A UAF1-Containing Multisubunit Protein Complex
Regulates the Fanconi Anemia Pathway
Martin A. Cohn,1Przemyslaw Kowal,1Kailin Yang,1Wilhelm Haas,2Tony T. Huang,1,3
Steven P. Gygi,2and Alan D. D’Andrea1,*
1Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA
2Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
3Present address: Department of Biochemistry, New York University, New York, NY 10016, USA.
The deubiquitinating enzyme USP1 controls the
cellular levels of the DNA damage response
protein Ub-FANCD2, a key protein of the Fan-
coni anemia DNA repair pathway. Here we
report the purification of a USP1 multisubunit
protein complex from HeLa cells containing
stoichiometric amounts of a WD40 repeat-
containing protein, USP1 associated factor 1
(UAF1). In vitro reconstitution of USP1 deubi-
quitinating enzyme activity, using either ubiqui-
purified monoubiquitinated FANCD2 protein
as substrates, demonstrates that UAF1 func-
tions as an activator of USP1. UAF1 binding
increases the catalytic turnover (kcat) but does
not increase the affinity of the USP1 enzyme
for the substrate (KM). Moreover, we show that
DNA damage results in an immediate shutoff
of transcription of the USP1 gene, leading to
a rapid decline in the USP1/UAF1 protein com-
plex. Taken together, our results describe a
mechanism of regulation of the deubiquitinat-
ing enzyme, USP1, and of DNA repair.
The Fanconi anemia DNA repair pathway is activated after
various types of DNA damage resulting from DNA cross-
links, ultraviolet light, or ionizing radiation (Kennedy and
D’Andrea, 2005; Niedernhofer et al., 2005). Central to
the Fanconi anemia pathway is the FANCD2 protein,
which is monoubiquitinated after DNA damage, a modifi-
cation activating the protein and enabling it to participate
in DNA damage repair (Garcia-Higuera et al., 2001; Tani-
guchi et al., 2002). Recent studies indicate that FANCD2,
and an interacting protein, FANCI, are cooperatively
monoubiquitinated (Smogorzewska et al., 2007). The deu-
biquitinating enzyme, USP1, limits the cellular levels of
Ub-FANCD2 (Nijman et al., 2005a). USP1 levels decrease
after DNA damage, leading to a transient elevation of Ub-
FANCD2 levels and to increased DNA repair (Nijman et al.,
2005a). USP1 also deubiquitinates Ub-PCNA, suggesting
that it plays a role in regulating Ub-PCNA-mediated trans-
lesion synthesis (TLS) (Huang et al., 2006). Interestingly,
the USP1 enzyme can undergo autocleavage, resulting
in a 100 kDa N-terminal fragment and a 14 kDa C-terminal
fragment (Huang et al., 2006). The mechanisms that
govern the activity of USP1 following DNAdamage remain
unknown, and several models are possible. DNA damage
may regulate the expression level, protein stability, or the
the USP1 enzyme.
Recent studies indicate that deubiquitinating enzymes
associated with the proteasome are regulated through
their association with other noncatalytic protein subunits.
of the proteasome base, thus achieving a high level of en-
zymatic activity (Hanna et al., 2006; Leggett et al., 2002).
Yeast and human Uch37 bind the noncatalytic protein
Adrm1 and thereby become more active (Qiu et al.,
2006; Yao et al., 2006). Also, yeast Ubp3 interacts with
(Cohen et al., 2003). However, it has remained unknown
whether USP1 also exists in a complex with an analogous
To address this question and to better understand the
mechanism of USP1-mediated DNA damage response,
we purified the USP1 enzyme with associated proteins
from human HeLa cells. We isolated a native multisubunit
protein complex containing stoichiometric amounts of an
80 kDa protein, which we named USP1 associated factor
1 (UAF1). UAF1 is a WD40 repeat-containing protein,
which regulates both the stability and the activity of USP1.
