A genetic screen identifies FAN1, a Fanconi anemia-associated nuclease necessary for DNA interstrand crosslink repair.
ABSTRACT The Fanconi anemia (FA) pathway is responsible for interstrand crosslink repair. At the heart of this pathway is the FANCI-FAND2 (ID) complex, which, upon ubiquitination by the FA core complex, travels to sites of damage to coordinate repair that includes nucleolytic modification of the DNA surrounding the lesion and translesion synthesis. How the ID complex regulates these events is unknown. Here we describe a shRNA screen that led to the identification of two nucleases necessary for crosslink repair, FAN1 (KIAA1018) and EXDL2. FAN1 colocalizes at sites of DNA damage with the ID complex in a manner dependent on FAN1's ubiquitin-binding domain (UBZ), the ID complex, and monoubiquitination of FANCD2. FAN1 possesses intrinsic 5'-3' exonuclease activity and endonuclease activity that cleaves nicked and branched structures. We propose that FAN1 is a repair nuclease that is recruited to sites of crosslink damage in part through binding the ubiquitinated ID complex through its UBZ domain.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: The application of new proteomics and genomics technologies support a view in which few drugs act solely by inhibiting a single cellular target. Indeed, drug activity is modulated by complex, often incompletely understood cellular mechanisms. Therefore efforts to decipher mode of action through genetic perturbation such as RNAi typically yields "hits" that fall into several categories. Of particular interest to the present study, we aimed to characterize secondary activities of drugs on cells. Inhibiting a known target can result in a clinically relevant synthetic phenotypes. In one scenario, drug perturbation could, for example, improperly activates a protein which normally inhibits a particular kinase. In other cases, additional, lower affinity targets can be inhibited as in the example of inhibition of c-Kit observed in Bcr-Abl-positive cells treated with Glivec. Drug transport and metabolism also play an important role in the way any chemicals act within the cells. Finally, RNAi per se can also affect cell fitness by more general off-target effects, e.g. via the modulation of apoptosis or DNA damage repair. Regardless of the root cause of these unwanted effects, understanding the scope of a drug's activity and polypharmacology is essential for better understanding its mechanism(s) of action, and such information can guide development of improved therapies. We describe a rapid, cost-effective approach to characterize primary and secondary effects of small-molecules using small-scale libraries of virally integrated shRNAs. We demonstrate this principle using a "minipool" comprised of shRNAs that target the genes encoding the reported protein targets of approved drugs. Among the 28 known reported drug-target pairs, we successfully identify 40% of the targets described in the literature and uncover several unanticipated drug-target interactions based on drug-induced synthetic lethality. We provide a detailed protocol for performing such screens and for analyzing the data. This cost-effective approach to mammalian knockdown screens, combined with the increasing maturation of RNAi technology will expand the accessibility of similar approaches in academic settings.G3-Genes Genomes Genetics 06/2013; · 1.79 Impact Factor
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ABSTRACT: The Fanconi anemia (FA) network is important for the repair of interstrand DNA cross-links. A key event in FA pathway activation is the monoubiquitylation of the FA complementation group I (FANCI)-FANCD2 (ID) complex by FA complementation group L (FANCL), an E3 ubiquitin ligase. In this study, we show that RAD18, another DNA damage-activated E3 ubiquitin ligase, also participates in ID complex activation by ubiquitylating proliferating cell nuclear antigen (PCNA) on Lys164, an event required for the recruitment of FANCL to chromatin. We also found that monoubiquitylated PCNA stimulates FANCL-catalyzed FANCD2 and FANCI monoubiquitylation. Collectively, these experiments identify RAD18-mediated PCNA monoubiquitination as a central hub for the mobilization of the FA pathway by promoting FANCL-mediated FANCD2 monoubiquitylation.The Journal of Cell Biology 10/2010; 191(2):249-57. · 10.82 Impact Factor
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ABSTRACT: Several proteins in the BRCA-Fanconi anemia (FA) pathway, such as FANCJ, BRCA1, and FANCD2, interact with mismatch repair (MMR) pathway factors, but the significance of this link remains unknown. Unlike the BRCA-FA pathway, the MMR pathway is not essential for cells to survive toxic DNA interstrand crosslinks (ICLs), although MMR proteins bind ICLs and other DNA structures that form at stalled replication forks. We hypothesized that MMR proteins corrupt ICL repair in cells that lack crosstalk between BRCA-FA and MMR pathways. Here, we show that ICL sensitivity of cells lacking the interaction between FANCJ and the MMR protein MLH1 is suppressed by depletion of the upstream mismatch recognition factor MSH2. MSH2 depletion suppresses an aberrant DNA damage response, restores cell cycle progression, and promotes ICL resistance through a Rad18-dependent mechanism. MSH2 depletion also suppresses ICL sensitivity in cells deficient for BRCA1 or FANCD2, but not FANCA. Rescue by Msh2 loss was confirmed in Fancd2-null primary mouse cells. Thus, we propose that regulation of MSH2-dependent DNA damage response underlies the importance of interactions between BRCA-FA and MMR pathways.The EMBO Journal 06/2014; · 9.82 Impact Factor
A Genetic Screen Identifies FAN1,
a Fanconi Anemia-Associated Nuclease
Necessary for DNA Interstrand Crosslink Repair
Agata Smogorzewska,1,2,3,* Rohini Desetty,3Takamune T. Saito,4Michael Schlabach,1Francis P. Lach,3
Mathew E. Sowa,5Alan B. Clark,6Thomas A. Kunkel,6J. Wade Harper,5Monica P. Colaia ´covo,4and Stephen J. Elledge1,*
1Howard Hughes Medical Institute, Department of Genetics, Harvard Medical School, Department of Medicine, Division of Genetics,
Brigham and Women’s Hospital, Boston, MA 02115, USA
2Department of Pathology, Massachusetts General Hospital, Boston MA 02114, USA
3Laboratory of Genome Maintenance, The Rockefeller University, New York, NY 10065, USA
4Department of Genetics
5Department of Pathology
Harvard Medical School, Boston MA 02115, USA
6Laboratory of Molecular Genetics and Laboratory of Structural Biology, National Institute of Environmental Health Sciences,
National Institutes of Health, DHHS, Research Triangle Park, NC 27709, USA
*Correspondence: firstname.lastname@example.org (A.S.), email@example.com (S.J.E.)
The Fanconi anemia (FA) pathway is responsible for
interstrand crosslink repair. At the heart of this
pathway is the FANCI-FAND2 (ID) complex, which,
upon ubiquitination by the FA core complex, travels
to sites of damage to coordinate repair that includes
nucleolytic modification of the DNA surrounding the
a shRNA screen that led to the identification of two
nucleases necessary for crosslink repair, FAN1
(KIAA1018) and EXDL2. FAN1 colocalizes at sites of
dent on FAN1’s ubiquitin-binding domain (UBZ),
the ID complex, and monoubiquitination of FANCD2.
FAN1 possesses intrinsic 50-30exonuclease activity
and endonuclease activity that cleaves nicked and
branched structures. We propose that FAN1 is
a repair nuclease that is recruited to sites of crosslink
damage in part through binding the ubiquitinated ID
complex through its UBZ domain.
Cells in all organisms experience massive amounts of sponta-
neous DNA damage each day. A failure to properly respond to
this genotoxic stress can lead to both developmental abnormal-
ities and tumorigenesis. Organisms have evolved a complex
signal transduction pathway called the DNA damage response
(DDR) that senses genotoxic stress and orchestrates a response
by activating specific types of repair, arresting the cell cycle and
altering transcription. At the core of this signal transduction
pathway are two PI-3 kinase-like protein kinases, ATM and
ATR (Bakkenist and Kastan, 2004; Bartek et al., 2004; Harper
and Elledge, 2007), which support the damage-induced phos-
phorylation of hundreds of substrates to coordinate DNA repair
(Matsuoka et al., 2007; Stokes et al., 2007).
