BRAF (gene)

BRAF is a human gene that makes a protein called BRaf. The gene is also referred to as proto-oncogene
B-Raf and v-Raf murine sarcoma viral oncogene homolog B, while the protein is more formally known as
serine/threonine-protein kinase B-Raf.[2][3]

ing, it is composed of three conserved domains characteristic of the Raf kinase family: conserved region
1 (CR1), a Ras-GTP-binding[7] self-regulatory domain,
conserved region 2 (CR2), a serine-rich hinge region, and
conserved region 3 (CR3), a catalytic protein kinase doa consensus sequence on proThe B-Raf protein is involved in sending signals inside main that phosphorylates
[8]
In its active conformation, B-Raf forms
tein
substrates.
cells, which are involved in directing cell growth. In
and electrostatic interac2002, it was shown to be faulty (mutated) in some human dimers via hydrogen-bonding
[9]
tions
of
its
kinase
domains.
[4]
cancers.
Certain other inherited BRAF mutations cause birth defects.
2.1
Drugs that treat cancers driven by BRAF mutations have
been developed. Two of these drugs, vemurafenib[5] and
dabrafenib are approved by FDA for treatment of latestage melanoma. Vemurafenib was the rst drug to come
out of fragment-based drug discovery.

Chemokines,
Hormones,
Transmitters
(e.g., interleukins,
serotonin, etc.)

Growth Factors
(e.g., TGF, EGF)

RTK

PKC
NF-B

Dishevelled

FAK
Src

Raf

Adenylate
cyclase
PKA

IB

GSK-3

MEK
MEKK

MKK

-catenin
TCF

Myc: Mad:
Max Max

ERK JNKs
Jun

CREB

Caspase 9

Gene Regulation
Apoptosis

Caspase 8
FADD

Bcl-2

p53

Bad
FasR

ARF
mdm2

Abnormality
Sensor

Mt

Cell
Proliferation

2.3 CR3

-catenin:TCF
Gli
CycID
p16
Rb CDK4
p15
E2F
CyclE
CDK2

SMO

Fos
Cytochrome C

Hedgehog

APC

MAPK

STAT3,5
Bcl-xL

Conserved Region 2 (CR2) provides a exible linker that

connects CR1 and CR3 and acts as a hinge.

Wnt

Fyn/Shc

Ras

Patched

Cytokine Receptor

Akk
JAKs

cdc42

Grb2/SOS
G-Protein

Frizzled

Akt

Integrins

RTK
PLC

PI3K

2.2 CR2

Extracellular
Matrix

GPCR

Cytokines
(e.g., EPC)

Conserved region 1 autoinhibits B-Rafs kinase domain (CR3) so that B-Raf signaling is regulated rather
than constitutive.[8] Residues 155227[10] make up the
Ras-binding domain (RBD), which binds to Ras-GTPs
eector domain to release CR1 and halt kinase inhibition. Residues 234280 comprise a phorbol ester/DAGbinding zinc nger motif that participates in B-Raf membrane docking after Ras-binding.[10][11]

Function
Survival Factors
(e.g., IGF1)

CR1

Conserved Region 3 (CR3), residues 457717,[10] makes

up B-Rafs enzymatic kinase domain. This largely conserved structure[12] is bi-lobal, connected by a short hinge
region.[13] The smaller N-lobe (residues 457530) is primarily responsible for ATP binding while the larger Clobe (residues 535717) binds substrate proteins.[12] The
active site is the cleft between the two lobes, and the catalytic Asp576 residue is located on the C-lobe, facing the
inside of this cleft.[10][12]

p27
p21

Bax

Bim

Death factors
(e.g. FasL, Tnf)

Overview of signal transduction pathways involved in apoptosis.

The role of Raf proteins, like B-Raf is indicated in the center.