Purification of the Native USP1/UAF1 Complex
To further explore the mechanism of USP1-mediated
deubiquitination of Ub-FANCD2, wepurified the USP1 en-
from HeLa cells. An HeLa cell line stably expressing
a Flag- and HA-epitope tagged fusion protein of USP1
(e-USP1) was generated by retroviral transduction. The
786 Molecular Cell 28, 786–797, December 14, 2007 ª2007 Elsevier Inc.
exogenous e-USP1 protein was expressed at levels com-
parable to the endogenous protein and also underwent
autocleavage, a feature previously reported for the USP1
protein (Huang et al., 2006; Nijman et al., 2005a). To verify
the functionality of the e-USP1 fusion protein, we demon-
strated that it is able to complement USP1-deficient cells
(see Figure S1 available online) and that it possesses
deubiquitinating enzyme activity in vitro (Figure S2). Taken
together, our results demonstrate that the epitope-tagged
USP1 protein is functional.
Nuclear extract was prepared from HeLa cells, and the
native USP1 complex was purified by a two-step immu-
noaffinity purification scheme (Nakatani and Ogryzko,
2003). SDS-PAGE analysis of the purified complex
demonstrated the presence of multiple polypeptides
(Figure 1A, lane 2). No polypeptides were observed in
a mock purification from untransduced HeLa cells,
indicating that all polypeptides copurifying with e-USP1
were bona fide subunits of the USP1 complex (Figure 1A,
lane 1). Mass spectrometric analysis of the polypeptides
identified full-length USP1, the N-terminal cleavage prod-
uct of USP1, and the C-terminal cleavage product of
of 80 kDa, was identified as the previously studied p80
protein (Park et al., 2002). We now refer to this protein
as UAF1. UAF1 contains 677 amino acids and harbors
seven or eight potential WD40 repeats in the N-terminal
half and a predicted coiled-coil domain structure in the
C-terminal half. Tertiary structure prediction using the
Phyre software (http://www.sbg.bio.ic.ac.uk/?phyre) sug-
gests the presence of a complete propeller structure
comprised by the WD40 repeats. The intensities of
Coomassie blue-stained USP1 and UAF1 proteins in
the SDS-PAGE were nearly identical, suggesting stoi-
chiometric amounts of the two proteins in the complex
and a possible functional relationship.
plex by immunoblotting, using antibodies to the USP1
protein (Nijman et al., 2005a) and newly generated anti-
bodies against the UAF1 protein. The results confirmed
Figure 1. Purification of a Native USP1
Complex Containing UAF1
(A) The USP1 complex was purified from HeLa
nuclearextract and stained by Coomassieblue
stain. The polypeptides identified by mass
spectrometry are indicated.
(B) Western blot analysis of the USP1 complex
using anti-USP1 (top) or anti-UAF1 (bottom)
(C) Endogenous USP1 interacts with UAF1.
Flag-HA-tagged UAF1 was expressed in HeLa
bodies. The immunoprecipitate was analyzed
by immunoblotting using anti-USP1 antibodies
(top) and anti-UAF1 antibodies (bottom).
(D) The majority of cellular USP1 is in complex
with UAF1. Flag-HA-tagged UAF1 was ex-
pressed in HeLa cells and immunoprecipitated
by anti-Flag antibodies. The immunoprecipi-
tates were analyzed by immunoblotting using
the indicated antibodies. FT, flowthrough from
the immunoprecipitation. IP, immunoprecipi-
Molecular Cell 28, 786–797, December 14, 2007 ª2007 Elsevier Inc. 787
USP1/UAF1 Regulates the Fanconi Anemia Pathway
damage. Inhibition of transcription is followed by a rapid
decay in USP1 protein levels, involving an active degrada-
tion of the enzymatic competent N-terminal fragment of
USP1. The lowered USP1 levels allow cells to accumulate
monoubiquitinated FANCD2 protein, which is essential for
an effective DNA damage response.
Various types of DNA damage lead to the activation of
the Fanconi anemia DNA repair pathway, including the
UV irradiation employed in the current study. It is unclear
exactly which regulatory factors mediate the shutdown
of USP1 gene transcription and how the type and dose
of damage affect the cellular response and survival.
