FEBS Letters 585 (2011) 2853–2860

journal homepage: www.FEBSLetters .org

Review

Ubiquitylation and the Fanconi anemia pathway

Elizabeth Garner, Agata Smogorzewska ⇑Laboratory of Genome Maintenance, The Rockefeller University, New York, NY 10065, United States

a r t i c l e i n f o

Article history:Received 12 April 2011Revised 29 April 2011Accepted 29 April 2011Available online 19 May 2011

Edited by Ashok Venkitaraman andWilhelm Just

Keywords:MonoubiquitylationFANCLFANCD2FANCISLX4FAN1

0014-5793/$36.00 � 2011 Federation of European Biodoi:10.1016/j.febslet.2011.04.078

⇑ Corresponding author.E-mail address: asmogorzewska@rockefeller.edu (

a b s t r a c t

The Fanconi anemia (FA) pathway maintains genome stability through co-ordination of DNA repairof interstrand crosslinks (ICLs). Disruption of the FA pathway yields hypersensitivity to interstrandcrosslinking agents, bone marrow failure and cancer predisposition. Early steps in DNA damagedependent activation of the pathway are governed by monoubiquitylation of FANCD2 and FANCIby the intrinsic FA E3 ubiquitin ligase, FANCL. Downstream FA pathway components and associatedfactors such as FAN1 and SLX4 exhibit ubiquitin-binding motifs that are important for their DNArepair function, underscoring the importance of ubiquitylation in FA pathway mediated repair.Importantly, ubiquitylation provides the foundations for cross-talk between repair pathways, whichin concert with the FA pathway, resolve interstrand crosslink damage and maintain genomicstability.� 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction

First described as a signal for protein degradation [1] modifica-tion by ubiquitin functions extensively as a regulatory signal tocontrol diverse biological pathways. As seen by the reviews in thisissue, DNA damage is a fertile ground for identification of ubiquitinfunction. Here we concentrate on Fanconi anemia (FA), a rare butvery instructive disease for ubiquitin signaling. FA patients showdefects in DNA interstrand crosslink repair and have biallelic muta-tions in one of fourteen genes that code among others, for a ubiq-uitin ligase (FANCL), proteins modified by monoubiquitylation(FANCD2 and FANCI), and a protein with ubiquitin-binding do-mains (FANCP/SLX4). The functional implications of ubiquitylationand de-ubiquitylation within the FA pathway and the regulation ofinterstrand crosslink repair will be the focus of this review.

2. The Fanconi anemia pathway

Fanconi anemia (FA) is a pathologically diverse, recessivelyinherited disease that typically culminates in bone marrow failurein affected individuals. Patients also display profound genomeinstability that correlates with cancer predisposition and may havedevelopmental defects [2]. On a molecular level, the FA pathwayrepresents a facet of the cellular DNA repair strategy employed

chemical Societies. Published by E

A. Smogorzewska).

to resolve DNA damage, particularly interstrand crosslinks (ICLs),which covalently link the Watson and Crick strands of the DNA.ICLs particularly affect processes that inherently require DNAunwinding and strand separation such as DNA replication andtranscription [3] (Fig. 1A). Consequently, ICLs are particularly del-eterious if unresolved. Besides being activated by exogenous dam-age inflicted by DNA crosslinking agents such as diepoxybutane(DEB), mitomycin C (MMC) or cisplatin, FA proteins are activatedduring normal S phase suggesting that they might participate in re-pair of replication errors [4,5].

Identification of mutated genes in FA patients has paved theway for the identification of a diverse set of proteins necessaryfor crosslink repair. A ‘genuine FANC protein’ must be rooted inthe human disorder. This is usually a consequence of mutationleading to loss of gene function. Currently biallelic mutations inany one of the 14 genes is responsible for FA with the 15th gene(RAD51C) being mutated in an FA-like syndrome [6] (Fig. 1). Theknown patients with RAD51C mutations have not yet presentedsigns of bone marrow failure and as such RAD51C has not yet beenassigned an official FANC name. A FANCO designation has been setaside if more evidence is gathered to firmly establish RAD51C as anFA gene. Eight proteins (FANCA, FANCB, FANCC, FANCE, FANCF,FANCG, FANCL, FANCM) form a complex commonly referred to asthe core complex which monoubiquitylates both components ofthe ID complex, FANCD2 and FANCI [4,7–9] (Fig. 1B). AlthoughFANCM has been first reported as an essential component of thecore complex [10], it is now clear that FANCM is important forresistance to interstrand crosslinks but not at the step of

lsevier B.V. All rights reserved.

C Activation of the nucleases; UBZ domain containing effectors

D Translesion step in ICL repair

Symbol KeyUb monoubiquitylation

UBZ domain

interstrand crosslink

B Activation of the ID complex

phosphorylationP

E HR proteins in FA

?SLX4

FAN1

I D2Ub

P

P

SLX4

?