B-Raf is a member of the Raf kinase family of growth

signal transduction protein kinases. This protein plays
a role in regulating the MAP kinase/ERKs signaling 2.3.1 Subregions
pathway, which aects cell division, dierentiation, and
secretion.[6]
P-Loop

The P-loop of B-Raf (residues 464471) stabilizes the

non-transferable phosphate groups of ATP during en2 Structure
zyme ATP-binding. Specically, S467, F468, and G469
backbone amides hydrogen-bond to the -phosphate of
B-Raf is a 766-amino acid, regulated signal transduction ATP to anchor the molecule. B-Raf functional moserine/threonine-specic protein kinase. Broadly speak- tifs have been determined by analyzing the homology of
1

ENZYMOLOGY

nase, locking the kinase in its inactive state until the activation loop is phosphorylated, destabilizing these interactions with the presence of negative charge. This triggers
the shift to the active state of the kinase. Specically,
L597 and V600 of the activation loop interact with G466,
F468, and V471 of the P-loop to keep the kinase domain
inactive until it is phosphorylated.[13]

3 Enzymology

Figure 1: Inactive conformation of B-Raf kinase (CR3) domain.

P-Loop (orange) hydrophobic interactions with activation loop
(gray) residues that stabilize the inactive kinase conformation are
shown with sticks. F595 (red) blocks the hydrophobic pocket
where the ATP adenine binds (yellow). D576 (orange) is shown
as part of the catalytic loop (magenta). Figure modied from
PDB id 1UWH.

PKA analyzed by Hanks and Hunter to the B-Raf kinase

domain.[12]
Nucleotide-Binding Pocket
V471, C532, W531, T529, L514, and A481 form a hydrophobic pocket within which the adenine of ATP is
anchored through Van der Waals attractions upon ATP
binding.[12][14]
Catalytic Loop
Residues 574581 compose a section of the kinase domain responsible for supporting the transfer of the phosphate of ATP to B-Rafs protein substrate. In particular, D576 acts as a proton acceptor to activate the
nucleophilic hydroxyl oxygen on substrate serine or threonine residues, allowing the phosphate transfer reaction
to occur mediated by base-catalysis.[12]
DFG Motif

B-Raf is a serine/threonine-specic protein kinase. As

such, it catalyzes the phosphorylation of serine and
threonine residues in a consensus sequence on target proteins by ATP, yielding ADP and a phosphorylated protein
as products.[12] Since it is a highly regulated signal transduction kinase, B-Raf must rst bind Ras-GTP before becoming active as an enzyme.[11] Once B-Raf is activated,
a conserved protein kinase catalytic core phosphorylates
protein substrates by promoting the nucleophilic attack
of the activated substrate serine or threonine hydroxyl
oxygen atom on the -phosphate group of ATP through
bimolecular nucleophilic substitution.[12][16][17][18]

3.1 Activation
3.1.1 Relieving CR1 autoinhibition
The kinase (CR3) domain of human Raf kinases is inhibited by two mechanisms: autoinhibition by its own
regulatory Ras-GTP-binding CR1 domain and a lack
of post-translational phosphorylation of key serine and
tyrosine residues (S338 and Y341 for c-Raf) in the CR2
hinge region. During B-Raf activation, the proteins
autoinhibitory CR1 domain rst binds Ras-GTPs eector
domain to the CR1 Ras-binding domain (RBD) to release the kinase CR3 domain like other members of the
human Raf kinase family. The CR1-Ras interaction is
later strengthened through the binding of the cysteinerich subdomain (CRD) of CR1 to Ras and membrane
phospholipids.[8] Unlike A-Raf and C-Raf, which must
be phosphorylated on hydroxyl-containing CR2 residues
before fully releasing CR1 to become active, B-Raf is
constituitively phosphorylated on CR2 S445.[19] This allows the negatively charged phosphoserine to immediately repel CR1 through steric and electrostatic interactions once the regulatory domain is unbound, freeing the
CR3 kinase domain to interact with substrate proteins.