A life-threatening lesion is the DNA double-strand crosslink,
which covalently connects the Watson and Crick strands of
known as the Fanconi anemia (FA) pathway has evolved to
specifically deal with these types of lesions. FA is a recessive
developmental and cancer predisposition syndrome whose
dence of malignancies (Alter et al., 2003). Cells from FA patients
are hypersensitive to DNA interstrand crosslinking (ICL) agents
such as mitomycin C (MMC) (Auerbach and Wolman, 1976). To
date, 13 proteins have been implicated in FA. At the center of
this pathway is the FANCI/FANCD2 (ID) complex, which loads
onto sites of crosslinks to direct DNA repair. The ID complex is
chromatin bound, and when it encounters a DNA replication
structure stalled due to a DNA crosslink, it becomes phosphory-
lated by the ATR/ATRIP kinase, which is localized through
recognition of RPA at the lesion (Zou and Elledge, 2003). Phos-
phorylation of both I and D2 is required for ID function (Andreas-
monoubiquitination of both subunits by a multisubunit E3 ligase
formed by eight FA proteins (FANCA/B/C/E/F/G/L/M) and the
E2-conjugating enzyme UBE2T (Cole et al., 2010; Machida
et al., 2006; Meetei et al., 2004). Ubiquitinated ID then accumu-
lates at the damage site and directs repair (Smogorzewska
et al., 2007; Knipscheer et al., 2009; Raschle et al., 2008).
The repair of a crosslink is thought to involve two incision
events on a single DNA strand flanking the lesion, followed by
bypass synthesis over the lesion on the remaining intact strand
using a translesion polymerase, possibly Rev1 (Niedzwiedz
et al., 2004; Simpson and Sale, 2003) in combination with Rev3
and Rev7 (Lehmann et al., 2007; Raschle et al., 2008). A recently
identified A family nuclear DNA polymerase, PolN, might also
36 Molecular Cell 39, 36–47, July 9, 2010 ª2010 Elsevier Inc.
After bypass synthesis, two more incision events flanking the
can then be repaired by gene conversion using homologous
recombination (HR). Both the incision step and the bypass poly-
merase step are dependent upon ubiquitination of the ID
complex (Knipscheer et al., 2009; Raschle et al., 2008). The
nucleases responsible for the incision and excision events
are not precisely known, although XPF-ERCC1 and Mus81
complexes have been implicated (Ciccia et al., 2008). Recently,
SLX4, a scaffold for various DNA repair nucleases, has been
identified to be necessary for resistance to crosslinking agents.
SLX4 interacts with both Mus81 and XPF and together with
SLX1 forms a Holliday junction resolvase, although it is unclear
which SLX4 activity is responsible for conferring resistance to
DNA crosslinks (Fekairi et al., 2009; Munoz et al., 2009; Saito
et al., 2009; Svendsen et al., 2009). After bypass synthesis, the
two strands liberated by the first two incision events constitute
a double-strand break and are repaired by HR. Since HR
requires 30overhangs to initiate recombination, it is likely these
strands are processed by a 50-30exonuclease to generate the
recombination substrate that can gap repair off the strand from
which the lesion was removed.
A number of outstanding issues remain unresolved in the FA
pathway. First and foremost is how the ID complex orches-
trates repair and the role of ubiquitination. Does it play a struc-
tural role in simply maintaining the complex at sites of damage,
or does it recruit repair factors? In addition, the identities of the
enzymatic factors involved in manipulation of the DNA at the
site of the lesion are still unknown. Furthermore, it is unknown
if there are additional backup pathways that can also function
to repair crosslinks in the absence of FA. The FA pathway
appears to be conserved in metazoans, but is missing from
prokaryotes and unicellular eukaryotes such as S. cerevisiae
and S. pombe. Thus, alternative repair pathways do exist in
nature. To address these issues and identify new genes
involved in crosslink repair, we performed an RNAi screen for
genes required for resistance to the crosslinking agent MMC
and discovered two nucleases required for crosslink repair,
one of which, FAN1, possesses both endonuclease and exonu-
clease activities and is recruited to lesions by the monoubiqui-
tinated ID complex.
Genome-wide shRNA Screen to Identify Proteins
Necessary for Resistance to Interstrand Crosslink
To identify proteins involved in resistance to crosslinking agents,
we screened U2OS cells transduced with a library of 74,905
retroviral shRNAs targeting 32,293 unique human transcripts
(Figure 1A). Using competitive hybridization of probes that
detect the half-hairpins (HHs) of the shRNAs, we compared the
relative abundance of shRNAs between untreated cells and cells
treated with low levels of MMC. Cells bearing shRNAs that
conferred sensitivity were depleted from the treated population.
About 2173 hairpins targeting 2017 genes conferred sensitivity
to MMC using the criteria of an average loss of 2-fold from
the treated population (log2 > 1) (see Table S1 available online).
Among these were previously known DDR proteins including
BRCA1, TOPBP1, RAD18, RAD17, RAD51, RAD54, FANCE,
and others. We employed the multicolor competition assay
(MCA) (Smogorzewska et al., 2007) with 379 shRNAs against
a selected group of genes that made the cutoff in the primary
screen to retest for MMC sensitivity. Eighty-four shRNAs tested
conferred MMC sensitivity (Table S2). Figure 1B shows the top
38 scoring hairpins in which resistance to MMC was below
80% of control shRNA. To further examine damage sensitivity,
pools of siRNAs were also tested (Figure 1C). Based on the
results of this assay and domain analysis, two genes, EXDL2
and KIAA1018, were chosen for further study.
EXDL2—A Putative 30-50Exonuclease Necessary
for Resistance to MMC
EXDL2 is an uncharacterized 621 amino acid protein with
a nuclease domain most similar to the WRN-exo domain in the
WRN protein (Figures S1A and S1B) (Perry et al., 2006). Based
on the conservation of the four key negatively charged residues
(DEDD) that serve as ligands for the metal ions, as well as a tyro-
sine residue that has been shown to be important for the catal-
ysis, EXDL2 is predicted to be a 30-50exonuclease. A mutation
in a D. melanogaster ortholog of EXDL2, CG6744, displays
a phenotype of hyperrecombination (Cox et al., 2007). In human
cells, depletion of EXDL2 using three different siRNAs led to
sensitivity to MMC, the Topo1 inhibitor camptothecin (CPT),
and the alkylating agent MMS (Figure 1D and Figure S1C). This
spectrum of sensitivities is similar to mutants in the FA pathway.
Therefore, we tested if FANCD2 ubiquitination was affected in
the EXDL2-depleted cells. Based on the normal ubiquitination
of FANCD2 before and after damage (Figure S1D), we conclude
that EXDL2 is either downstream of FANCD2 in the Fanconi
pathway or in a parallel pathway of crosslink repair.
FAN1 (KIAA1018) Is Required for the Resistance
to Crosslinking Agents
A second protein with an interesting domain structure is
KIAA1018, which we renamed FAN1 (Fanconi-associated
nuclease 1) based on the data presented below. FAN1 has a
nuclease-like fold called DUF994 (later renamed VRR-NUC) at
its C terminus, potential DNA-binding (SAP) and protein-protein
interaction (TPR) motifs in its midsection, and a Rad18-like
ZnF domain at the N terminus (Kinch et al., 2005) (Fig-
ure 4A). Depletion of FAN1 with multiple siRNAs leads to sensi-
tivity to crosslinking agents including MMC, chlorambucil,
carboplatin, and oxaliplatin, as well as CPT and MMS (Figures
2A and 2B).
