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FEBS Letters 585 (2011) 2853–2860

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Review Ubiquitylation and the pathway ⇑ Elizabeth Garner, Agata Smogorzewska

Laboratory of Genome Maintenance, The Rockefeller University, New York, NY 10065, United States

article info abstract

Article history: The Fanconi anemia (FA) pathway maintains genome stability through co-ordination of DNA repair Received 12 April 2011 of interstrand crosslinks (ICLs). Disruption of the FA pathway yields hypersensitivity to interstrand Revised 29 April 2011 crosslinking agents, bone marrow failure and cancer predisposition. Early steps in DNA damage Accepted 29 April 2011 dependent activation of the pathway are governed by monoubiquitylation of FANCD2 and FANCI Available online 19 May 2011 by the intrinsic FA E3 ubiquitin ligase, FANCL. Downstream FA pathway components and associated Edited by Ashok Venkitaraman and factors such as FAN1 and SLX4 exhibit ubiquitin-binding motifs that are important for their DNA Wilhelm Just repair function, underscoring the importance of ubiquitylation in FA pathway mediated repair. Importantly, ubiquitylation provides the foundations for cross-talk between repair pathways, which Keywords: in concert with the FA pathway, resolve interstrand crosslink damage and maintain genomic Monoubiquitylation stability. FANCL Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. FANCD2 FANCI SLX4 FAN1

1. Introduction to resolve DNA damage, particularly interstrand crosslinks (ICLs), which covalently link the Watson and Crick strands of the DNA. First described as a signal for protein degradation [1] modifica- ICLs particularly affect processes that inherently require DNA tion by ubiquitin functions extensively as a regulatory signal to unwinding and strand separation such as DNA replication and control diverse biological pathways. As seen by the reviews in this transcription [3] (Fig. 1A). Consequently, ICLs are particularly del- issue, DNA damage is a fertile ground for identification of ubiquitin eterious if unresolved. Besides being activated by exogenous dam- function. Here we concentrate on Fanconi anemia (FA), a rare but age inflicted by DNA crosslinking agents such as diepoxybutane very instructive disease for ubiquitin signaling. FA patients show (DEB), mitomycin C (MMC) or cisplatin, FA proteins are activated defects in DNA interstrand crosslink repair and have biallelic muta- during normal S phase suggesting that they might participate in re- tions in one of fourteen genes that code among others, for a ubiq- pair of replication errors [4,5]. uitin ligase (FANCL), proteins modified by monoubiquitylation Identification of mutated genes in FA patients has paved the (FANCD2 and FANCI), and a protein with ubiquitin-binding do- way for the identification of a diverse set of proteins necessary mains (FANCP/SLX4). The functional implications of ubiquitylation for crosslink repair. A ‘genuine FANC protein’ must be rooted in and de-ubiquitylation within the FA pathway and the regulation of the human disorder. This is usually a consequence of mutation interstrand crosslink repair will be the focus of this review. leading to loss of gene function. Currently biallelic mutations in any one of the 14 genes is responsible for FA with the 15th gene 2. The Fanconi anemia pathway (RAD51C) being mutated in an FA-like syndrome [6] (Fig. 1). The known patients with RAD51C mutations have not yet presented Fanconi anemia (FA) is a pathologically diverse, recessively signs of bone marrow failure and as such RAD51C has not yet been inherited disease that typically culminates in bone marrow failure assigned an official FANC name. A FANCO designation has been set in affected individuals. Patients also display profound genome aside if more evidence is gathered to firmly establish RAD51C as an instability that correlates with cancer predisposition and may have FA gene. Eight proteins (FANCA, FANCB, FANCC, FANCE, FANCF, developmental defects [2]. On a molecular level, the FA pathway FANCG, FANCL, FANCM) form a complex commonly referred to as represents a facet of the cellular DNA repair strategy employed the core complex which monoubiquitylates both components of the ID complex, FANCD2 and FANCI [4,7–9] (Fig. 1B). Although FANCM has been first reported as an essential component of the ⇑ Corresponding author. core complex [10], it is now clear that FANCM is important for E-mail address: [email protected] (A. Smogorzewska). resistance to interstrand crosslinks but not at the step of