D594, F595, and G596 compose a motif central to BRafs function in both its inactive and active state. In
the inactive state, F595 occupies the nucleotide-binding
pocket, prohibiting ATP from entering and decreasing
the likelihood of enzyme catalysis.[9][14][15] In the active
state, D594 chelates the divalent magnesium cation that 3.1.2 CR3 domain activation
stabilizes the - and -phosphate groups of ATP, orientAfter the autoinhibitory CR1 regulatory domain is reing the -phosphate for transfer.[12]
leased, B-Rafs CR3 kinase domain must change to its
Activation Loop
ATP-binding active conformer before it can catalyze proResidues 596600 form strong hydrophobic interactions tein phosphorylation. In the inactive conformation, F595
with the P-loop in the inactive conformation of the ki- of the DFG motif blocks the hydrophobic adenine bind-

3.3

Inhibitors

ing pocket while activation loop residues form hydrophobic interactions with the P-loop, stopping ATP from accessing its binding site. When the activation loop is phosphorylated, the negative charge of the phosphate is unstable in the hydrophobic environment of the P-loop. As a
result, the activation loop changes conformation, stretching out across the C-lobe of the kinase domain. In this
process, it forms stabilizing -sheet interactions with the
6 strand. Meanwhile, the phosphorylated residue approaches K507, forming a stabilizing salt bridge to lock
the activation loop into place. The DFG motif changes
conformation with the activation loop, causing F595 to
move out of the adenine nucleotide binding site and into
a hydrophobic pocket bordered by the C and E helices. Together, DFG and activation loop movement upon
phosphorylation open the ATP binding site. Since all
other substrate-binding and catalytic domains are already
in place, phosphorylation of the activation loop alone activates B-Rafs kinase domain through a chain reaction
that essentially removes a lid from an otherwise-prepared
active site.[13]

3.2

Mechanism of catalysis

3
3.2.1 ATP binding
B-Raf binds ATP by anchoring the adenine nucleotide
in a nonpolar pocket (yellow, Figure 1) and orienting the molecule through hydrogen-bonding and electrostatic interactions with phosphate groups. In addition to
the P-loop and DFG motif phosphate binding described
above, K483 and E501 play key roles in stabilizing nontransferable phosphate groups. The positive charge on
the primary amine of K483 allows it to stabilize the negative charge on ATP - and -phosphate groups when ATP
binds. When ATP is not present, the negative charge of
the E501 carboxyl group balances this charge.[12][13]
3.2.2 Phosphorylation
Once ATP is bound to the B-Raf kinase domain, D576 of
the catalytic loop activates a substrate hydroxyl group, increasing its nucleophilicity to kinetically drive the phosphorylation reaction while other catalytic loop residues
stabilize the transition state.(Figure 2). N581 chelates
the divalent magnesium cation associated with ATP to
help orient the molecule for optimal substitution. K578
neutralizes the negative charge on the -phosphate group
of ATP so that the activated ser/thr substrate residue
won't experience as much electron-electron repulsion
when attacking the phosphate. After the phosphate group
is transferred, ADP and the new phosphoprotein are
released.[12]

3.3 Inhibitors
Since constitutively active B-Raf mutants commonly
cause cancer (see Clinical Signicance) by excessively
signaling cells to grow, inhibitors of B-Raf have been
developed for both the inactive and active conformations of the kinase domain as cancer therapeutic
candidates.[13][14][15]
3.3.1 Sorafenib
BAY43-9006 (Sorafenib, Nexavar)is a V600E mutant BRaf and C-Raf inhibitor approved by the FDA for the
Figure 2: Base-catalyzed nucleophilic attack of a ser- treatment of primary liver and kidney cancer. Bay43ine/threonine substrate residue on the -phosphate group of ATP. 9006 disables the B-Raf kinase domain by locking the enStep 1: chelation of secondary magnesium ion by N581 and de- zyme in its inactive form. The inhibitor accomplishes this
protonation of substrate Ser/Thr by D576. Step 2: nucleophilic by blocking the ATP binding pocket through high-anity
attack of activated substrate hydroxyl on ATP -phosphate. Step for the kinase domain. It then binds key activation loop
3: magnesium complex breaks down and D576 deprotonates. and DFG motif residues to stop the movement of the acStep 4: release of products.
tivation loop and DFG motif to the active conformation.
Finally, a triuoromethyl phenyl moiety sterically blocks
To eectively catalyze protein phosphorylation via the bi- the DFG motif and activation loop active conformation
kinase domain to shift
molecular substitution of serine and threonine residues site, making it impossible for the
[13]
conformation
to
become
active.
with ADP as a leaving group, B-Raf must rst bind ATP
and then stabilize the transition state as the -phosphate The distal pyridyl ring of BAY43-9006 anchors in the
of ATP is transferred.[12]
hydrophobic nucleotide-binding pocket of the kinase N-

4 CLINICAL SIGNIFICANCE

Figure 4: Structures of Vemurafenib (right) and its precursor,

PLX 4720 (left), two inhibitors of the active conformation of the
B-Raf kinase domain

selectively inhibits the proliferation of cells with unregulated B-Raf, normally those that cause cancer.