To examine the evolutionary conservation of FAN1’s role
in crosslink repair, we examined the crosslink sensitivity of a
C. elegans FAN1 mutant (tm423) that carries a deletion of the
SAP domain (Figure S2). fan-1 mutants lay normal numbers of
eggs and show normal larval development as well asno increase
in either embryonic lethality or the percent of males among their
progeny that would suggest a meiotic phenotype (Figure 2D).
However, treatment with either MMC or nitrogen mustard
(HN2) results in decreased embryonic viability compared to
wild-type as judged by decreased hatching (p < 0.0001,
The FAN1 Nuclease Functions in Crosslink Repair
Molecular Cell 39, 36–47, July 9, 2010 ª2010 Elsevier Inc. 37
respectively, chi-square test) (Figure 2E). Thus, FAN1 plays an
evolutionarily conserved role in resistance to crosslink damage.
Since the tm423 mutant may be a hypomorph, a meiotic pheno-
type could emerge in null animals.
FAN1 Associates with Mismatch Repair Proteins
To identify FAN1-associated proteins, a HA-tagged FAN1 was
purified from 293TREX cells, and interacting proteins were iden-
tified by LC-MS/MS. Proteomic data were processed using the
Comparative Proteomic Analysis Software Suite (CompPASS)
(Sowa et al., 2009). The top-scoring interacting proteins were
the mismatch repair proteins MLH1, MLH3, PMS1, and PMS2
(Figure 3A). KIAA1018 was previously detected in a proteomic
analysis of MLH1-interacting proteins (Cannavo et al., 2007);
however, the interaction was not independently confirmed. To
confirm these interactions, we performed immunoprecipitations.
HA-FAN1 was identified in immunoprecipitates of MLH1, MLH3,
and PMS2 (Figure 3B). Reciprocal immunoprecipitations identi-
fied all four proteins in precipitation reactions with anti-HA anti-
bodies (Figure 3C). Endogenous FAN1 immunoprecipitations
brought down both FAN1 and MLH1 in a FAN1-dependent
manner (Figure 3D). The interaction with the mismatch repair
machinery raised the possibility that FAN1’s role in ICL resis-
FAN1 from the HCT116 cells, which carries inactivating muta-
tions in both alleles of MLH1 (Figure 3E). FAN1 depletion with
four different siRNAs still increased sensitivity of these cells to
Figure 1. Whole-Genome shRNA Screen to Identify Genes Necessary for Crosslink Resistance
(A) Schematic of the primary screen. Changes in hairpin abundance after transduction with the shRNA library and MMC treatment were followed by competitive
DNA array hybridization.
(B) MCA in U2OS cells transduced with the indicated shRNAs. Of the tested hairpins, only those that showed less than 80% resistance to 15 or 50 nM MMC
in Table S2.
(C) MCA in U2OS cells transfected with the indicated siRNAs (poolsof four siGENOME siRNAs from Dharmacon). Cells transfected with siRNAs against ATMand
ATR were used as a control.
(D) MCA in U2OS cells transfected with three separate siRNAs against EXDL2. Cells transfected with siRNAs against FANCI were used as a control. Error bars
represent standard deviation across three technical replicates.
The FAN1 Nuclease Functions in Crosslink Repair
38 Molecular Cell 39, 36–47, July 9, 2010 ª2010 Elsevier Inc.
FAN1 Colocalizes to Sites of Damage with FANCD2 via
Its UBZ Domain
and nuclease domains (Figures 4A and 4B) led us to test FAN1
localization to sites of DNA damage. We subjected GFP-tagged
FAN1 cells to laser microirradiation, which results in localized
DNA damage tracks (Bekker-Jensen et al., 2006). Within
15 min of microirradiation, GFP-FAN1 localized to sites of DNA
(Rogakou et al., 1999). The GFP tracks were also seen in the
absence of g-H2AX staining (data not shown). We next asked if
FAN1 localized to sites of crosslinked DNA damage caused by
MMC. Most MMC-induced foci contained both FAN1 and
FANCD2 (Figure 4D). We next tested various FAN1 domain
(Figure 5A). Mutants in the nuclease domain behaved like wild-
type. The N-terminal 373 amino acids lacking SAP or a protein
with a SAP domain mutation (L477P) still localized to sites of
damage, but the strength of the GFP-FAN1 signal was substan-
tially diminished. Only the mutant with two conserved cysteine
residues of the UBZ domain substituted by alanines (C44A,
C47A) completely failed to localize to microirradiation tracks
(Figure 5B). The same UBZ mutant did not form foci in MMC-
treated cells (Figure 5C) but was expressed at similar levels as
the other alleles (Figure 5C, top panel ‘‘no triton’’). Several other
mutant alleles localized to sites of damage with reduced effi-
ciency (Figure S3). Interestingly, the N-terminal 90 amino acids
although with diminished efficiency. Based on these experi-
ments, we conclude that the UBZ domain is critical for localizing
assist in localization. Careful kinetic analysis of the behavior of
the different mutants will be necessary to fully understand the
role of all domains in localization of FAN1 to sites of damage.
FANCI and FANCD2 Are Required for FAN1 Localization
to Sites of DNA Damage
To examine possible dependency on the ID complex, we
assessed formation of GFP-FAN1 foci after MMC treatment
in cells depleted of FANCD2 and FANCI. In FANCI- or FANCD2-
depleted cells, FAN1 was no longer able to form foci (Figure 6A
and Figure S4B). The percentage of cells with foci in cells
Figure 2. Sensitivity to Crosslinking Agents and Camptotecin in Human Cells Depleted of FAN1 and in a FAN1 Mutant C. elegans Strain
(A) MCA in U2OS cells transfected with six separate siRNAs against FAN1 and treated with different types of DNA damaging agents.
(B) MCA in U2OS cells transfected siRNAs against FAN1 and treated with different DNA crosslinking agents.
(C) RT-qPCR in U2OS cells transfected with the different siRNAs against FAN1.
(A–C) Error bars represent the standard deviation (SD) across three technical replicates.
scored,dL1-L4 worms. ND, not determined due to n = 0. Wild-type data are from Saito et al. (2009).
(E) Relative hatching of wild-type, fan-1, and him-18 mutants after treatment with the indicated doses of MMC and nitrogen mustard (HN2). Hatching is plotted as
a fraction of the hatching observed in untreated animals. Error bars indicate standard error of the mean for at least 20 animals in each of three independent
The FAN1 Nuclease Functions in Crosslink Repair
Molecular Cell 39, 36–47, July 9, 2010 ª2010 Elsevier Inc. 39
tively, while 93% of control depleted cells showed FAN1 foci.
FAN1 also fails to form foci in PD20 cells, which lack FANCD2
protein (Figure 6B), but FAN1 foci reappear upon complementa-
quitination-defective FANCD2 K561R mutant also fail to
form FAN1 foci (Figure 6B). We next asked if FAN1 is required
for FANCD2 monoubiquitination. Cells transfected with four
different siRNAs against FAN1 were treated with MMC. FANCD2
in these cells was ubiquitinated to the same extent as in cells
treated with siRNAs against luciferase (Figure 6C). We conclude
the crosslink sensitivity of FA-defective cells is in part due to
a failure to recruit FAN1 to sites of DNA damage.
