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BRCApathway• Transcriptional regulation• Molecular interactions• DNA damage & response

Transcriptional Regulation Direct response to DNA damage Related BRCA products

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TRANSCRIPTIONAL REGULATION

IFNγ targetgenes

DNA damage responsegenes

Growth arrest andDNA repair genes

Chromatinremodeling

Estrogen induciblegenes

Growth/transforminggenes

DNA damage repair,checkpoint activation

ER-α c-Myc

ZBRK1p53 GADD45

BACH1

ATF-1

STAT1HDACs

SWI/SNF

BRCA1

ATM-ATR

CHK1

BRCA1

BRCA2

T DIRECT RESPONSE TO DNA DAMAGE

Chromatin remodeling

HomologousRecombination

X-chromosomesilencing

Non-homologouend joining

heterodimerizationubiquitination

Cell cycle checkpoints

S phase and G2 arrest DNA repairHomologous recombination

BACH1

HDACs

SWI/SNF

MSH2-MSH6

MRN Complax

p53 FANCD2

BARD1

Xist

RAD51

Chk1

BLM

RbPLK1

GADD45

WAF1

ATM-ATR

CHK1

Featured antibodies

ATM (phospho S1981) (81292)

Clonality Applications Host Species cross reactivityM ICC/IF, IHC-P, IP, WB Rb Hu

Chk1 (47574)

Clonality Applications Host Species cross reactivityP ELISA, IHC-P, WB Rb Hu, Ms, Rat, Dm

c-Myc (86356)

Clonality Applications Host Species cross reactivityP ICC/IF, WB Rb Hu

p53 (phospho S315) (1647)

Clonality Applications Host Species cross reactivityP ICC/IF, IHC-P, WB Rb Hu

p53 (phospho S392) (33889)

Clonality Applications Host Species cross reactivityM ICC/IF, IHC-P, IP, WB Rb Hu, Rat

More information available at:www.abcam.com/cancer

Associated reagents and products

Abcam offers a variety of associatedreagents and products that complement ourprimary antibodies in a wide range ofapplications.

Our catalog has been extended toinclude:

• Over 300 rapid ELISAs covering 360targets

• Thousands of secondary antibodiesalso available under pre-adsorbed andfragment formats

• EXPOSE biotin-free IHC kits with highsensitivity

• A wide range of recombinant andpurified native proteins and peptides

Product Clonality Applications Host Species Antibody Proteins PeptidesReactivity www.abcam.com/ab…

ATF1 P ELISA, IHC-P, WB Rb Hu 78903 51448 -ATM P WB Rb Hu 82512 - 90257ATM M IHC-P, IP, WB Ms Hu, Ms, Rat, Mk 78 - -ATM P IP, WB Rb Hu 17995 - -ATM (phospho S1981) M ICC/IF, IHC-P, IP, WB Rb Hu 81292 - 13767ATR M WB Ms Hu 4471 - -ATR P IF, IP, WB Rb Hu, Ms 2905 - -BARD1 P WB Rb Hu 64164 - 74356BRCA1 M ICC/IF, IHC-Fr, IHC-P, IP, WB Ms Hu 16780 82204 -BRCA1 P IHC-P, WB Rb Hu 48790 - -BRCA1 P ELISA, IHC-P Rb Hu 47429 - -BRCA1 P IP, WB Rb Hu 9141 - 40076BRCA1 (phospho S1423) P WB Rb Hu 2838 - -BRCA2 P IHC-Fr, IHC-P, WB Rb Hu 75335 - -BRCA2 P IP, WB Rb Hu, Ms 90541 - -BRCA2 (phospho T3349) P NULL Rb - 36459 - 36509Chk1 P ELISA, IHC-P, WB Rb Hu, Ms, Rat, Dm 47574 69918 -Chk1 (phospho S296) M ICC/IF, WB Rb Hu 79758 - -Chk1 (phospho S345) P ELISA, ICC/IF, IHC-P Rb Hu, Ms, Rat, Dm 47318 - -c-Myc P ICC/IF, WB Rb Hu 86356 84132 -c-Myc P ELISA, IHC-P, WB Rb Hu, Ms, Rat 51154 - -c-Myc M Flow Cyt, ICC/IF, IHC-P Ms Hu 64478 - -c-Myc (phospho S74) P NULL Rb Hu 46848 - -Estrogen Receptor alpha p ELISA, ICC/IF, IHC-P, WB Rb Hu 31315 82606 -Estrogen Receptor alpha P ICC/IF, IHC-P, WB Rb Hu, Ms, Rat 37438 38041 -FANCD2 P ICC/IF, WB Rb Hu 31577 - 31576p53 M ELISA, ICC, ICC/IF, IHC-Fr,