Figure 3: B-Raf kinase domain locked in the inactive conformation by bound BAY43-9006. Hydrophobic interactions anchor
BAY43-9006 in the ATP binding site while urea group hydrogenbonding traps D594 of the DFG motif. The BAY43-9006 triuoromethyl phenyl ring further prohibits DFG motif and activation
loop movement to the active confermer via steric blockage.

lobe, interacting with W531, F583, and F595. The hydrophobic interactions with catalytic loop F583 and DFG
motif F595 stabilize the inactive conformation of these
structures, decreasing the likelihood of enzyme activation. Further hydrophobic interaction of K483, L514,
and T529 with the center phenyl ring increase the anity
of the kinase domain for the inhibitor. Hydrophobic
interaction of F595 with the center ring as well decreases the energetic favorability of a DFG conformation switch further. Finally, polar interactions of BAY439006 with the kinase domain continue this trend of increasing enzyme anity for the inhibitor and stabilizing
DFG residues in the inactive conformation. E501 and
C532 hydrogen bond the urea and pyridyl groups of the
inhibitor respectively while the urea carbonyl accepts a
hydrogen bond from D594s backbone amide nitrogen to
lock the DFG motif in place.[13]

Since Vemurafenib only diers from its precursor,

PLX4720, in a phenyl ring added for pharmacokinetic
reasons,[15] PLX4720s mode of action is equivalent to
Vemurafenibs. PLX4720 has good anity for the ATP
binding site partially because its anchor region, a 7azaindole bicyclic, only diers from the natural adenine
that occupies the site in two places where nitrogen atoms
have been replaced by carbon. This enables strong intermolecular interactions like N7 hydrogen bonding to
C532 and N1 hydrogen bonding to Q530 to be preserved. Excellent t within the ATP-binding hydrophobic pocket (C532, W531, T529, L514, A481) increases
binding anity as well. Ketone linker hydrogen bonding
to water and diuoro-phenyl t in a second hydrophobic
pocket (A481, V482, K483, V471, I527, T529, L514,
and F583) contribute to the exceptionally high binding
anity overall. Selective binding to active Raf is accomplished by the terminal propyl group that binds to
a Raf-selective pocket created by a shift of the C helix. Selectivity for the active conformation of the kinase is further increased by a pH-sensitive deprotonated
sulfonamide group that is stabilized by hydrogen bonding
with the backbone peptide NH of D594 in the active state.
In the inactive state, the inhibitors sulfonamide group interacts with the backbone carbonyl of that residue instead,
creating repulsion. Thus, Vemurafenib binds preferentially to the active state of B-Rafs kinase domain.[14][15]

The triuoromethyl phenyl moiety cements the thermodynamic favorability of the inactive conformation when
the kinase domain is bound to BAY43-9006 by sterically 4 Clinical signicance
blocking the hydrophobic pocket between the C and E
helices that the DFG motif and activation loop would in- Mutations in the BRAF gene can cause disease in two
habit upon shifting to their locations in the active confor- ways. First, mutations can be inherited and cause birth
defects. Second, mutations can appear later in life and
mation of the protein.[13]
cause cancer, as an oncogene.
Inherited
mutations
in
this
gene
cause
cardiofaciocutaneous syndrome, a disease characmental retardation and a
PLX4032 (Vemurafenib) is a V600 mutant B-Raf in- terized by heart defects, [20]
distinctive
facial
appearance.
hibitor approved by the FDA for the treatment of latestage melanoma.[9] Unlike BAY43-9006, which inhibits Acquired mutations in this gene have been found in canthe inactive form of the kinase domain, Vemurafenib cers, including non-Hodgkin lymphoma, colorectal caninhibits the active DFG-in form of the kinase,[14][15] cer, malignant melanoma, papillary thyroid carcinoma,
rmly anchoring itself in the ATP-binding site. By in- non-small-cell lung carcinoma, and adenocarcinoma of
hibiting only the active form of the kinase, Vemurafenib the lung.[6]
3.3.2