0014-5793/$36.00 Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2011.04.078 2854 E. Garner, A. Smogorzewska / FEBS Letters 585 (2011) 2853–2860

A Stalling of replication by ICL D Translesion step in ICL repair

ATR ATRIP RPA core RPA RPA Ub TLS RPA complex RPA Ub RPA Ub RPA Ub Ub ? ICL Ub ? ? PCNA ? RAD18

Ub Ub B PCNA Activation of the ID complex Ub

FAAP24 P M P P B G A E HR proteins in FA C FAAP100 F E P L Ub Ub UbUb P I D2 ID2 ID2 UBE2T P Ub ? I Ub

D2 ATR ? P

Homologous recombination PALB2 (HR) Activation of the nucleases; FANCJ C BRCA2 RAD51C UBZ domain containing effectors

SNM1A FAN1 ? Ub Ub P ID2 PCNA SLX4 P Symbol Key ? interstrand crosslink Ub monoubiquitylation Ub P Ub I D2 Ub P Ub chromatin Ub Ub K63-Ub linked chains ? XPF MUS81 Ub P phosphorylation UBZ domain SLX4 SLX1

Fig. 1. Ubiquitylation in response to interstrand crosslinks, highlighting Fanconi anemia proteins and the associated factors necessary for repair. Particular attention is drawn to some of the unknown factors in interstrand crosslink repair. (A) Interstrand crosslinks prevent separation of the DNA helix. During replication this can lead to replication fork 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 of the ATR and the downstream DNA damage response. (B) Activation of the ID complex. ATR phosphorylates many FA proteins including FANCI. FANCI phosphorylation acts as a molecular 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 ID complex 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. Downstream effectors FAN1, SLX4, and SNM1A harbor ubiquitin-binding motifs that are thought to target them to the site of damage through ubiquitin mediated interactions. FAN1 binds the monoubiquitylated FANCD2. SLX4 displays binding affinity for ubiquitin chains though the docking sites for SLX4 are not currently known. SLX4 could potentially interact with ubiquitylated FANCD2 or PCNA. Interestingly all three proteins harbor nuclease activity or are associated with nucleases. Thus, control of ubiquitylation events regulates the 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 interstrand crosslink repair. Additional, non-PCNA and non-RAD18-dependent events are likely to allow translesion synthesis to occur. (HR) concludes the repair 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 context of FA, it clearly has other functions, which are not yet fully explored. monoubiquitylation of the ID complex [11]. Furthermore, unlike with mutations in FAN1. The downstream proteins in the pathway the other components of the core complex, FANCM has FA pathway include FANCD1/BRCA2 and the BRCA2 interacting partner, independent function including ATR-mediated checkpoint signal- FANCN/PALB2 [17–19], both of which are essential components ing [12]. Monoubiquitylated ID complex interacts with FAN1 (Fan- of homologous recombination repair, and a FANCJ / coni anemia Associated Nuclease 1), which in vitro has both endo- BRIP1/BACH1 [20–23]. FANCJ interacts with BRCA1 but this inter- and exo-nuclease activity [13–16]. There are no known FA patients 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 to the reaction. Interestingly, presence of FANCI conferred specificity crosslink damage but no individuals with biallelic BRCA1 muta- to the reaction so that K561 of FANCD2 was now correctly tions have been found to date. The newest addition to the Fanconi monoubiquitylated. This suggests that FANCI might direct the cor- anemia protein family is FANCP/SLX4 [25–27]. SLX4 acts as a rect lysine residue to the core complex or perhaps mask alternative scaffold that interacts with multiple proteins including nucleases surface lysines. Reconstitution of the whole core complex might be XPF, MUS81 and SLX1 [28–30]. XPF and MUS81 have been previ- necessary for robust in vitro ubiquitylation. In addition, since phos- ously implicated in crosslink repair [31,32] and the SLX4-SLX1 phorylation plays an important role in activating the pathway, interaction is responsible for Holliday junction resolution activity using pre-activated FANCI (see below) might be required. in vitro. It is unclear at this point which activity of SLX4 is essential FANCD2 and FANCI are the only known substrates of the core for crosslink repair. Besides the genuine FANC proteins discussed complex. There remains a possibility that there will be other sub- above, numerous proteins have been demonstrated to be associ- strates for this complicated E3 ligase (Fig. 1B and D). A hint