SNM1A

PCNA?

SLX1XPF MUS81Ub

PALB2

FANCJ

Homologous recombination (HR)

RAD51CBRCA2

A Stalling of replication by ICL

Ub

MMMM

GCGGACCFFFF

E

AAB

UUbb

FEEEL

UBE2TUb

Ub

ATR

P

P

P P

P

FAAP24

FAAP100

?

?I

I

I

D2

I D2P

P

UbUb

Ub

Ub

UbUb

UbRPA

RPA

ICL

ATR

RPA

ATRIP

PCNA

RPARPA

RPA

RPA

I D2P

P

UbUb

chromatin K63-Ub linked chainsUbUb

UbUb

Ub

TLS

Ub

Ub

Ub

RAD18

PCNA

?

core complex

?Ub

??

D2

D2

Fig. 1. Ubiquitylation in response to interstrand crosslinks, highlighting Fanconi anemia proteins and the associated factors necessary for repair. Particular attention is drawnto some of the unknown factors in interstrand crosslink repair. (A) Interstrand crosslinks prevent separation of the DNA helix. During replication this can lead to replicationfork collapse when forks encounter lesions from one or both directions. For a full model of DNA transactions, see [63,64]. This leads to RPA-ssDNA binding and activation ofthe ATR and the downstream DNA damage response. (B) Activation of the ID complex. ATR phosphorylates many FA proteins including FANCI. FANCI phosphorylation acts as amolecular switch in the FA pathway. The FA core complex is recruited to chromatin at sites of damage where it functions as an E3 ubiquitin ligase to monoubiquitylate the IDcomplex at the damage site. It is currently unknown if there are other substrates for ubiquitylation by the FA core complex. (C) UBZ domain containing effectors. Downstreameffectors FAN1, SLX4, and SNM1A harbor ubiquitin-binding motifs that are thought to target them to the site of damage through ubiquitin mediated interactions. FAN1 bindsthe monoubiquitylated FANCD2. SLX4 displays binding affinity for ubiquitin chains though the docking sites for SLX4 are not currently known. SLX4 could potentially interactwith ubiquitylated FANCD2 or PCNA. Interestingly all three proteins harbor nuclease activity or are associated with nucleases. Thus, control of ubiquitylation events regulatesthe localization of nucleases at the site of damage. One of the remaining questions is why there are so many nucleases at the repair sites. (D) Translesion synthesis step.Chromatin bound PCNA is monoubiquitylated by RAD18 in response to interstrand crosslinking damage and coordinates translesion synthesis steps during interstrandcrosslink repair. Additional, non-PCNA and non-RAD18-dependent events are likely to allow translesion synthesis to occur. Homologous recombination (HR) concludes therepair process. FA proteins BRCA2, PALB2, and the Fanconi anemia-like syndrome protein RAD51C are involved in this step. FANCJ is also important for HR but in the contextof FA, it clearly has other functions, which are not yet fully explored.

2854 E. Garner, A. Smogorzewska / FEBS Letters 585 (2011) 2853–2860

monoubiquitylation of the ID complex [11]. Furthermore, unlikethe other components of the core complex, FANCM has FA pathwayindependent function including ATR-mediated checkpoint signal-ing [12]. Monoubiquitylated ID complex interacts with FAN1 (Fan-coni anemia Associated Nuclease 1), which in vitro has both endo-and exo-nuclease activity [13–16]. There are no known FA patients

with mutations in FAN1. The downstream proteins in the pathwayinclude FANCD1/BRCA2 and the BRCA2 interacting partner,FANCN/PALB2 [17–19], both of which are essential componentsof homologous recombination repair, and a helicase FANCJ /BRIP1/BACH1 [20–23]. FANCJ interacts with BRCA1 but this inter-action is apparently dispensable in the context of FA pathway

E. Garner, A. Smogorzewska / FEBS Letters 585 (2011) 2853–2860 2855

[24]. Depletion of BRCA1 results in exquisite sensitivity of cells tocrosslink damage but no individuals with biallelic BRCA1 muta-tions have been found to date. The newest addition to the Fanconianemia protein family is FANCP/SLX4 [25–27]. SLX4 acts as ascaffold that interacts with multiple proteins including nucleasesXPF, MUS81 and SLX1 [28–30]. XPF and MUS81 have been previ-ously implicated in crosslink repair [31,32] and the SLX4-SLX1interaction is responsible for Holliday junction resolution activityin vitro. It is unclear at this point which activity of SLX4 is essentialfor crosslink repair. Besides the genuine FANC proteins discussedabove, numerous proteins have been demonstrated to be associ-ated with FA pathway components and as such need to be studiedin parallel. Technological advances including RNAi continue tofacilitate the identification of proteins whose absence yields agenome instability phenotype analogous to that of FA and the nextgeneration sequencing approaches should soon identify theremaining FANC genes in FA patients with thus far unidentifiedgenetic mutations.