Vemurafenib

4.2

BRAF inhibitors in the clinic

The V600E mutation of the BRAF gene has been associ- 4.1.1 BRAF-V600E
ated with hairy cell leukemia in numerous studies and has
BRAF V600E is a determinant of sensitivity to
been suggested for use in screening for Lynch syndrome
proteasome inhibitors. Vulnerability to proteasome
to reduce the number of patients undergoing unnecessary
inhibitors is dependent on persistent BRAF signalMLH1 sequencing.[21]
ing, because BRAF-V600E blockade by PLX4720
reversed sensitivity to carlzomib in BRAF-mutant
colorectal cancer cells. Proteasome inhibition might
represent a valuable targeting strategy in BRAF
V600E-mutant colorectal tumors.[35]

4.1

Mutants

More than 30 mutations of the BRAF gene associated

with human cancers have been identied. The frequency
of BRAF mutations varies widely in human cancers, from
more than 80% in melanomas and nevi, to as little as 0
18% in other tumors, such as 13% in lung cancers and
5% in colorectal cancer.[22] In 90% of the cases, thymine
is substituted with adenine at nucleotide 1799. This leads
to valine (V) being substituted for by glutamate (E) at
codon 600 (now referred to as V600E) in the activation segment that has been found in human cancers.[23]
This mutation has been widely observed in papillary
thyroid carcinoma, colorectal cancer, melanoma and
non-small-cell lung cancer.[24][25][26][27][28][29][30] BRAFV600E mutation are present in 57% of Langerhans
cell histiocytosis patients.[31] The V600E mutation is a
likely driver mutation in 100% of cases of hairy cell
leukaemia.[32] High frequency of BRAF V600E mutations have been detected in ameloblastoma, a benign but
locally inltrative odontogenic neoplasm.[33] The V600E
mutation may also be linked, as a single-driver mutation
(a genetic 'smoking gun') to certain cases of papillary
craniopharyngioma development.[34]

4.2 BRAF inhibitors in the clinic

As mentioned above, some pharmaceutical rms are developing specic inhibitors of mutated B-raf protein for
anticancer use because BRAF is a well-understood, high
yield target.[14][36] Vemurafenib (RG7204 or PLX4032),
licensed by the US Food and Drug Administration as
Zelboraf for the treatment of metastatic melanoma in August 2011, is the current state-of-the-art example for why
active B-Raf inhibitors are being pursued as drug candidates. Vemurafenib is biochemically interesting as a
mechanism to target cancer due to its high ecacy and
selectivity.[9] In Phase II[37] and Phase III[38] clinical trials, B-Raf not only increased metastatic melanoma patient chance of survival but raised the response rate to
treatment from 7-12% to 53% in the same amount of time
compared to the former best chemotherapeutic treatment:
dacarbazine. In spite of the drugs high ecacy, 20% of
tumors still develop resistance to the treatment. In mice,
20% of tumors become resistant after 56 days.[39] While
the mechanisms of this resistance are still disputed, some
hypotheses include the overexpression of B-Raf to compensate for high concentrations of Vemurafenib[39] and
Other mutations which have been found are R461I, upstream upregulation of growth signaling.[40]
I462S, G463E, G463V, G465A, G465E, G465V,
G468A, G468E, N580S, E585K, D593V, F594L, More general B-raf inhibitors include GDC-0879, PLXG595R, L596V, T598I, V599D, V599E, V599K, 4720, Sorafenib Tosylate. dabrafenib, LGX818
V599R, V600K, A727V, etc. and most of these mutations are clustered to two regions: the glycine-rich P
loop of the N lobe and the activation segment and ank- 5 Interactions
ing regions.[1] These mutations change the activation segment from inactive state to active state, for example in BRAF (gene) has been shown to interact with:
the previous cited paper it has been reported that the
aliphatic side chain of Val599 interacts with the phenyl
AKT1,[41]
ring of Phe467 in the P loop. Replacing the medium sized
hydrophobic Val side chain with a larger and charged
C-Raf,[42]
residue as found in human cancer(Glu, Asp, Lys, or Arg)
HRAS,[43][44] and
would be expected to destabilize the interactions that
maintain the DFG motif in an inactive conformation, so
YWHAB.[45][46]
ipping the activation segment into the active position.
Depending on the type of mutation the kinase activity towards MEK may also vary. Most of the mutants stimulate enhanced B-Raf kinase activity toward MEK. How- 6 References
ever, a few mutants act through a dierent mechanism because although their activity toward MEK is reduced, they [1] Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz
adopt a conformation that activates wild-type C-RAF,
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Davis N, Dicks E, Ewing R, Floyd Y, Gray K, Hall S,
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EXTERNAL LINKS

