DSceGFP

hyg

I-Sce I B c g I

HR

iGFP

hyg

B c g I

GFP+ iGFP

B c g I

HomologousR

ecombination(%)

0. 1

1

10

10 0

*

*

Figure 3. Deletion of 53BP1 Restores HR in

Brca1 but Not Xrcc2Mutant Cells

(A) Immunofluorescence images showing Rad51

foci (red) with DAPI counterstain (blue) in cells of

the indicated genotypes after treatment with

ionizing radiation. Chart shows the percentage ofcells with Rad51 foci (n = 100 counted for each

genotype).

(B) B cells grown for 36 hr in BrdUTP were fixed

and metaphases prepared to visualize individual

sister chromatids. Sister chromatid exchanges

(SCEs) in metaphase chromosomes are indicated

with arrowsin theimage inthe toppanel.The chart

shows the meanSCEs in metaphase spreads from

B cells of the indicated genotypestreated withand

without PARP inhibitor. Error bars show the SD.

(C) Top: Structure of the HR reporter substrate

DR-GFPhyg is shown, along with the HR product

expressing a functional GFP+ gene. Bottom:

Frequency of HR relative to total transfected cells

in WT, Brca1D11/D11, and Brca1D11/D1153BP1/

MEFs, as measured withthe DR-GFPhygreporter.

*p < 0.0001 between WT and Brca1D11/D11, *p