Besides the major pathway of replication-dependent ICL repair,vertebrate cells also have a replication-independent repair path-way. This minor repair pathway is active during G0/G1 stages andis independent of replication and homologous recombination butdependent upon the nucleotide excision repair components XPCand XPA [33–37]. Many of the components of the FA pathwayincluding the core complex, FANCI and FANCD2 seem to be requiredfor this repair [38]. The key to understanding crosslink repair willbe careful molecular and biochemical dissection of function of theproteins involved in repair including crystallographic studies ofthe pathway’s components. It is clear that characterization of theubiquitin-driven events and nuclease regulation in the pathwaywill be key to understanding the Fanconi anemia pathway.

3. The E3 ubiquitin ligase activity of FANCL and the FA corecomplex

Ubiquitylation of FANCD2 and FANCI is absent in cells withmutations in the core complex components (except for FANCM).Prior to the identification of FANCL, the core complex was notshown to harbor proteins with any recognizable domains. At thattime, other known ubiquitin ligases such as BRCA1 were underscrutiny in efforts to identify the components required for thiscentral step in FA pathway regulation [7,39]. It was not until theidentification of FANCL that it was possible to attribute monoubiq-uitylation of FANCD2 as an intra-FA pathway step in the responseto ICLs [40].

FANCL is considered a key part of the FA core complex and rep-resents the E3 ligase necessary for ubiquitin conjugation to lysine561 of human FANCD2 and lysine 523 of FANCI. Structurally FANCLhas three distinct domains; an ELF, DRWD and RING domain [41].Functionally, the DRWD domain coordinates substrate bindingwhile the RING domain is required for E2 protein interactions.Yeast two hybrid experiments using full-length FANCL have iden-tified two E2 proteins capable of interacting with FANCL, UBE2Tand UBE2W [42]. The C-terminal RING domain is both necessaryand sufficient for FANCL binding of UBE2T [43]. The importanceof the RING domain is reflected in the evolutionary conservationof this domain in FANCL homologs [41]. UBE2T is considered tobe the most functionally important E2 enzyme required forFANCL-mediated ubiquitylation of FANCD2 in vivo and is requiredfor cellular resistance to ICLs [43]. In vitro data differ from in vivo-derived data as to the minimal requirements for FANCD2 monoub-iquitylation, since E1, UBE2T, and FANCL are shown to be sufficientfor FANCD2 monoubiquitylation in an in vitro context [42]. How-ever, this reaction was quite inefficient and ubiquitylationoccurred only on FANCD2, even when FANCI was also present in

the reaction. Interestingly, presence of FANCI conferred specificityto the reaction so that K561 of FANCD2 was now correctlymonoubiquitylated. This suggests that FANCI might direct the cor-rect lysine residue to the core complex or perhaps mask alternativesurface lysines. Reconstitution of the whole core complex might benecessary for robust in vitro ubiquitylation. In addition, since phos-phorylation plays an important role in activating the pathway,using pre-activated FANCI (see below) might be required.

FANCD2 and FANCI are the only known substrates of the corecomplex. There remains a possibility that there will be other sub-strates for this complicated E3 ligase (Fig. 1B and D). A hint of thatmight be seen in the result showing that monoubiquitylatedFANCD2 on the chromatin is not sufficient for crosslink resistancein DT40 cells that lack the FA core complex [44]. Since FANCImonoubiquitylation in DT40 cells is almost non-contributory (seenext section), it is likely that there will be other substrates ofFANCL.

4. Regulation of ID complex monoubiquitylation

Monoubiquitylated ID complex is at the heart of the Fanconianemia pathway. The monoubiquitylation of FANCD2 at lysine561 is essential for the downstream function of the FA pathwayin repair. Cells expressing K561R FANCD2 (which cannot bemonoubiquitylated) as the only FANCD2 allele are as sensitive tocrosslinking agents as null cells [7]. The contribution of monoubiq-uitylated FANCI to crosslink resistance seems to be less importantthan that of monoubiquitylated FANCD2 in human cells [4], and al-most non-contributing in DT40 cells [45]. Monoubiquitylation ofFANCD2 occurs following the localization of the core complex tosites of damage (Fig. 1B). Core complex localization requires thecombined activity of FANCM and its interacting factor FAAP24 thatfacilitates the recruitment of the core complex to regions of dam-age [46,47]. The recruitment of FANCM to sites of damage is en-hanced further by the association of FANCM with additionalFAAP proteins, MHF1 and MHF2 [48]. MHF1 and MHF2 containputative histone fold domains and form a dimeric complex withDNA binding affinity. MHF1 and 2 are found in complex withFANCM and FAAP24 in mammalian cells and MHF1 is critical forthe integrity of the complex. The MHF dimer complex augmentsthe association of FANCM with chromatin and enhances FANCMdependent processing of branched DNA. Destabilization of theMHF1/2 dimer complex with FANCM by silencing of MHF1 expres-sion leads to a decreased efficiency of FANCD2 monoubiquitylationin response to DNA damage and increased genome instability.