FAN1 Interacts with FANCD2
The dependence of FAN1 foci formation on the presence of
monoubiquitinated FANCD2 raised a possibility that FANCD2
and FAN1 interact. The chromatin fraction from cells expressing
GFP-FAN1 were subjected to immunoprecipitation with anti-
FANCD2 antibodies. GFP-FAN1 strongly coimmunoprecipitated
with FANCD2 (Figure 6D).Therefore, FAN1 and FANCD2 interact
Figure 3. FAN1 Associates with Mismatch Repair Proteins
(A) HA-FAN1 was expressed in 293 TREX cells using an inducible retroviral system and cell extracts were subjected to immunoprecipitation and LC-MS/MS as
described inthe ExperimentalProcedures.High-confidence candidate-interacting proteinsare shown as determined usingCompPASS to derive normalized WD
score for individual proteins in the immune complex. TSC, total spectral count. This experiment was done in duplicate, and average values are reported. In both
experiments all listed proteins were identified.
(B)Cellextractsof293TREXcells expressing HA-FAN1weresubjected toimmunoprecipitationusingtheindicated antibodies. HA-FAN1wasidentifiedbyimmu-
noblotting with an anti-HA antibody. IN represents 5% input.
(C) Cellextractsof 293TREX cells expressing HA-FAN1weresubjected toimmunoprecipitationusing ananti-HAantibody, andimmunoprecipitateswere probed
with the indicated antibodies. HC, heavy chain of the antibody used in the immunoprecipitation.
(D) Endogenous FAN1 was immunoprecipitated (± antigenic peptide) from HeLa cells with or without FAN1 depletion using two separate shRNAs and immuno-
blotted for FAN1 and MLH1. shRNAs #1–739 and shRNA #2–600. Note that the FAN1 antibody does not recognize endogenous protein in straight western, only
(E) MMC and camptotecin sensitivity caused by depletion of FAN1 are not dependent on MLH1 or MSH3. MCA in HCT116 cells transfected with the indicated
siRNAs. Error bars represent the standard deviation (SD) across three technical replicates.
The FAN1 Nuclease Functions in Crosslink Repair
40 Molecular Cell 39, 36–47, July 9, 2010 ª2010 Elsevier Inc.
FAN1 Has an Endonuclease and 50Exonuclease Activity
FAN1 contains a conserved nuclease domain. To assess its
well as a nicked substrate (Figure 7A, lane 17). The second
nicked substrate (Figure 7A, lane 20). The endonuclease activity
on the 50FLAP substrate was seen on the top strand at the junc-
tion of the two DNA duplexes, not on the single strand flap itself.
Neither the exonuclease nor the endonuclease activities were
seen in the immunoprecipitates from cells expressing control
HA protein or a mutant FAN1 with the key residues predicted to
be necessary for the catalysis substituted to alanines (Q864A,
D960A, E975A, K977A) (Figure 7A, lanes 3, 9, 12, 18, and 21),
although the wild-type and mutantproteins were immunoprecip-
itated to the same extent (Figure S5A). To further show that this
activity is intrinsic to FAN1 as opposed to a factor like MLH1/
PMS2 associated with FAN1 in mammalian cells, we expressed
the last 644 amino acids of human FAN1 (aa 373–1017) as
a His6fusion protein in bacteria (Figure 7B, Figures S5B and
S5C). Indeed, the purified protein had the activities seen with
the mammalian FAN1. More robust activity of FAN1 purified
from bacteria gave us an opportunity to define the substrate
specificity. Using a 30-labeled substrate, we have confirmed
100830 67469 509893
Figure 4. FAN1 Is an Evolutionarily Conserved Protein that Localizes to Sites of DNA Damage
(A) Schematic of the domain architecture of FAN1. Conserved domains are indicated: UBZ, ubiquitin-binding zinc finger; SAP, SAF-A/B, Acinus and PIAS; TPR,
tetratricopeptide repeat; Nuc (VRR-NUC) virus type replication-repair nuclease.
able UBZ domain. Stars indicate residues mutated in subsequent experiments.
(C) U2OS cells expressing GFP-FAN1 were laser microirradiated and after 30 min were processed for imaging of GFP-FAN1 and g-H2AX. GFP was visualized
directly. Nuclei were stained with DAPI.
(D) U2OS cells expressing GFP-FAN1 were treated with 1 mM MMC for 24 hr and processed for indirect immunofluorescence with antibodies against GFP and
FANCD2. Nuclei are stained with DAPI. Images were captured and deconvolved using the DeltaVision Image Restoration Microscope.
The FAN1 Nuclease Functions in Crosslink Repair
Molecular Cell 39, 36–47, July 9, 2010 ª2010 Elsevier Inc. 41
26). The major endonuclease activity of FAN1 was observed on
the top strand of the 50FLAP substrate at the junction of the
DNA duplexes (Figure 7B, lane 8) and across from a nick (Fig-
ure 7B, lane 35). A weak 50FLAP endonuclease activity was
seen on the bottom strand (Figure 7B, lane 11). On a replication
fork, the major activity was on a top strand (Figure 7B, lane 29),
with some activity seen on the bottom strand (Figure 7B,
lane 32). FAN1 activity was stimulated by ATP (Figure S5E). We
noted a well-conserved motif in FAN1 (GFDQGIHGEGST, amino
FAN1 with the key residues predicted to be necessary for the
catalysis substituted to alanines (Q864A, D960A, E975A,
K977A) (Figure 7B) or a mutant with just two mutations (E975A,
K977A) (Figure S5F) lacked the 50-30exonuclease and the endo-
nuclease activities. The bacterially purified proteins did have
some contaminating 30exonuclease activity (Figure 7B, lane
27). Based on these in vitro experiments, we conclude that
FAN1 possesses an intrinsic nuclease activity that participates
in DNA repair.
An shRNA Screen Identifies Many Putative Players
in Crosslink Resistance
ICLs are among the most lethal lesions to cells, and their repair
pathway(s) remain poorly understood. Given the difficulty that
ICLs create during replication and transcription, it is critical to
identify all the participants in the repair process. Therefore we
performed an shRNA screen to identify new components of
crosslink repair. Despite the limitations of RNAi, we were able
to confirm a number of proteins involved in resistance to cross-
linking agents using shRNAs and siRNAs. Among these were
C4orf21, which has both a ZF-GRF, a presumed DNA-binding
domain, and a domain with similarities to helicases and
FLJ25006, which has a kinase domain with predicted serine/
threonine activity. Among the known DNA repair proteins was
POLQ, an error-prone translesion DNA polymerase (Arana
et al., 2008; Seki et al., 2004). POLQ in chicken cells has been
shown to be involved in repair of oxidative damage, but not in
crosslink repair (Yoshimura et al., 2006). However, in C. elegans,
POLQ has been implicated in ICL repair, in a pathway distinct
UBZ mut NUC mut
N term SAP mut
UBZ mut NUC mut N term SAP mut
Figure 5. Localization of FAN1 to Damage Sites Depends on the UBZ Domain
(A) Schematic of mutant proteins used in FAN1 localization experiments.
(B) U2OS cells expressing the indicated GFP-FAN1 mutants were laser microirradiated and after 30 min were processed for imaging of GFP-FAN1 and g-H2AX.
(C) U2OS expressing the indicated GFP-FAN1 mutants were treated with 1 mM MMC for 24 hr and processed for indirect immunofluorescence with antibodies
against GFPand FANCD2. Cellswere either directlyfixed withformaldehyde orpre-extracted withTritonX-100 before fixation. Exposures wereadjustedtoshow
the presence of foci even if foci were dim. For the comparison of the intensity of foci, see Figure S3.
The FAN1 Nuclease Functions in Crosslink Repair
42 Molecular Cell 39, 36–47, July 9, 2010 ª2010 Elsevier Inc.
from the FA pathway but in the same pathway as the C. elegans
BRCA1 ortholog (Muzzini et al., 2008).