IHC-P, IP, WB Ms Hu 7757 84768 -p53 M ELISA, Flow Cyt, IHC-Fr,

IHC-P, IP, RIA, WB Ms Hu, Rat 28 82199 -p53 M ELISA, Flow Cyt, ICC, ICC/IF,

IHC (Methanol fixed), IHC-Fr, IHC-P, IP, WB Ms Hu, Ms, Rat 26 82201 -

p53 (acetyl K382) P WB Rb Hu 37318 - 37536p53 (acetyl K386) P ELISA, ICC/IF, IHC-P Rb Hu, Ms, Rat 52172 - -p53 (mono methyl K372) P IP, WB Rb Hu 16033 - 16033p53 (phospho S315) P ICC/IF, IHC-P, WB Rb Hu 1647 - 28896p53 (phospho S392) M ICC/IF, IHC-P, IP, WB Rb Hu, Rat 33889 - -p53 (phospho S6) M ICC/IF, IHC-P, IP, WB Rb Hu 32132 - -PALB2 P WB Rb Hu 38587 - 38740PLK1 M I-ELISA, ICC/IF, IP, WB Ms Hu, Ms, Rat 17057 51426 -PLK1 M ICC, ICC/IF, IF, IP, WB Ms Hu, Ms, Rat 17056 82064 -PLK1 P ICC/IF, IHC-P, WB Rb Hu, Ms, Rat 47867 - 48102Rad51 P WB Rb Hu 84304 63808 -Rad51 (phospho T309) P WB Rb Hu 31769 - 33130Rb P ICC/IF, WB Rb Hu 85607 83205 -Rb P ELISA, IHC-P, WB Rb Hu, Ms, Rat 39689 73767 -Rb (phospho S795) P ELISA, IHC-P, WB Rb Hu, Ms, Rat 47474 - -STAT1 p ELISA, IHC-P, IP, WB Rb Hu, Ms, Rat 31369 82610 -STAT1 M Flow Cyt, IP, WB Rb Hu, Ms 92506 - -STAT1 M IP, WB Ms Hu, Ms 3987 - -

BRCA Leaflet01:Layout 1 01/12/2010 10:06 Page 1

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BRCA1-B Complex | via BRCT domainsDNA Replication, S-Phase Progression

BRCA1-A Complex | via BRCT domainsDNA Damage Signaling | G2/M checkpoint

BRCA1-C Complex | via BRCT domainsDNA end resection and G2-M checkpoint control

Linkage to the Fanconi anaemia pathwayoverlapping functions

Functional domains of BRCA1 andBRCA2 and sites of interaction withselected binding partners

BRCA1 and BRCA2Maintenance of genomic stability

Regulate centrosome

number

Regulate DNAdecatenation

Regulate transcription

Regulate spindle

microtubuleorganization

Regulate spindle

assembly= ubiquitin

D

Disrupts BRCA1/BARD1interaction

B

BRCA1

U

BARD1

TopollA

UUU

TPX2UUU

RNA Pol IIUUU

BAP1

γ-tubulinUUU

HMMRUUU

BRCA1-A complex

DNA Damage:double strand break

H2AX

H2AX

Lys63

RAP80 binds K63 chains,recruits BRCA1-A complex

CHK1 PhosphorylationG2-M checkpointactivation

ATM-ATR phosphorylationof H2AX marks sites ofDNA damage

MDC1 binds phospho-H2AX, recruits ubiquitinE3 ligase complex

E3 ligase RNF168 binds ubiquitinated histonesand adds further K63linked ubiquitination

a

= phosphorylation

1

R

1

RAP80

UBC13

RNF168

UBC13

RNF8

ATM-ATR

P

P

UU

PUU

UUU

UUU

UUU

H2AX

PUU

UUU

MDC1

P

H2AX

P

Inclusion in the BRCA1-C complex in late S and G2 phases directs CtIP toward homologous recombination (HR) instead of microhomology mediated end joining (MMEJ)

G1 PHASE S-G2 PHASE

CtIP stimulates the activity of the MRN complex, leading to DNA end resection (with exonuclease Exo I, and the helicase BLM) .

RPAG2-M Checkpoint

RPA is recruited to single-stranded DNA and the RPA-DNA complex recruits ATR, which activates theCHK1 and the G2-M checkpoint.