It is unclear how the ID complex localizes to chromatin before itis monoubiquitylated. It does posses intrinsic DNA-binding activity[49–51] with preference for branched structures, so it is possiblethat it gets to the sites of damage on its own via DNA binding. Oncemonoubiquitylated, it clearly resides at chromatin where it can befound in damage-induced foci [4,7]. It has been previously pro-posed that a chromatin receptor for the monoubiquitylated IDcomplex might be present at the chromatin [44,52,53], althoughsuch a receptor has not yet been identified. In the light of FAN1binding to the chromatin bound monoubiquitylated ID complex(see below), existence of such a receptor is less likely, althoughnot impossible. If such a receptor does exist, understanding ofthe re-modeling of the ID complex architecture from interactingwith a receptor via monoubiquitin to interacting with FAN1 andother effector proteins at chromatin will be very important.

Fanconi anemia proteins are regulated by DNA damage signal-ing involving ATM and ATR kinases. ATM is activated in responseto double-strand breaks and in a manner that involves DNA resec-tion while ATR signals replicative stress and is activated by bindingof ATR/ATRIP to the RPA coated ssDNA [54,55] (Fig. 1A). Multiple

2856 E. Garner, A. Smogorzewska / FEBS Letters 585 (2011) 2853–2860

components of the FA pathway are phosphorylated includingFANCA, FANCG, FANCM, FANCD1 and both constituents of the IDcomplex [4,5,10,45,56–62]. The importance of many of the phos-phorylation events is not entirely clear but some of them areimportant for crosslink resistance. In particular, phosphorylationof FANCI on SQ/TQ sites close to the monoubiquitylation site ofFANCI is necessary for the monoubiquitylation and localization ofboth FANCI and FANCD2 to damage-induced foci [45]. FANCI carry-ing phosphomimic mutations of these sites showed constitutiveactivation of the pathway even in the absence of damage. Basedon these findings, it has been proposed that FANCI phosphorylationfunctions as a molecular switch to turn on the FA pathway.Although it is unclear how this switch my work, one possibilityis that phosphorylation of FANCI drives an interaction of the IDcomplex with a protein that interacts with the core complex orwith a protein in the core complex itself. It will be important tounderstand how phosphorylation of FANCI triggers monoubiquity-lation of FANCD2.

5. Ubiquitin mediated interaction of the ID complex and FAN1

The chromatin bound monoubiquitylated ID complex facilitatessubsequent steps of ICL repair in ways that are still being eluci-dated. The ID complex and monoubiquitylation of FANCD2 is re-quired for ICL unhooking and translesion DNA synthesis tofacilitate bypass of the lesion [63,64]. It is likely that monoubiqui-tylation of the ID complex forms a platform for at least some ofthese events and that the protein components involved in makingthe incisions and translesion synthesis may harbor ubiquitin-bind-ing motifs themselves (Fig. 1C). The newly identified nuclease re-quired for ICL repair, FAN1, is one such protein. FAN1 has a UBZdomain at its N terminus. UBZ (ubiquitin binding zinc finger) do-main is one of many ubiquitin recognition motifs (reviewed in[65]). Mutation of the UBZ domain precluded FAN1 from localizingto damage made by laser irradiation or by crosslinking damage[13–16]. The ID complex was also required for foci formation byFAN1. Moreover, cells with endogenous FANCD2 replaced withthe K561R FANCD2 mutant lost FAN1 localization. Since FANCD2and FAN1 were able to interact in immunoprecipitation experi-ments, monoubiquitylated FANCD2 is considered a platform forFAN1 binding through the UBZ domain. Other regions of FAN1 pro-tein might also be required for interaction with FANCD2 analogousto REV1 interacting not only with the monoubiquitin on PCNA butalso with PCNA itself through the BRCT motifs [66]. Since FAN1 dis-plays both endonuclease and exonuclease activity this ubiquitin-mediated recruitment focuses the nuclease activity of FAN1 tothe site of the ICL. Thus far, in vitro experiments have been per-formed on non-crosslinked substrates so it is unclear at which stepof repair FAN1 acts. It may work in concert with the MUS81-EME1and XPF-ERCC1 nuclease that have been implicated in crosslinkunhooking [31,32]. It might also act at the later stages of excisionof the unhooked crosslink or even later during preparation forhomologous recombination events. The concept of ubiquitin-mediated recruitment via the ID complex will undoubtedly be animportant aspect of understanding the downstream processes ofFA pathway-mediated DNA repair. It will be interesting to uncoverhow the ID complex co-ordinates the downstream effectors of thepathway, some of which may yet to be identified.