In conclusion, we present a mechanism of regulation of
deubiquitinating enzymes targeting monoubiquitinated
substrates (Figure 7A). Weshow that the USP1 protein ex-
ists in a catalytically active protein complex with the UAF1
protein, which in addition to activating USP1 also serves
to maintain its stability in vivo. The active USP1/UAF1
complex ensures that the Fanconi anemia pathway is
kept at an adequate state under normal cell growth.
When cells experience genotoxic stress, transcription of
the USP1 gene is rapidly shut off, leading to a decrease
of USP1/UAF1 complex, thereby activating the Fanconi
anemia pathway through increased protein levels of Ub-
FANCD2, ensuring maintenance of genomic stability.
Cell Lines, Antibodies, and Plasmids
HeLa and HEK293T cells were grown in DMEM (Invitrogen) supple-
mented with 10% FBS. Antibodies used were as follows: anti-USP1
antibody (Nijman et al., 2005a); anti-FANCD2 (sc-20022; Santa Cruz
Biotechnology); anti-PCNA (sc-56; Santa Cruz Biotechnology); anti-
gamma-tubulin (CP06; Calbiochem); anti-c-Myc (sc-40; Santa Cruz
Biotechnology); and anti-HA (mouse monoclonal antibody clone
12CA5). Anti-UAF1 rabbit polyclonal antibodies were raised by immu-
nizing a rabbit with an N-terminal His-tagged fusion protein containing
amino acids 400–677 of UAF1 according to standard immunology
methods (Harlow and Lane, 1988).
Flag-HA-tagged USP1, UAF1, and FANCD2 were expressed using
the pOZ-N plasmid (Nakatani and Ogryzko, 2003). Flag-tagged
UAF1 deletion constructs were expressed using the pcDNA3.1 plas-
mid (Invitrogen). UAF1-DWD2, UAF1-DWD2–4, UAF1-D507–546,
UAF1-D546–585, UAF1-D635–677 carry deletions of aa 65–104, 65–
198, 507–546, 546–585, and 635–677, respectively. shRNA-mediated
knockdown of the UAF1 gene was achieved by expressing the target
sequences 50-GGACCGAGATTATCTTTC-30(#1) and 50-CAAGCAA
GATCCATATATA-30(#2) in the pSuper.retro vector (Clontech).
Mass Spectrometric Analysis
Proteins were reduced with DTT, cysteine residues were derivatized
with iodoacetamide, and the proteins were separated by SDS-
PAGE. Proteins from Coomassie-stained gel bands were in-gel di-
gested with trypsin (Shevchenko et al., 1996). The generated peptide
mixtures were subjected to LC-MS/MS using a hybrid linear ion trap/
FT-ICR mass spectrometer (LTQ FT, Thermo Electron) essentially as
described previously (Haas et al., 2006). MS/MS spectra were as-
signed by searching them with the SEQUEST algorithm (Eng et al.,
1994) against the human International Protein Index sequence data-
The USP1 complex was purified from nuclear extracts prepared from
HeLa cells expressing N-terminal Flag- and HA-epitope-tagged
USP1 as described (Nakatani and Ogryzko, 2003). N-terminal Flag-
and HA-epitope-tagged UAF1 and FANCD2 proteins were purified
by the same method, although more stringent washes including 0.5 M
KCl were applied during the purification procedures to obtain proteins
purified to homogeneity. Monoubiquitinated FANCD2 protein was pu-
rified from cells treated with 2 mM hydroxyurea for 24 hr.