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7 Further reading
Garnett MJ, Marais R (2004). Guilty as charged:
B-Raf is a human oncogene. Cancer Cell 6 (4):
3139. doi:10.1016/j.ccr.2004.09.022. PMID
15488754.
Quiros RM, Ding HG, Gattuso P, Prinz RA, Xu X
(2005). Evidence that one subset of anaplastic thyroid carcinomas are derived from papillary carcinomas due to BRAF and p53 mutations. Cancer
103 (11): 22618. doi:10.1002/cncr.21073. PMID
15880523.
Karbowniczek M, Henske EP (2006). The role
of tuberin in cellular dierentiation: are B-Raf
and MAPK involved?". Ann N Y Acad Sci 1059
(1): 16873. doi:10.1196/annals.1339.045. PMID
16382052.
Ciampi R, Nikiforov YE (2007). RET/PTC rearrangements and BRAF mutations in thyroid tumorigenesis. Endocrinology 148 (3): 93641.
doi:10.1210/en.2006-0921. PMID 16946010.
Espinosa AV, Porchia L, Ringel MD
(2007).
Targeting BRAF in thyroid cancer. British Journal of Cancer 96 (1): 1620.
doi:10.1038/sj.bjc.6603520.
PMC 2360215.
PMID 17179987.

8 External links
Allanson, Judith E; Roberts, Amy E (2011-08-04).
Noonan Syndrome. NBK1124. In Pagon RA, Bird
TD, Dolan CR et al., eds. (1993). GeneReviews
[Internet]. Seattle WA: University of Washington,
Seattle. Check date values in: |date= (help)
Rauen,
Katherine
A
Cardiofaciocutaneous Syndrome.
In GeneReviews

(2012-09-06).
NBK1186.

Gelb, Bruce D; Tartaglia, Marco (2010-11-16).

LEOPARD Syndrome. NBK1383. In GeneReviews
BRAF gene. NCI Dictionary of Cancer Terms.
Retrieved 2007-11-25.
Finding faults in BRAF Cancer Research UK
blog post about the discovery of cancer-causing
BRAF mutations (incl video)

9
This article incorporates public domain material from the
U.S. National Cancer Institute document Dictionary of
Cancer Terms. This article incorporates text from the
United States National Library of Medicine, which is in
the public domain.

10

9 TEXT AND IMAGE SOURCES, CONTRIBUTORS, AND LICENSES

Text and image sources, contributors, and licenses

9.1

Text

BRAF (gene) Source: http://en.wikipedia.org/wiki/BRAF_(gene)?oldid=659842841 Contributors: The Anome, Ronz, Nurg, Rich Farmbrough, Arcadian, Rjwilmsi, Ground Zero, RussBot, RDBrown, Niels Olson, Eastlaw, Patho~enwiki, Agathman, Alaibot, Hb2019, Nono64,
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9.2

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File:B-Raf_Phosphorylation_Mechanism.png
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Contributors: http://www.ebi.ac.uk/pdbe-srv/view/images/entry/1uwh600.png, displayed on http://www.ebi.ac.uk/pdbe-srv/view/entry/
1uwh/summary Original artist: Jawahar Swaminathan and MSD sta at the European Bioinformatics Institute
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File:Protein_BRAF_PDB_1uwh.png Source: http://upload.wikimedia.org/wikipedia/commons/a/af/Protein_BRAF_PDB_1uwh.png
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