6. Deubiquitylation and FA pathway regulation

Ubiquitylation of the ID complex is an activating event neces-sary for the repair of ICLs. Equally important is the event of turningthat signal off once the repair is completed. Failure to de-ubiquity-late the ID complex results, somewhat counter intuitively, in

sensitivity to crosslinking agents [67,68]. This implies that the cy-cling of the ID complex between ubiquitylated and non-ubiquity-lated forms is essential for the activity of the pathway. USP1 isthe deubiquitylating enzyme that interacts with FANCD2 in chro-matin and removes its monoubiquitin [69] (Fig. 2). USP1 is regu-lated on many levels. USP1 levels are tightly regulated at atranscriptional level to peak during S-phase, presumably whenits activity is most required. It is also regulated by polyubiquityla-tion and proteasome-dependent degradation at the end of S phaseonce it is no longer needed. USP1 levels during S phase howeverare fairly constant [69]. This implies that there will be as of yetunidentified regulatory steps in the pathway, possibly at the chro-matin level, which will allow deubiquitylation of FANCD2 andresetting of the pathway. Interestingly, upon damage USP1transcription is shut off with a subsequent rapid decline of USP1protein [70]. It is not known what happens to the chromatin-associated USP1 pool after DNA damage and it is this fraction ofUSP1 that is of particular interest. It is unlikely that it disappears;rather its activity is probably regulated in some way to allow forde-ubiquitylation of the ID complex only after the damage is re-paired. USP1 activity is greatly enhanced by its partner, a WD40domain containing protein, USP1 Associated Factor 1 (UAF1) [70].Future work should concentrate on regulation of USP1 since ourappreciation of the mechanisms of deactivation, lags behind ourunderstanding of the activation of the Fanconi anemia pathway.Interestingly, USP1 is also a de-ubiquitylating enzyme for PCNA(Fig. 2), suggesting that regulation of de-ubiquitylation of PCNAand the ID complex might be co-regulated. Clearly there will alsobe separate regulatory networks, for example ELG1 interacts withUSP1-UAF1 and affects PCNA deubiquitylation, but not FANCD2ubiquitylation status [71].

7. The Ubiquitin binding motifs of FANCP/SLX4

FANCP/SLX4 represents the newest member of the FA pathwayto date and constitutes the 14th Fanconi anemia complementationgroup FA-P [26,27]. The SLX4 protein is a large scaffold with multi-ple recognizable domains. At the N terminus, it has two UBZ do-mains followed by an MLR (MUS312/MEI9 interaction likeregion), BTB domain, SAP domain, and finally a helix-turn-helixmotif at the C terminus [28–30]. Human SLX4 interacts with multi-ple proteins including XPF-ERCC1, MUS81-EME1, SLX1, MSH2,MSH3, TRF2, RAP1, and PLK1 among others. It appears that mostif not all of the cellular pool of MUS81 and SLX1 interact withSLX4 while only a small portion of the cellular XPF pool interactswith SLX4 [30]. In vitro SLX4 is able to enhance the activity ofthe associated nucleases. Cells derived from FA-P patients displaynormal monoubiquitylation of FANCD2 following DNA damagebut genomic instability and sensitivity to inter-strand crosslinkingagents characteristic of an FA phenotype. The early steps of ICL re-pair that requires the core components of the FA pathway are unaf-fected by a loss of SLX4 suggesting that it is a downstream effectorof the pathway [26,27]. Obvious candidates for the essential com-ponents of SLX4 in the setting of FA are XPF and MUS81 but the ex-act role of SLX4 in FA pathway still needs to be determined. Patientmutations suggest an important role for the UBZ domains. Two ofthe four families with SLX4 mutations have a very interesting in-frame internal deletion of amino acids 317–387, correspondingto half of the first UBZ domain and the whole second part of UBZdomain. Although the level of expression of this allele was lowerthan wild type, it was able to interact with XPF and MUS81.

The UBZ domains of SLX4 interact efficiently with K63-linkedubiquitin chains in vitro in a manner that requires intact UBZdomains [26]. Mutation of the two cysteines in both of the UBZdomains resulted in abrogation of ubiquitin chain binding. These

I D2

PCNA

ID complex

USP1

USP1 proteasome

I D2P

P

UbUb

I D2UbUb

?

Ub

UAF1

Ub

Ub

ELG1UAF1

P

P

Ub

Ub

UbUb

Ub

Ub

UbUb

Ub

Ub

D2

Fig. 2. Deactivation of the Fanconi anemia pathway by deubiquitylation. The signaling that takes place in the activation of DNA repair must be attenuated when DNA repairhas concluded. The ID complex is deubiquitylated by USP1 whose activity is enhanced by interaction with UAF1. USP1 has a parallel role in the deubiquitylation of PCNA.USP1/UAF1 substrate affinity is regulated by auxiliary factors such as ELG1, which is required for the deubiquitylation of PCNA specifically. The DUBs are themselves subjectto regulation on both a transcriptional and post-transcriptional level. Importantly, USP1 is polyubiquitylated as DNA replication concludes. Polyubiquitylated USP1 is targetedfor proteasomal degradation.