Among the validated genes with strong phenotypes were two
putative nucleases. We validated involvement of one, EXDL2, in
crosslink sensitivity using multiple siRNAs. EXDL2 has a WRN-
like exonuclease domain and promises to shed light on the
mechanism of crosslink resistance. The other protein FAN1,
with an intriguing domain structure, has now been placed as
a bona fide nuclease necessary for crosslink repair in the FA
Evolutionary Conservation of FAN1 Function
FAN1 is easily identifiable in Dictyostelium discoideum, which
has orthologs of several FA proteins (Zhang et al., 2009), but
no obvious orthologs are apparent in Drosophila or Xenopus.
Interestingly, an S. pombe ortholog of FAN1 has a SAP domain,
aTRPdomain,andanucleasedomain,andthusit ispredicted to
also function in DNA repair transactions. Fission yeast does not
possess the classical FA proteins except for an ortholog of
FANCM, Fml1, which promotes Rad51-dependent gene conver-
sion at stalled replication forks and limits crossing over during
mitotic double-strand break repair (Sun et al., 2008). It will be
interesting to test if the S. pombe FAN1 mutant is sensitive to
crosslinking agents and, if so, how it functions without the other
proteins present in human cells. This could shed light on alterna-
tive pathways for crosslink repair in human cells.
Interaction of FAN1 with Mismatch Repair Proteins
We identified all human MutL proteins in nearly stoichiometric
complexes with FAN1, suggesting a highly conserved function.
Despite the strong interaction with MutLs, we have yet to identify
a mismatch repair defect in extracts derived from cells depleted
of FAN1 using shRNAs. This could be due to the limitations of
RNAi to create the equivalent of a null mutation. Alternatively,
the MutL complexes may play roles outside of mismatch repair.
During mismatch repair in bacteria, MutL complexes are
recruited to sites of mismatches by MutS complexes that sense
the mismatch lesions. MutL complexes then recruit the UvrD
+K>R FANCD2+WT FANCD2+vector
siRNAs (U2OS cells)
MMC: - + - + - + - + - + - +
Figure 6. Localization of FAN1 Depends on the Fanconi Anemia Proteins FANCI and FANCD2
(A) U2OS cells expressing GFP-FAN1 and transfected with the indicated siRNAs were treated with 1 mM MMC for 24 hr, fixed, and processed for indirect immu-
nofluorescence with antibodies against GFP and FANCD2. The corresponding Triton X-100 extraction experiment is shown in Figure S4B.
GFP-FAN1 were treated with 1 mM MMC for 24 hr, fixed, and processed for indirect immunofluorescence with antibodies against GFP and FANCD2. The
corresponding Triton X-100 extraction experiment is shown in Figure S4C.
(C) U2OS cells transfected with the indicated siRNAs were treated with 1 mM MMC for 24 hr and collected for western blotting with anti-FANCD2 antibodies.
(D) Cell extracts of 293 cells expressing GFP-FAN1 were subjected to immunoprecipitation using FANCD2 antibody. GFP-FAN1 and FANCD2 were identified by
The FAN1 Nuclease Functions in Crosslink Repair
Molecular Cell 39, 36–47, July 9, 2010 ª2010 Elsevier Inc. 43
helicase and the MutH endonuclease, which it also activates, to
initiate repair. MutL complexes have also been shown to have an
endogenous endonuclease activity that could participate in
mismatch repair (Kadyrov et al., 2006). It is possible that MutL
complexes could play a similar role in ICL repair. In place of
MutS complexes, the monoubiquitinated ID complex would
play the analogous role as an ICL lesion sensor to recruit the
FAN1-MutL complex to introduce one or more of the four inci-
sions needed for ICL repair. In addition, this complex could
recruit additional factors to aid in repair. The MutLa (MLH1-
PMS2 heterodimer) complex has been identified as an interactor
of FANCJ (BRIP1), a helicase involved in crosslink repair (Peng
et al., 2007). A direct MLH1-FANCJ interaction was required
for FANCJ to complement the crosslink sensitivity of a patient
cell line with a mutation in the FANCJ gene. Thus, it is possible
that FAN1 and MutLa might act as a bridge between the ID
complex and FANCJ allowing the nuclease activity of FAN1
to pair with FANCJ’s helicase activity during the crosslink
repair process. It will be important to determine if the interaction
of MLH1 with FAN1 and FANCJ is mutually exclusive and
whether FAN1 or MLH1 is required to localize FANCJ to the sites
FAN1 Localization to Sites of Crosslink Damage
and Interaction with FANCD2
FAN1 localizes to laser microirradiation sites as well as to cross-
link-induced damage foci, where it colocalized with FANCD2.
Localization of FAN1 to foci was dependent on the UBZ domain
of FAN1, the presence of FANCI and FANCD2, and the monou-
biquitination of FANCD2. Based on these findings, we
5’ FLAP3’ FLAP 3’ FLAP
WT FAN1WT FAN1 WT FAN1
MUT FAN1 MUT FAN1MUT FAN1
WT FAN1WT FAN1
MUT FAN1MUT FAN1
5’ FLAP3’ FLAP
BACTERIAL HIS-FAN1 aa373-1017
ICL DNA REPLICATION BLOCK
Figure 7. FAN1 Possesses an Intrinsic Endonuclease and Exonuclease Activity
(A) Control HA empty vector (CONTROL), HA-FAN1, or HA-FAN1 nuclease mutant (MUT FAN1) (Q864A_D960A_E975A_K977A) complexes were precipitated
from 293 TREX cells and incubated with the indicated32P-end-labeled substrates prior to electrophoresis on denaturing gels. Asterisk (*) indicates the position
of label on the labeled strand.
(B) Bacterial His6-tagged FAN1 aa 373–1017 (wild-type or mutant Q864A_D960A_E975A_K977A) were incubated with32P-end-labeled substrates prior to
electrophoresis on denaturing gels.
(C) A model of FAN1 activity in the FA pathway. Right-hand side is based on Knipscheer et al. (2009), Raschle et al. (2008). See text for details.
(D) Summary of FAN1’s endonucleaolytic activity. The size of the arrows corresponds to the strength of the endonuclease.
The FAN1 Nuclease Functions in Crosslink Repair
44 Molecular Cell 39, 36–47, July 9, 2010 ª2010 Elsevier Inc.
hypothesize that FAN1 is recruited to sites of damage by the ID
complex using monoubiquitinated FANCD2 and FANCI as an
interaction platform for FAN1’s UBZ domain.