Transient transfection of 293T cells for immunoprecipitation experi-
ments was performed using Fugene 6 (Roche) according to the man-
Proteins purified from Sf9 cells were expressed using either the
pFastBac-HTa vector (Invitrogen) containing an N-terminal His tag
(for USP1) or the pFastBac-1 vector (Invitrogen) with an engineered
C-terminal Strep II tag (for UAF1). For USP1 and USP1/UAF1 complex
purification, cell pellets were resuspended in lysis buffer (50 mM Tris-
HCl [pH 8.0], 150 mM NaCl, 10 mM BME, 10 mM imidazole, 10% glyc-
erol, and 0.2% Triton X-100) and sonicated to lyse. Lysates were
centrifuged, and the supernatants were incubated with Ni-NTA aga-
rose resin (QIAGEN) for 1 hr. The resin was washed extensively, and
the proteins were eluted in elution buffer (50 mM Tris-HCl [pH 8.0],
100 mM NaCl, 10 mM BME, 10% glycerol, and 250 mM imidazole).
Eluted protein was bound to a 5 ml HiTrap Q-FF cartridge (GE Biosci-
ences), washed with washing buffer (50 mM Tris-HCl [pH 8.0], 100 mM
KCl, 5 mM DTT, 0.1 mM EDTA, and 10% glycerol), and eluted in the
same buffer containing 500 mM KCl. For UAF1 purification, cells
were resuspended in lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM
NaCl, 2 mM DTT, and 10% glycerol) and centrifuged and the clarified
lysate was incubated for 1 hr with the Strep-Tactin resin (Novagen).
Following incubation, the resin was washed extensively and the pro-
tein was eluted in the same buffer containing 2.5 mM desthiobiotin.
In Vitro Enzymatic Assays
Transcription/translation in rabbit reticulocyte was performed accord-
ing to the manufacturer’s recommendations with the exception that
the reactions were performed at 30?C (Promega). In vitro enzymatic
assays in the presence of 5 mM ubiquitin vinyl sulfone (Ub-VS;
U-202; Boston Biochem) were performed at 30?C. In vitro enzymatic
assays using ubiquitin-7-amido-4-methylcoumarin (Ub-AMC; U-550;
Boston Biochem) were performed in 50–100 ml reaction buffer
(20 mM HEPES-KOH [pH 7.8], 20 mM NaCl, 0.1 mg/ml ovalbumin
was monitored in a FluoStar Galaxy Fluorometer (BMG Labtech).
In vitro deubiquitination reactions of Ub-FANCD2 were performed in
10 ml reaction buffer (90 mM Tris-HCl [pH 8.0], 50 mM NaCl, 0.03%
NP-40, 4 mg/ml BSA, and 2 mM DTT) for 2 hr at 30?C.
Northern Blot Analysis
Northern blot analysis was performed as described using the 394 nt
PstI-EcoRV fragment of the USP1 cDNA (Cohn et al., 1997).
Supplemental Data include three figures and can be found with this ar-
The authors would like to thank members of the D’Andrea laboratory
for helpful discussions; Drs. Fred Goldberg and Robert Cohen for con-
structive comments and suggestions; Dr. David Smith for help with the
fluorometricanalysis inthe Ub-AMC assays; Dr.Yoshihiro Nakatani for
the pOZ-N vector; and Dr. Jae Jung for kindly providing the cDNA for
UAF1. K.Y. is a Harvard University Presidential Scholar. This work was
supported by NIAID grant 1U19A1067751 and NIH grants HL52725
796 Molecular Cell 28, 786–797, December 14, 2007 ª2007 Elsevier Inc.
USP1/UAF1 Regulates the Fanconi Anemia Pathway
Received: April 10, 2007
Revised: June 13, 2007
Accepted: September 22, 2007
Published: December 13, 2007
Cohen, M., Stutz, F., Belgareh,N., Haguenauer-Tsapis, R., and Darge-
mont, C. (2003). Ubp3 requires a cofactor, Bre5, to specifically
de-ubiquitinate the COPII protein, Sec23. Nat. Cell Biol. 5, 661–667.
Cohn, M.A., Kramerov, D., Hulgaard, E.F., and Lukanidin, E.M. (1997).
The differentiation antigen Ly-6E.1 is expressed in mouse metastatic
tumor cell lines. FEBS Lett. 403, 181–185.