E. Garner, A. Smogorzewska / FEBS Letters 585 (2011) 2853–2860 2857

data led to a proposal that FANCP might localize to sites of damagevia ubiquitin mediated interactions. The substrates of such interac-tions are unknown at this time (Fig. 1C). It is still possible that SLX4interacts with monoubiquitylated ID complex as suggested for thechicken FANCP in DT40 cells [72]. However, the presence of tan-dem UBZ domains would be consistent with SLX4 binding to a tar-get that is endowed with ubiquitin chains. Identity of ubiquitylatedsubstrates is a hot subject and identification of these in the contextof the FA pathway will be truly exciting. It is appealing to thinkthat different nucleases will arrive at the sites of a crosslink via dif-ferent, albeit ubiquitin-dependent interactions. FAN1 would arrivevia monoubiquitylated ID complex, SLX4/XPF/MUS81 complex, viaa different, unknown interactor. This would give an opportunity toregulate these nucleases separately and make sure that the inci-sions are made only at the correct sites and at the right time.

8. Translesion polymerases in the ICL repair

Translesion synthesis (TLS) polymerases are specialized en-zymes that are able to insert a nucleotide across a damaged sitein DNA. Multiple translesion polymerases have been implicatedin the FA pathway. Knockout of REV1, and components of POLf(REV3, REV7) in chicken DT40 cells leads to exquisite sensitivityto crosslinking agents [73–77]. Moreover REV1 and REV3 are epi-static with FANCC for crosslink sensitivity suggesting that theyare in the same pathway [73,75]. The current model for ICL repairbased on the repair of a crosslinked plasmid in Xenopus egg extractproposes that translesion polymerases are essential for the bypassof the crosslink that was freed, commonly referred to as ‘‘un-hooked’’, by endonucleolytic incisions [63,64]. It has been pro-posed that REV1 inserts one nucleotide against the unhookedcrosslink, which is further extended by the REV3 catalytic subunitof POLf [63,64]. Depletion of FANCD2 affected the translesion stepas well as the ‘‘unhooking’’ step. It is not yet known whether bothof these events are direct consequences of FANCD2 depletion. It ispossible that both of these activities are co-regulated by the IDcomplex or that the ID complex regulates only one of them andthat the second depends upon the initiation (or even completion)of the first. Interestingly, REV1 focus formation due to ICLs isdependent on the functional FA core complex but does not seemto involve FANCD2 or RAD18-dependent PCNA monoubiquityla-tion [78] (Fig. 1D). How the translesion polymerases get to the

ICL is an exciting topic. Many others polymerases might also be in-volved in ICL repair including POLg, POLj, POLi, POLh, and POLm.The involvement of these polymerase was recently extensively re-viewed in [79]. Of particular interest to the FA pathway is POLm(POLN), which is an A type DNA polymerase. Depletion of POLNin human cells results in DNA crosslink sensitivity with defectsin homologous recombination [80]. POLN was found to interactwith monoubiquitylated FANCD2 on chromatin after mitomycinC treatment, although it is unclear if monoubiquitylation ofFANCD2 is required for the interaction, Furthermore, the POLNdependent requirements for this interaction are not yet known.

9. RAD18 and FA pathway activation

Although RAD18 does not seem to be involved in REV1 localiza-tion to ICLs, lack of RAD18 has been reported by several laborato-ries to be involved in activation of the FA pathway since depletionor lack of RAD18 led to slower kinetics of FANCD2 and FANCImonoubiquitylation [81–84]. The mechanism of how RAD18 mightaffect ID complex monoubiquitylation is not clear at this time sincethe published data from different groups do not agree with eachother. RAD18 is an E3 ligase and together with RAD6 it has beenshown to catalyze monoubiquitylation of PCNA on lysine 164and this modification promotes error-prone translesion bypass[85,86]. According to two papers [82,83], PCNA ubiquitination onlysine 164 is required for activation of the FA pathway upon cis-platin or benzo[a]pyrenedihydrodiol epoxide (BPDE) damage andthat ubiquitylated PCNA recruits the core complex which in turnubiquitylates the ID complex. Findings of Williams et al. [81] con-tradict those conclusions. They show that RAD18 co-immunopre-cipitates with FANCD2 and that the RING domain of RAD18 isnecessary for this interaction. However, they show that FANCD2ubiquitylation is normal after mitomycin C treatment of cells con-taining an ubiquitylation-resistant form of PCNA, and that thechromatin loading of FA core complex proteins appears normalin RAD18-knockout cells. The authors suggest that RAD18 couldbe responsible for modifying the core complex itself or otherunidentified DNA repair proteins upstream of the ID complex.These discrepancies need to be addressed by the labs that reportedthese findings. There were some obvious differences in cell linesand in damaging agents used between all the reports, includingthe use of BPDE and cisplatin in reports describing PCNA

2858 E. Garner, A. Smogorzewska / FEBS Letters 585 (2011) 2853–2860

monoubiquitylation-dependent mechanism, and mitomycin C inthe report that did not. Regardless of the mechanism, RAD18 is cer-tainly a factor that regulates the FA pathway.