1

ATR CHK1

BLM

CtIP

CtIP

BLM

BRCA1-C complex

BRCA1-C complex

BRCA1-C complex

P

P

U

t

ATRCHK1

stalled replication fork

ATR

UPSTREAMNETWORK

D

monoubiquitinationlocalizes complexesto sites of damage atchromatin

A

FA core complex

FA ID complex

PFA ID complex

R

1,863 aa

RING(E3) NLS Coiledcoil

BRCT

BAP1BARD1

D

BRCA2PALB2

RNA Pol IIp53HDAC1/2

AbraxasBACH1

CtIP

3,418 aa

BRCA1

BRCA2

RAD51

BRC Repeats

OB Folds TR2/NLS

RAD51ssDNA

p53RB SWI/SNF

EMSY

Helical Domain

DMC1

B

BRCA2

PALB2

t

R

1

BARD1

BRCA1

Additional interactions(e.g. BRCA1-A, -B or -C complexes)for localization, functionality.

HR REPAIR

Concentration of BRCA2 at double-strand break sites, loading of RAD51

RAD51

WD40

Coiled-Coil

BRCTRING

Coiled-Coil BRCTRING

BRCA1

P

P

P

U

t

DOWNSTREAMNETWORK

ATR

?

Translesionbypass

?

Homologousrecombination

?

BARD1

BLM

FA core complex

P

BRIP1/FANCJ

BRCA2/FANCD1

PALB2/FANCN

FA ID complex

MDC1

P

H2AX

P

Preinitiation Complex

BRCA1-B complex

BRCA1-A complex

BRCA1-B complex

BRCA1

BRCA2

P

P

P

P

P

U

U

PALB2

Regulate centrosome

number

Regulate DNAdecatenation

Regulate transcription

Regulate spindle

microtubuleorganization

Regulate spindle

assembly= ubiquitin

DNA Damage:double strand break

H2AX

H2AX

Lys63

RAP80 binds K63 chains,recruits BRCA1-A complex

CHK1 PhosphorylationG2-M checkpointactivation

ATM-ATR phosphorylationof H2AX marks sites ofDNA damage

MDC1 binds phospho-H2AX, recruits ubiquitinE3 ligase complex

E3 ligase RNF168 binds ubiquitinated histonesand adds further K63linked ubiquitination

adapted from Huen et al., 2010

Stalled replication fork= phosphorylation

1. Timely S-phase progression throughloading of licensing factor CDC45L

2. Activation of replication checkpoint in response to stalled forks.

G1-S checkpointIntra-S phase checkpoint

Inclusion in the BRCA1-C complex in late S and G2 phases directs CtIP toward homologous recombination (HR) instead of microhomology mediated end joining (MMEJ)

G1 PHASE S-G2 PHASE

CtIP stimulates the activity of the MRN complex, leading to DNA end resection (with exonuclease Exo I, and the helicase BLM) .

RPAG2-M Checkpoint

RPA is recruited to single-stranded DNA and the RPA-DNA complex recruits ATR, which activates theCHK1 and the G2-M checkpoint.

1,863 aa

RING(E3) NLS Coiledcoil

BRCT

BAP1BARD1

Disrupts BRCA1/BARD1interaction

BRCA2PALB2

RNA Pol IIp53HDAC1/2

AbraxasBACH1

CtIP

3,418 aa

BRCA1

BRCA2

RAD51

BRC Repeats

OB Folds TR2/NLS

RAD51ssDNA

p53RB SWI/SNF

EMSY

Helical Domain

DMC1

BARD1

BRCA1

Additional interactions(e.g. BRCA1-A, -B or -C complexes)for localization, functionality.

HR REPAIR

Concentration of BRCA2 at double-strand break sites, loading of RAD51

ATRCHK1

stalled replication fork

ATR

UPSTREAMNETWORK

DOWNSTREAMNETWORK

monoubiquitinationlocalizes complexesto sites of damage atchromatin

ATR

?

Translesionbypass

?

Homologousrecombination

?

adapted from Wong et al., 2007

BARD1

BRCA1

U

BARD1

CDC45L

ATR

RAP80

TopollA

UBC13

RNF168

UBC13

RNF8

CHK1

BLM

BLM

CtIP

CtIP

BLM

FA core complex

BRCA1-C complex

BRCA1-C complex

BRCA1-C complex

FA core complex

ATM-ATR

RAD51

PP

P

BRIP1/FANCJ

BRCA2/FANCD1

PALB2/FANCN

FA ID complex

FA ID complex

PFA ID complex

WD40

Coiled-Coil

BRCTRING

Coiled-Coil BRCTRING

UU

PUU

UUU

UUU

TPX2UUU

RNA Pol IIUUU

BAP1

γ-tubulinUUU

HMMRUUU

UUU

UUU

H2AX

PUU

UUU

MDC1

P

H2AX

P

Preinitiation Complex

BRCA1-B complex

BRCA1-A complex

BRCA1-B complex

BRCA1

BRCA2

P

P

P

P

P

U

U

PALB2

Regulate centrosome

number

Regulate DNAdecatenation

Regulate transcription

Regulate spindle

microtubuleorganization

Regulate spindle

assembly= ubiquitin

DNA Damage:double strand break

H2AX

H2AX

Lys63

RAP80 binds K63 chains,recruits BRCA1-A complex

CHK1 PhosphorylationG2-M checkpointactivation

ATM-ATR phosphorylationof H2AX marks sites ofDNA damage

MDC1 binds phospho-H2AX, recruits ubiquitinE3 ligase complex

E3 ligase RNF168 binds ubiquitinated histonesand adds further K63linked ubiquitination