Since several mutant alleles of FAN1 examined, including the
truncation allele lacking the SAP domain and the TPR domain,
showed decreased GFP-FAN1 staining at the sites of DNA
the ubiquitinated ID complex and the UBZ domain, FAN1 is
stabilized in damage foci by interactions with the DNA or other
FAN1 was also identified by our group as a phosphoprotein in
a proteomic analysis of DDR (C. Zhou and S.J.E., unpublished
data). The identified phosphorylated SQ site, S210, in the human
protein is evolutionarily conserved in mouse, chicken, and fish
and is the only SQ/TQ site in FAN1 with such a high extent of
conservation. Phosphorylation plays a pivotal role in activating
FA pathway, and it is likely that the activity of enzymes partici-
pating in repair will be tightly regulated. Therefore, it will be
importantto establish thefunctional consequences of abolishing
FAN1 Is a Nuclease in the Fanconi Anemia Pathway
Biochemical analysis of FAN1 isolated from human cells
revealed that FAN1 acts both as an endonuclease and as
an exonuclease. The major endonucleolytic activity of FAN1
appears to act opposite a nick and at branched structures with
a 30end at the branch point (Figure 7C). FAN1’s minor activities
are toward a 30FLAP and toward replication forks across a 50end
at the branch point. The 50to 30exonuclease activity is active on
most 50ends, although its strength varies depending on the
specific substrate, with those substrates processed efficiently
by the endonuclease activity being poorer substrates for the
exonuclease than those inefficiently processed by the endonu-
clease. The ultimate test of FAN1’s activity will come from exam-
ining its function on crosslinked substrates in the setting of DNA
replication, since this is where ATR is activated and where the
interaction with the monoubiquitinated ID complex positions
FAN1 at the sites of damage. However, the in vitro activities of
FAN1 seen so far fit well with the repair activities hypothesized
to be present at the crosslink and other lesions that necessitate
replication restart (Figure 7D). FAN1 may be involved in the
unhooking of the ICL in the setting of replication. The ID complex
is required for this process, and since it is also necessary for
FAN1 localization at the site of DNA damage, FAN1 is a good
candidate for this activity. The exonuclease activity of FAN1
might also be important for the processing of the DNA strands
to generate 30overhangs for the homologous recombinational
repair that is necessary to restart the replication fork after the
crosslink is removed or to gap repair unreplicated regions
when both forks reach the ICL. Lastly, the final steps of removal
of the fully unhooked crosslink might also rely on FAN1 activity.
This is among the first instances for which we have an under-
standing of how a nuclease important for crosslink resistance
localizes to sites of damage. Although other nucleases including
XPF-ERCC1, MUS81-EME1, and SLX1 have been implicated in
et al., 2009), we still do not understand how they are recruited
to the sites of DNA damage or the nature of their relationship
to FA proteins.
FAN1 Is a Candidate Tumor Suppressor
Based on FAN1’s function in ICL resistance, colocalization,
dependence on FANCI and FANCD2 function, and interaction
with FANCD2, FAN1 is a candidate FA gene. Since three of the
FA genes, FANCD1 (BRCA2), FANCN (PALB2), and FANCJ
(BRIP1),arealsomutated infamilial breastcancerpredisposition
syndrome (Rahman et al., 2007; Wooster et al., 1995), FAN1
should be sequenced in the appropriate cohort of patients who
display familiar predisposition to breast cancer but lack identi-
fied predisposing mutations.
The identification of FAN1 brings us closer to an under-
standing of the biochemical pathway involved in DNA crosslink
repair and sets the stage for more precise examination of the
repair process in reconstituted crosslink repair systems.
Cell Culture, Plasmids, Antibodies, RNAi, RT-qPCR
U2OS, DR-U2OS, HeLa, 293T, 293TREX, and HCT116 cells were grown in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v)
FBS (Invitrogen), 100 units of penicillin per ml, and 0.1 mg streptomycin per
ml. PD20 cells were grown as above but with 15% FBS. Plasmids were con-
structed using recombinational cloning via the Gateway system (Invitrogen).
KIAA1018 clone was obtained from Origene, and the wild-type or truncation
mutants were amplified and recombined into pDONR223 (Lamesch et al.,
2007). pDONR223 derivatives were recombined into appropriate recipient
vectors using LR clonase (Invitrogen). Mutagenesis was performed using
multisite mutagenesis kit (Agilent) (primers are listed in Table S4). Antibodies
against FAN1 were raised in rabbits against peptide CGQSDSAKREVKQKIS
were FANCD2 (Novus NB100-182), GFP (Roche 11814460001), HA (Covance
MMS-101R), MLH1 (Santa Cruz sc-582), MLH3 (Bethyl A301-849A and A301-
850A), PMS1 (Santa Cruz sc-615), and PMS2 (BD Pharmigen 556415). siRNA
transfections were performed using Lipofectamine RNAiMAX as suggested
by the manufacturer with the final siRNA concentration of 50 nM. siRNA
sequences are listed in Table S5. For RT-qPCR, Superscript III reverse tran-
scriptase followed by Platinum cybergreen super mix (Invitrogen) were used
according to the instructions. GAPDH or actin was used as control.
Whole-Genome shRNA Screen
The pool-based shRNA screen using HH barcode deconvolution was per-
formed as described previously (Schlabach et al., 2008).
Protein Purification and Mass Spectrometry
The 293 TREX cells expressing FAN1 were lysed and immunoprecipitated
using anti-HA antibodies (Sowa et al., 2009; Svendsen et al., 2009). Com-
plexes were either used for DNA cleavage assays, subjected to immunoblot-
ting, or eluted with HA peptide and trypsinized prior to mass spectrometry
(Sowa et al., 2009; Svendsen et al., 2009). Processing of samples for mass
spectrometry as well as analysis of proteomic data using CompPASS were
as described (Sowa et al., 2009). For immunoprecipitations with FANCD2,
chromatin fraction was prepared as described (Moldovan et al., 2009). For
DNA cleavage assays, the immune complexes were washed three times in
buffer containing 20 mM Tris HCl (pH 8.0), 5 mM MgCl2, 1 mM DTT. Bacterial
proteins (FAN1 373–1017) were expressed using pDEST17 (Invitrogen),
induced with L-arabinose, and purified on a Ni-NTA column. The proteins
were dialyzed against a buffer containing 50 mM Tris HCl (pH 7.5), 100 mM
NaCl, 0.01%NP40, 10% glycerol, 1 mM DTT, 0.5 mM EDTA and were stored
The FAN1 Nuclease Functions in Crosslink Repair
Molecular Cell 39, 36–47, July 9, 2010 ª2010 Elsevier Inc. 45
Multicolor Competition Assay
Experiments were done as described (Smogorzewska et al., 2007).
Laser-Induced Damage and Immunofluorescence
Microirradiation wasperformedas described previously (Bekker-Jensen et al.,
2006). Immunofluorescence experiments were done as described (Smogor-
zewska et al., 2007).
In Vitro Cleavage Assays
In vitro cleavage of DNA substrates was performed using the FAN1 immune
complexes or bacterially purified FAN1 in conjunction with previously
described DNA substrates (Ciccia et al., 2003; Ip et al., 2008; Rass and
West, 2006; Svendsen et al., 2009). DNA cleavage assays were performed
generated by annealing oligonucleotides and were purified by polyacrylamide
gel electrophoresis as described previously (Ciccia et al., 2003; Ip et al.,
2008; Rass and West, 2006; Svendsen et al., 2009). The sequences of
substrates are provided in Table S4. Radiolabeled substrates were incubated
with the indicated immune complexes or bacterially purified FAN1. DNA
cleavage assays were performed in 20 mM Tris HCl (pH 8.0), 5 mM MgCl2,
1 mM ATP, and 1 mM DTT. For bacterially purified FAN1, 20 ng of wild-type
protein or 40 ng of mutant protein was used with each substrate. After
30 min (for the bacterial protein) or 2 hr (for the immunoprecipitated proteins)
at 37?C, reaction mixtures were treated with 1% Proteinase K in SDS prior
to electrophoresis on either 12% polyacrylamide gels (native) or 16% poly-
acrylamide-urea gels (denaturing). Reaction products were visualized by
32P- or 30
32P-end-labeled DNA substrates. Substrates were
C. elegans Genetics
C. elegans strains were cultured at 20?C under standard conditions (Brenner,
1974). The N2 Bristol strain was used as the wild-type background. The
following mutationsand chromosomerearrangements wereused inthisstudy:
LGIII, him-18(tm2181) (Saito et al., 2009), qC1[dpy-19(e1259) glp-1(q339)
qIs26] (III); LGIV, fan-1(tm423), nT1[ unc-?(n754) let-? qIs50] (IV; V), nT1[qIs51]
The fan-1(tm423) mutant, obtained from the Japanese National Bioresource
Project, carries a 411 bp in-frame deletion encompassing parts of exons 5–8.