10. Ubiquitin binding motif of SNM1A

Saccharomyces cerevisiae SNM1 (sensitivity to nitrogen mus-tard), also known as PSO2 (sensitivity to psoralen), has been iden-tified in screens for selective sensitivity to ICLs [87,88]. The 50 to 30

exonuclease activity of Pso2p is thought to be important for pro-cessing of incisions made by nucleotide excision repair in yeast.In vertebrates, there are three orthologs of SNM1; SNM1A,SNM1B/Apollo, and SNM1C/Artemis (for review see [89]) SNM1Aand SNM1B both seem to participate in ICL repair, and the doubleknockout in DT40 cells is more sensitive to cisplatin than either ofthe single knockouts, suggesting that they function in separatepathways or that they are redundant for cisplatin resistance [90].SNM1A is also not epistatic in crosslink resistance with FANCC,RAD18 or XRCC3 (homologous recombination pathway) in DT40cells. SNMA1A, but not SNM1B was able to complement crosslinksensitivity of the yeast Pso2 mutant [91] suggesting that SNM1 isa functional ortholog of Pso2. Recent work led to the identificationof a PIP box motif in vertebrate SNM1 required for binding to PCNAirrespective of DNA damage [71]. A newly identified UBZ domain inthe N-terminus of SNM1A, which is conserved even in S. cerevisiaePso2, was required for damage induced foci formation by a GFP-tagged SNM1 in human cells (Fig. 1C). SNM1A foci formation afterDNA damage was dependent on RAD18 [71] though the functionalconsequences of these interactions are not yet clear. It remains tobe determined whether SNM1B functions in crosslink repair in hu-man cells.

11. Evolutionary conservation of proteins in the Fanconianemia pathway

The most conserved of the FA proteins are FANCM, SLX4 andBRCA2, orthologs of which are present from yeast to human [28–30,92–94]. Such an exquisite conservation; however, may reflectfunctions of these proteins outside of ICL repair. Vertebrates, flies,frogs, worms, plants and even a slime mold D. discoideum, have rec-ognizable orthologs of some of the components of the core com-plex, importantly FANCL [41,95–98]. Conservation of thecomponents of the core complex, other than FANCL, fluctuatethroughout evolution with some components lost or gained in dif-ferent species (reviewed in [98]). All organisms that have FANCLalso have FANCD2 and FANCI, which have been shown to bemonoubiquitylated in some [64,95,97,98]. S. cerevisiae and Saccha-romyces pombe have no core complex nor ID complex components.However, S. pombe does have a FAN1 ortholog whose function isunknown. S. pombe Fan1 has a well-conserved nuclease domainand a SAP domain but no recognizable ubiquitin binding domain[16]. SAP (SAF-A/B, Acinus and PIAS) domains have been postulatedto be modules involved in targeting proteins to chromatin [99]. Assuch, it is possible that the SAP domain in the S. pombe FAN1 mightbe involved in the localization of FAN1 to sites of damage.

Both S. cerevisiae and S. pombe have Slx4 orthologs. Indeed, Slx4and Slx1 were first identified in yeast in a screen for factors re-quired for the viability of S. cerevisiae lacking RecQ helicase Sgs1[100]. There are clear similarities between the yeast and humanorthologs of Slx4 in that they both interact with XPF-ERCC1 andSLX1 orthologs [101]. SLX4 from yeast to human also containsSAP domains and the C-terminal Helix-turn-helix domains. TheSlx4 orthologs in yeast do not have the UBZ domains, BTB domainsnor do they interact with MUS81. These differences suggest thatthere has been an evolutionary expansion of SLX4 function, but

also adoption of MUS81 into the SLX4 complex, offering an oppor-tunity for coordination of multiple nuclease activities through theSLX4 protein.

12. Proteolysis and DNA damage signaling

Ubiquitylation clearly plays pan-cellular roles that are critical forsignaling and, as discussed here, crucial for FA pathway regulationand DNA repair. Monoubiquitylation and polyubiquitin chainsmay be of primary importance in signaling events, as platforms forcomplex interactions. However, the importance of proteolytic deg-radation in response to polyubiquitylation and proteasome target-ing cannot be underestimated in terms of its importance for DNArepair. Many of the regulatory factors discussed here may be tar-geted for proteasomal degradation as is the case for the FA regula-tory deubiquitylating enzyme USP1 (discussed above, Fig. 2) andFANCM, which is degraded during M phase, releasing the core com-plex from chromatin [102]. It has also been shown that proteasomefunction is in fact required for monoubiquitylation and foci forma-tion of FANCD2 [103]. The proteasome has also been linked to vari-ous aspects of DSB repair and homologous recombination [104,105].Understanding how the proteasome balances the accumulation ofrepair factors with their degradation will involve a more thoroughunderstanding of polyubiquitylation targets. In turn, how the pro-teasome is localized at sites of damage and its regulation is animportant aspect of DNA damage signaling and repair.