adapted from Huen et al., 2010

Stalled replication fork= phosphorylation

1. Timely S-phase progression throughloading of licensing factor CDC45L

2. Activation of replication checkpoint in response to stalled forks.

G1-S checkpointIntra-S phase checkpoint

Inclusion in the BRCA1-C complex in late S and G2 phases directs CtIP toward homologous recombination (HR) instead of microhomology mediated end joining (MMEJ)

G1 PHASE S-G2 PHASE

CtIP stimulates the activity of the MRN complex, leading to DNA end resection (with exonuclease Exo I, and the helicase BLM) .

RPAG2-M Checkpoint

RPA is recruited to single-stranded DNA and the RPA-DNA complex recruits ATR, which activates theCHK1 and the G2-M checkpoint.

1,863 aa

RING(E3) NLS Coiledcoil

BRCT

BAP1BARD1

Disrupts BRCA1/BARD1interaction

BRCA2PALB2

RNA Pol IIp53HDAC1/2

AbraxasBACH1

CtIP

3,418 aa

BRCA1

BRCA2

RAD51

BRC Repeats

OB Folds TR2/NLS

RAD51ssDNA

p53RB SWI/SNF

EMSY

Helical Domain

DMC1

BARD1

BRCA1

Additional interactions(e.g. BRCA1-A, -B or -C complexes)for localization, functionality.

HR REPAIR

Concentration of BRCA2 at double-strand break sites, loading of RAD51

ATRCHK1

stalled replication fork

ATR

UPSTREAMNETWORK

DOWNSTREAMNETWORK

monoubiquitinationlocalizes complexesto sites of damage atchromatin

ATR

?

Translesionbypass

?

Homologousrecombination

?

adapted from Wong et al., 2007

BARD1

BRCA1

U

BARD1

CDC45L

ATR

RAP80

TopollA

UBC13

RNF168

UBC13

RNF8

CHK1

BLM

BLM

CtIP

CtIP

BLM

FA core complex

BRCA1-C complex

BRCA1-C complex

BRCA1-C complex

FA core complex

ATM-ATR

RAD51

PP

P

BRIP1/FANCJ

BRCA2/FANCD1

PALB2/FANCN

FA ID complex

FA ID complex

PFA ID complex

WD40

Coiled-Coil

BRCTRING

Coiled-Coil BRCTRING

UU

PUU

UUU

UUU

TPX2UUU

RNA Pol IIUUU

BAP1

γ-tubulinUUU

HMMRUUU

UUU

UUU

H2AX

PUU

UUU

MDC1

P

H2AX

P

Preinitiation Complex

BRCA1-B complex

The breast and ovarian cancer susceptibility proteins BRCA1 andBRCA2 were discovered in the 1990s through genetic studies offamilies with with high risk of breast and ovarian cancer. Indeed, thecumulative risk of developing breast cancer by age 70 yearsapproaches 50-70% in BRCA1 mutation carriers. Subsequentresearch has implicated these proteins in a variety of functionsassociated with maintenance of genomic stability. BRCA1, incombination with BARD1, has E3 ubiquitin ligase activty; BRCA2 hasno enzymatic activity. Both BRCA1 and BRCA2 are large proteins thatfulfill their tumor suppressive roles. as members of multiple, protein complexes with overlapping functions.Three BRCA1 complexes (A, B and C) are formed via phosphorylation-dependent interactions with the BRCT domains. A fourth complex, thePALB2/BRCA1/BRCA2 supercomplex, is formed through the coiled coildomain, and thus is likely not be exclusive of A, B and C complexpartners. Complicating matters, BRCA2 is also a Fanconi anaemia(FA) gene (FANCD1), while BRCA1 is not, though BRCA1 interactswith several FA gene products.

CORE COMPLEX | via RING finger domainsE3 Ligase Activity | example targets

PALB2/BRCA1 Supercomplex | viacoiled-coil domain Linking homologous recombinationfunctions of BRCA1/BRCA2

Produced in collaboration with AndrewHorwitz, Department of Molecular Pharmacology,University of California, San Francisco Adapted from Huen et. al., 2010 Adapted from Wong et. al., 2007

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