This deletion results in the loss of the predicted SAP motif.
DNA Interstrand Crosslink Sensitivity Assay in C. elegans
Young adult worms were treated with 0, 250, or 500 mM of MMC (Sigma) in M9
buffer containing E. coli OP50 with slow shaking in the dark for 19 hr. Treat-
ment with nitrogen mustard (mechlorethamine hydrochloride; Sigma) was
similar, but with doses of 0, 50, or 100 mM. Following treatment with MMC
or HN2, animals were plated to allow recovery for 3 hr. Twenty animals
were plated five per plate, and hatching was assessed for the time period
22–26 hr from the start of treatment. Each damage condition was replicated
at least three times in independent experiments. him-18/slx-4 mutants, shown
previously to be extremely sensitive to ICL-inducing agents (Saito et al., 2009),
were used as a control. Since untreated him-18(tm2181) mutants have
reduced hatching, embryonic viability after DNA damage treatment was
plotted as a percentage of the hatching after DNA damage normalized by
that in untreated animals (relative hatching) (Saito et al., 2009).
Supplemental Information includes five figures, four tables, Supplemental
Experimental Procedures, and Supplemental References and can be found
with this article at doi:10.1016/j.molcel.2010.06.023.
We thank Jen Svendsen and members of the Elledge lab for protocols and
discussion. Special thanks to Alberto Ciccia for insightful suggestions and
comments. This work is supported by grants from the National Institutes of
Health (NIH) and CMCR grant #1U19A1067751-01 to S.J.E., NIH grant
R01GM072551 and a Giovanni Armenise-Harvard Foundation award to
M.P.C., a NIH grant to J.W.H., and Project Z01 ES065089 to T.A.K., in the Divi-
sion of Intramural Research of the National Institute of Environmental Health
Sciences, NIH. A.S. was supported by T32CA09216 to the Pathology Depart-
ment at the Massachusetts General Hospital and by Burroughs Wellcome
Fund Career Award for Medical Scientists and is an Irma T. Hirschl scholar.
S.J.E. is an investigator of the Howard Hughes Medical Institute.
Received: April 13, 2010
Revised: June 7, 2010
Accepted: June 9, 2010
Published: July 8, 2010
Alter, B.P., Greene, M.H., Velazquez, I., and Rosenberg, P.S. (2003). Cancer in
Fanconi anemia. Blood 101, 2072.
Andreassen, P.R., D’Andrea, A.D., and Taniguchi, T. (2004). ATR couples
FANCD2 monoubiquitination to the DNA-damage response. Genes Dev. 18,
Arana, M.E.,Seki, M.,Wood, R.D.,Rogozin, I.B., and Kunkel, T.A.(2008). Low-
fidelity DNA synthesis by human DNA polymerase theta. Nucleic Acids Res.
Auerbach, A.D., and Wolman, S.R. (1976). Susceptibility of Fanconi’s anaemia
fibroblasts to chromosome damage by carcinogens. Nature 261, 494–496.
Bakkenist, C.J., and Kastan, M.B. (2004). Initiating cellular stress responses.
Cell 118, 9–17.
Bartek, J., Lukas, C., and Lukas, J. (2004). Checking on DNA damage in
S phase. Nat. Rev. Mol. Cell Biol. 5, 792–804.
Bekker-Jensen, S., Lukas, C., Kitagawa, R., Melander, F., Kastan, M.B.,
Bartek, J., and Lukas, J. (2006). Spatial organization of the Mamm. Genome
surveillance machinery in response to DNA strand breaks. J. Cell Biol. 173,
Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77,
Cannavo, E., Gerrits, B., Marra, G., Schlapbach, R., and Jiricny, J. (2007).
Characterization of the interactome of the human MutL homologues MLH1,
PMS1, and PMS2. J. Biol. Chem. 282, 2976–2986.
Ciccia, A., Constantinou, A., and West, S.C. (2003). Identification and charac-
terization of the human mus81-eme1 endonuclease. J. Biol. Chem. 278,
Ciccia, A., McDonald, N., and West, S.C. (2008). Structural and functional
relationships of the XPF/MUS81 family of proteins. Annu. Rev. Biochem. 77,
Cole, A.R., Lewis, L.P., and Walden, H. (2010). The structure of the catalytic
subunit FANCL of the Fanconi anemia core complex. Nat. Struct. Mol. Biol.
Cox, L.S., Clancy, D.J., Boubriak, I., and Saunders, R.D. (2007). Modeling
Werner Syndrome in Drosophila melanogaster: hyper-recombination in flies
lacking WRN-like exonuclease. Ann. N Y Acad. Sci. 1119, 274–288.
Fanconi, G. (1967). Familial constitutional panmyelocytopathy, Fanconi’s
anemia (F.A.). I. Clinical aspects. Semin. Hematol. 4, 233–240.
Fekairi, S., Scaglione, S., Chahwan, C., Taylor, E.R., Tissier, A., Coulon, S.,
Dong, M.Q., Ruse, C., Yates, J.R., 3rd, Russell, P., et al. (2009). Human
SLX4 is a Holliday junction resolvase subunit that binds multiple DNA repair/
recombination endonucleases. Cell 138, 78–89.
Harper, J.W., and Elledge, S.J. (2007). The DNA damage response: ten years
after. Mol. Cell 28, 739–745.
Ho, G.P., Margossian, S., Taniguchi, T., and D’Andrea, A.D. (2006). Phosphor-
ylation of FANCD2 on two novel sites is required for mitomycin C resistance.
Mol. Cell. Biol. 26, 7005–7015.
The FAN1 Nuclease Functions in Crosslink Repair
46 Molecular Cell 39, 36–47, July 9, 2010 ª2010 Elsevier Inc.
Ip, S.C., Rass, U., Blanco, M.G., Flynn, H.R., Skehel, J.M., and West, S.C.
(2008). Identification of Holliday junction resolvases from humans and yeast.
Nature 456, 357–361.
Ishiai, M., Kitao, H., Smogorzewska, A., Tomida, J., Kinomura, A., Uchida, E.,
Saberi, A., Kinoshita, E., Kinoshita-Kikuta, E., Koike, T., et al. (2008). FANCI
phosphorylationfunctions as amolecularswitchtoturnon theFanconi anemia
pathway. Nat. Struct. Mol. Biol. 15, 1138–1146.
Kadyrov, F.A., Dzantiev, L., Constantin, N., and Modrich, P. (2006). Endonu-
cleolytic function of MutLalpha in human mismatch repair. Cell 126, 297–308.
Kinch, L.N., Ginalski, K., Rychlewski, L., and Grishin, N.V. (2005). Identification
of novel restriction endonuclease-like fold families among hypothetical
proteins. Nucleic Acids Res. 33, 3598–3605.
Knipscheer, P., Raschle, M., Smogorzewska, A., Enoiu, M., Ho, T.V., Scharer,
O.D., Elledge, S.J., and Walter, J.C. (2009). The Fanconi anemia pathway
promotes replication-dependent DNA interstrand cross-link repair. Science
Lamesch, P., Li, N., Milstein, S., Fan, C., Hao, T., Szabo, G., Hu, Z., Venkate-
san, K., Bethel, G., Martin, P., et al. (2007). hORFeome v3.1: a resource of
human open reading frames representing over 10,000 human genes. Geno-
mics 89, 307–315.
Lehmann, A.R., Niimi, A., Ogi, T., Brown, S., Sabbioneda, S., Wing, J.F.,
Kannouche, P.L., and Green, C.M. (2007). Translesion synthesis: Y-family
polymerases and the polymerase switch. DNA Repair (Amst.) 6, 891–899.