13. Perspectives

There is a lot to be learned about the role of ubiquitylation inthe Fanconi anemia pathway. Many of the remaining issues werenoted in their respective sections above. Here we highlight a few,including the activation of ubiquitylation of the ID complex, roleof ubiquitin in regulation of nucleases, and a potential role of other,ubiquitin-like modifiers in the FA pathway.

13.1. Turning on ubiquitylation

Phosphorylation of FANCI turns on the FA pathway in responseto damage. Understanding how this takes place is an importantconcept in how ubiquitylation events occur in response to chal-lenges such as DNA damage. Are ubiquitin ligase complexes consti-tutively active such that the recruitment of substrates to the ligasecomplex is the regulated step? Are ubiquitin ligase complexesthemselves activated by post-translational modification? In thecase of damage-induced ubiquitylation of the ID complex it maybe that phosphorylation of both the core complex and the ID com-plex regulate the ‘activation’ of ID complex ubiquitylation. In thisrespect phospho-binding motifs may be the chromatin ‘receptors’that localize core complex with substrate leading to ubiquitylation.

13.2. Targets of FANCL monoubiquitylation

Thus far only FANCD2 and FANCI have been identified as targetsof FANCL monoubiquitylation. It is compelling to think that theremay be alternative targets for FANCL monoubiquitylation. Thishypothesis is supported by the observation that the core complexis not dispensable for crosslink repair even when FANCD2monoubiquitylation has been satisfied [44]. The missing monoub-iquitylated factor in that study could have been FANCI, however,FANCI monoubiquitylation appears not to be very important inthe DT40 cells. In light of these findings, there still might be somemissing substrates of the core complex. Experimentally, findingalternative targets of FANCL monoubiquitylation may not be sim-ple. Such a modification is not represented in abundance anddissecting monoubiquitylated targets from polyubiquitylated

E. Garner, A. Smogorzewska / FEBS Letters 585 (2011) 2853–2860 2859

targets may prove challenging. Additionally the interdependenceof posttranslational modification (like the phosphorylation switchof FANCI for the ID complex ubiquitylation) may complicate effortstowards examining this hypothesis.

13.3. Co-ordination of nucleases

Interstrand crosslink repair involves number of coordinatednucleolytic events. There is a plethora of nucleases implicated asbeing important in the repair of crosslink lesions. To advance ourunderstanding of how these events are regulated requires dissec-tion of the step-wise order of events that guides repair throughunhooking events, translesion synthesis and recombination events.Since there is likely to be redundancy between the functions ofthese nuclease complexes, dissecting these events in vivo maynot allow us to gain an accurate picture of how the nucleolytic re-pair events are regulated. In this instance in vitro recapitulation ofthese reactions may be the only way to know the absolute require-ments for, and order of, events at an interstrand crosslink lesion.Implicit in gaining an understanding of nuclease regulation is theidentification of how nucleases are organized at sites of repair.The identification of FANCP/SLX4 shed some light on the recruit-ment of nucleases to the sites of damage. The docking sites forthese recruitment factors will provide important insight intodynamics of these processes and understanding of the ubiquitinbinding motifs in these proteins will be essential.

13.4. Ubiquitin-like modifications

Ubiquitin modification modulates molecular interactions andlocalization of substrates. Other small molecule modifications havebeen identified that like ubiquitin are conjugated onto target sub-strates and can play a role in the regulation of cellular processessuch as cellular signal transduction, cell cycle regulation andDNA repair. SUMO (small ubiquitin-like modifier) modificationdynamics are similar to that of ubiquitylation in that they involvean enzymatic cascade to conjugate SUMO to target substrates. Thisequilibrium is maintained by desumoylating enzymes. In yeastPCNA is a platform for both ubiquitination and SUMOylation[106]. SUMOylation of PCNA takes place during S-phase creatinga binding site for down-stream effectors and can suppress homol-ogous recombination. SUMOylation motifs are well characterisedand various protein components of the FA pathway harbor putativeSUMOylation sites. It will be interesting to see if and howSUMOylation and indeed other ubiquitin-like modifications maybe involved in FA pathway function.

Acknowledgements

We thank Arleen Auerbach, Frank Lach, and Yonghwan Kim fortheir comments. Work in our lab is supported by the AndersonCancer Center at the Rockefeller University, Burroughs WellcomeFund Career Award for Medical Scientists, Starr Center Consortiumgrant, and a grant UL1RR024143 from the National Center for Re-search Resources (NCRR). A.S. is a Rita Allen Foundation and anIrma T. Hirschl scholar.

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