Driscoll, J., and Goldberg, A.L. (1990). The proteasome (multicatalytic
protease) is a component of the 1500-kDa proteolytic complex which
J. Biol. Chem.265,
Eng, J.K., McCormack, A.L., and Yates, J.R., III (1994). An approach to
correlate tandem mass spectral data of peptides with amino acid se-
quences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–
Fujiwara, T., Saito, A., Suzuki, M., Shinomiya, H., Suzuki, T., Takaha-
shi, E., Tanigami, A., Ichiyama, A., Chung, C.H., Nakamura, Y., and
Tanaka, K. (1998). Identification and chromosomal assignment of
USP1, a novel gene encoding a human ubiquitin-specific protease.
Genomics 54, 155–158.
Garcia-Higuera, I., Taniguchi, T., Ganesan, S., Meyn, M.S., Timmers,
C., Hejna, J., Grompe, M., and D’Andrea, A.D. (2001). Interaction of
the Fanconi anemia proteins and BRCA1 in a common pathway.
Mol. Cell 7, 249–262.
Gastwirt, R.F., Slavin, D.A., McAndrew, C.W., and Donoghue, D.J.
(2006). Spy1 expression prevents normal cellular responses to DNA
damage: inhibition of apoptosis and checkpoint activation. J. Biol.
Chem. 281, 35425–35435.
Gavin, A.C., Bosche, M., Krause, R., Grandi, P., Marzioch, M., Bauer,
A., Schultz, J., Rick, J.M., Michon, A.M., Cruciat, C.M., et al. (2002).
Functional organization of the yeast proteome by systematic analysis
of protein complexes. Nature 415, 141–147.
Gavin, A.C., Aloy, P., Grandi, P., Krause, R., Boesche, M., Marzioch,
M., Rau, C., Jensen, L.J., Bastuck, S., Dumpelfeld, B., et al. (2006).
Proteome survey reveals modularity of the yeast cell machinery. Na-
ture 440, 631–636.
Haas, W., Faherty, B.K., Gerber, S.A., Elias, J.E., Beausoleil, S.A., Ba-
kalarski, C.E., Li, X., Villen, J., and Gygi, S.P. (2006). Optimization and
use of peptide mass measurement accuracy in shotgun proteomics.
Mol. Cell. Proteomics 5, 1326–1337.
Hanna, J., Hathaway, N.A., Tone, Y., Crosas, B., Elsasser, S., Kirkpa-
trick, D.S., Leggett, D.S., Gygi, S.P., King, R.W., and Finley, D. (2006).
Deubiquitinating enzyme Ubp6 functions noncatalytically to delay pro-
teasomal degradation. Cell 127, 99–111.
Harlow, E., and Lane, D. (1988). Antibodies: a laboratory manual (Cold
Spring Harbor, N.Y.: Cold Spring Harbor Laboratory).
Ho, Y., Gruhler, A., Heilbut, A., Bader, G.D., Moore, L., Adams, S.L.,
Millar, A., Taylor, P., Bennett, K., Boutilier, K., et al. (2002). Systematic
identification of protein complexes in Saccharomyces cerevisiae by
mass spectrometry. Nature 415, 180–183.
Huang, T.T., Nijman, S.M., Mirchandani, K.D., Galardy, P.J., Cohn,
M.A., Haas, W., Gygi, S.P., Ploegh, H.L., Bernards, R., and D’Andrea,
A.D. (2006). Regulation of monoubiquitinated PCNA by DUB auto-
cleavage. Nat. Cell Biol. 8, 339–347.
Ingvarsdottir, K., Krogan, N.J., Emre, N.C., Wyce, A., Thompson, N.J.,
Emili, A., Hughes, T.R., Greenblatt, J.F., and Berger, S.L. (2005). H2B
ubiquitin protease Ubp8 and Sgf11 constitute a discrete functional
module within the Saccharomyces cerevisiae SAGA complex. Mol.
Cell. Biol. 25, 1162–1172.