Machida, Y.J., Machida, Y., Chen, Y., Gurtan, A.M., Kupfer, G.M., D’Andrea,
A.D., and Dutta, A. (2006). UBE2T is the E2 in the Fanconi anemia pathway
and undergoes negative autoregulation. Mol. Cell 23, 589–596.
Matsuoka, S., Ballif, B.A., Smogorzewska, A., McDonald, E.R., 3rd, Hurov,
K.E., Luo, J., Bakalarski, C.E., Zhao, Z., Solimini, N., Lerenthal, Y., et al.
(2007). ATM and ATR substrate analysis reveals extensive protein networks
responsive to DNA damage. Science 316, 1160–1166.
Meetei, A.R., Yan, Z., and Wang, W. (2004). FANCL replaces BRCA1 as the
likely ubiquitin ligase responsible for FANCD2 monoubiquitination. Cell Cycle
Moldovan, G.L., Madhavan, M.V., Mirchandani, K.D., McCaffrey, R.M.,
Vinciguerra, P., and D’Andrea, A.D. (2009). DNA polymerase POLN partici-
pates in crosslink repair and homologous recombination. Mol. Cell. Biol. 30,
Munoz, I.M., Hain, K., Declais, A.C., Gardiner, M., Toh, G.W., Sanchez-Pulido,
L., Heuckmann, J.M., Toth, R., Macartney, T., Eppink, B., et al. (2009). Coor-
dination of structure-specific nucleases by human SLX4/BTBD12 is required
for DNA repair. Mol. Cell 35, 116–127.
Muzzini, D.M., Plevani, P., Boulton, S.J., Cassata, G., and Marini, F. (2008).
Caenorhabditis elegans POLQ-1 and HEL-308 function in two distinct DNA
interstrand cross-link repair pathways. DNA Repair (Amst.) 7, 941–950.
Niedzwiedz, W., Mosedale, G., Johnson, M., Ong, C.Y., Pace, P., and Patel,
K.J. (2004). The Fanconi anaemia gene FANCC promotes homologous recom-
bination and error-prone DNA repair. Mol. Cell 15, 607–620.
Peng, M., Litman, R., Xie, J., Sharma, S., Brosh, R.M., Jr., and Cantor, S.B.
(2007). The FANCJ/MutLalpha interaction is required for correction of the
cross-link response in FA-J cells. EMBO J. 26, 3238–3249.
Perry, J.J., Yannone, S.M., Holden, L.G., Hitomi, C., Asaithamby, A., Han, S.,
Cooper, P.K., Chen, D.J., and Tainer, J.A. (2006). WRN exonuclease structure
and molecular mechanism imply an editing role in DNA end processing. Nat.
Struct. Mol. Biol. 13, 414–422.
Rahman, N., Seal, S., Thompson, D., Kelly, P., Renwick, A., Elliott, A., Reid, S.,
Spanova, K., Barfoot, R., Chagtai, T., et al. (2007). PALB2, which encodes
a BRCA2-interacting protein, is a breast cancer susceptibility gene. Nat.
Genet. 39, 165–167.
Raschle, M., Knipscheer, P., Enoiu, M., Angelov, T., Sun, J., Griffith, J.D.,
Ellenberger, T.E., Scharer, O.D., and Walter, J.C. (2008). Mechanism of
replication-coupled DNA interstrand crosslink repair. Cell 134, 969–980.
Rass, U., and West, S.C. (2006). Synthetic junctions as tools to identify and
characterize Holliday junction resolvases. Methods Enzymol. 408, 485–501.
Rogakou, E.P., Boon, C., Redon, C., and Bonner, W.M. (1999). Megabase
chromatin domains involved in DNA double-strand breaks in vivo. J. Cell
Biol. 146, 905–916.
Saito, T.T., Youds, J.L., Boulton, S.J., and Colaiacovo, M.P. (2009).
Caenorhabditis elegans HIM-18/SLX-4 interacts with SLX-1 and XPF-1 and
maintains genomicintegrityinthegermlineby processing recombination inter-
mediates. PLoS Genet. 5, e1000735. 10.1371/journal.pgen.1000735.
Schlabach, M.R., Luo, J., Solimini, N.L., Hu, G., Xu, Q., Li, M.Z., Zhao, Z.,
Smogorzewska, A., Sowa, M.E., Ang, X.L., et al. (2008). Cancer proliferation
gene discovery through functional genomics. Science 319, 620–624.
Schmid,W., and Fanconi,G. (1978). Fragility and spiralization anomalies of the
chromosomes in three cases, including fraternal twins, with Fanconi’s anemia,
type Estren-Dameshek. Cytogenet. Cell Genet. 20, 141–149.
Seki, M., Masutani, C., Yang, L.W., Schuffert, A., Iwai, S., Bahar, I., and
Wood, R.D. (2004). High-efficiency bypass of DNA damage by human DNA
polymerase Q. EMBO J. 23, 4484–4494.
Simpson, L.J., and Sale, J.E. (2003). Rev1 is essential for DNA damage
tolerance and non-templated immunoglobulin gene mutation in a vertebrate
cell line. EMBO J. 22, 1654–1664.
K.E., Luo, J., Ballif, B.A., Gygi, S.P., Hofmann, K., D’Andrea, A.D., et al. (2007).
Identification of the FANCI protein, a monoubiquitinated FANCD2 paralog
required for DNA repair. Cell 129, 289–301.
Sowa, M.E., Bennett, E.J., Gygi, S.P., and Harper, J.W. (2009). Defining the
human deubiquitinating enzyme interaction landscape. Cell 138, 389–403.
Stokes, M.P., Rush, J., Macneill, J., Ren, J.M., Sprott, K., Nardone, J., Yang,
V., Beausoleil, S.A., Gygi, S.P., Livingstone, M., et al. (2007). Profiling of UV-
induced ATM/ATR signaling pathways. Proc. Natl. Acad. Sci. USA 104,
Sun, W., Nandi, S., Osman, F., Ahn, J.S., Jakovleska, J., Lorenz, A., and
Whitby, M.C. (2008). The FANCM ortholog Fml1 promotes recombination at
stalled replication forks and limits crossing over during DNA double-strand
break repair. Mol. Cell 32, 118–128.
Svendsen, J.M., Smogorzewska, A., Sowa, M.E., O’Connell, B.C., Gygi, S.P.,
Elledge, S.J., and Harper, J.W. (2009). Mammalian BTBD12/SLX4 assembles
a Holliday junction resolvase and is required for DNA repair. Cell 138, 63–77.
Wooster, R., Bignell, G., Lancaster, J., Swift, S., Seal, S., Mangion, J., Collins,
N., Gregory, S., Gumbs, C., and Micklem, G.(1995). Identification of the breast
cancer susceptibility gene BRCA2. Nature 378, 789–792.
Yoshimura, M., Kohzaki, M., Nakamura, J., Asagoshi, K., Sonoda, E., Hou, E.,
Zhang, X.Y., Langenick, J., Traynor, D., Babu, M.M., Kay, R.R., and Patel, K.J.
(2009). Xpf and not the Fanconi anaemia proteins or Rev3 accounts for the
extreme resistance to cisplatin in Dictyostelium discoideum. PLoS Genet. 5,
Zietlow, L., Smith, L.A., Bessho, M., and Bessho, T. (2009). Evidence for the
involvement of human DNA polymerase N in the repair of DNA interstrand
cross-links. Biochemistry 48, 11817–11824.
Zou, L., and Elledge, S.J. (2003). Sensing DNA damage through ATRIP recog-
nition of RPA-ssDNA complexes. Science 300, 1542–1548.
The FAN1 Nuclease Functions in Crosslink Repair
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