Kennedy,R.D., and D’Andrea, A.D.(2005).The Fanconi Anemia/BRCA
pathway: new faces in the crowd. Genes Dev. 19, 2925–2940.
Krogan, N.J., Cagney, G., Yu, H., Zhong, G., Guo, X., Ignatchenko, A.,
Li, J., Pu, S., Datta, N., Tikuisis, A.P., et al. (2006). Global landscape of
protein complexes in the yeast Saccharomyces cerevisiae. Nature
Lee, K.K., Florens, L., Swanson, S.K., Washburn, M.P., and Workman,
J.L. (2005). The deubiquitylation activity of Ubp8 is dependent upon
Sgf11 and its association with the SAGA complex. Mol. Cell. Biol.
Leggett, D.S., Hanna, J., Borodovsky, A., Crosas, B., Schmidt, M.,
Baker, R.T., Walz, T., Ploegh, H., and Finley, D. (2002). Multiple asso-
ciated proteins regulate proteasome structure and function. Mol. Cell
Nakatani, Y., and Ogryzko, V. (2003). Immunoaffinity purification of
mammalian protein complexes. Methods Enzymol. 370, 430–444.
Niedernhofer, L.J., Lalai, A.S., and Hoeijmakers, J.H. (2005). Fanconi
anemia (cross)linked to DNA repair. Cell 123, 1191–1198.
Nijman, S.M., Huang, T.T., Dirac, A.M., Brummelkamp, T.R.,
Kerkhoven, R.M., D’Andrea, A.D., and Bernards, R. (2005a). The deu-
biquitinating enzyme USP1 regulates the Fanconi anemia pathway.
Mol. Cell 17, 331–339.
Nijman, S.M., Luna-Vargas, M.P., Velds, A., Brummelkamp, T.R.,
Dirac, A.M., Sixma, T.K., and Bernards, R. (2005b). A genomic and
functional inventory of deubiquitinating enzymes. Cell 123, 773–786.
Park, J., Lee, B.S., Choi, J.K., Means, R.E., Choe, J., and Jung, J.U.
(2002). Herpesviral protein targets a cellular WD repeat endosomal
protein to downregulate T lymphocyte receptor expression. Immunity
(2006). hRpn13/ADRM1/GP110 is a novel proteasome subunit that
binds the deubiquitinating enzyme, UCH37. EMBO J. 25, 5742–5753.
Rumpf, S.,and Jentsch, S.(2006). Functional division of substrate pro-
cessing cofactors of the ubiquitin-selective Cdc48 chaperone. Mol.
Cell 21, 261–269.
Shevchenko, A., Wilm,M., Vorm, O., and Mann, M. (1996).Mass spec-
trometric sequencing of proteins silver-stained polyacrylamide gels.
Anal. Chem. 68, 850–858.
Smogorzewska, A., Matsuoka, S., Vinciguerra, P., McDonald, E.R., III,
Hurov, K.E., Luo, J., Ballif, B.A., Gygi, S.P., Hofmann, K., D’Andrea,
A.D., and Elledge, S.J. (2007). Identification of the FANCI protein,
a monoubiquitinated FANCD2 paralog required for DNA repair. Cell
Taniguchi, T., Garcia-Higuera, I., Xu, B., Andreassen, P.R., Gregory,
R.C., Kim, S.T., Lane, W.S., Kastan, M.B., and D’Andrea, A.D.
(2002). Convergence of the fanconi anemia and ataxia telangiectasia
signaling pathways. Cell 109, 459–472.
Yao, T., Song, L., Xu, W., DeMartino, G.N., Florens, L., Swanson, S.K.,
Washburn, M.P., Conaway, R.C., Conaway, J.W., and Cohen, R.E.
(2006). Proteasome recruitment and activation of the Uch37 deubiqui-
tinating enzyme by Adrm1. Nat. Cell Biol. 8, 994–1002.
Molecular Cell 28, 786–797, December 14, 2007 ª2007 Elsevier Inc. 797
USP1/UAF1 Regulates the Fanconi Anemia Pathway