of that ated with FA pathway components and as such need to be studied might be seen in the result showing that monoubiquitylated in parallel. Technological advances including RNAi continue to FANCD2 on the chromatin is not sufficient for crosslink resistance facilitate the identification of proteins whose absence yields a in DT40 cells that lack the FA core complex [44]. Since FANCI genome instability phenotype analogous to that of FA and the next monoubiquitylation in DT40 cells is almost non-contributory (see generation sequencing approaches should soon identify the next section), it is likely that there will be other substrates of remaining FANC genes in FA patients with thus far unidentified FANCL. genetic mutations. Besides the major pathway of replication-dependent ICL repair, vertebrate cells also have a replication-independent repair path- 4. Regulation of ID complex monoubiquitylation way. This minor repair pathway is active during G0/G1 stages and is independent of replication and homologous recombination but Monoubiquitylated ID complex is at the heart of the Fanconi dependent upon the nucleotide excision repair components XPC anemia pathway. The monoubiquitylation of FANCD2 at lysine and XPA [33–37]. Many of the components of the FA pathway 561 is essential for the downstream function of the FA pathway including the core complex, FANCI and FANCD2 seem to be required in repair. Cells expressing K561R FANCD2 (which cannot be for this repair [38]. The key to understanding crosslink repair will monoubiquitylated) as the only FANCD2 allele are as sensitive to be careful molecular and biochemical dissection of function of the crosslinking agents as null cells [7]. The contribution of monoubiq- proteins involved in repair including crystallographic studies of uitylated FANCI to crosslink resistance seems to be less important the pathway’s components. It is clear that characterization of the than that of monoubiquitylated FANCD2 in human cells [4], and al- ubiquitin-driven events and nuclease regulation in the pathway most non-contributing in DT40 cells [45]. Monoubiquitylation of will be key to understanding the Fanconi anemia pathway. FANCD2 occurs following the localization of the core complex to sites of damage (Fig. 1B). Core complex localization requires the combined activity of FANCM and its interacting factor FAAP24 that 3. The E3 ubiquitin ligase activity of FANCL and the FA core facilitates the recruitment of the core complex to regions of dam- complex age [46,47]. The recruitment of FANCM to sites of damage is en- hanced further by the association of FANCM with additional Ubiquitylation of FANCD2 and FANCI is absent in cells with FAAP proteins, MHF1 and MHF2 [48]. MHF1 and MHF2 contain mutations in the core complex components (except for FANCM). putative histone fold domains and form a dimeric complex with Prior to the identification of FANCL, the core complex was not DNA binding affinity. MHF1 and 2 are found in complex with shown to harbor proteins with any recognizable domains. At that FANCM and FAAP24 in mammalian cells and MHF1 is critical for time, other known ubiquitin ligases such as BRCA1 were under the integrity of the complex. The MHF dimer complex augments scrutiny in efforts to identify the components required for this the association of FANCM with chromatin and enhances FANCM central step in FA pathway regulation [7,39]. It was not until the dependent processing of branched DNA. Destabilization of the identification of FANCL that it was possible to attribute monoubiq- MHF1/2 dimer complex with FANCM by silencing of MHF1 expres- uitylation of FANCD2 as an intra-FA pathway step in the response sion leads to a decreased efficiency of FANCD2 monoubiquitylation to ICLs [40]. in response to DNA damage and increased genome instability. FANCL is considered a key part of the FA core complex and rep- It is unclear how the ID complex localizes to chromatin before it resents the E3 ligase necessary for ubiquitin conjugation to lysine is monoubiquitylated. It does posses intrinsic DNA-binding activity 561 of human FANCD2 and lysine 523 of FANCI. Structurally FANCL [49–51] with preference for branched structures, so it is possible has three distinct domains; an ELF, DRWD and RING domain [41]. that it gets to the sites of damage on its own via DNA binding. Once Functionally, the DRWD domain coordinates substrate binding monoubiquitylated, it clearly resides at chromatin where it can be while the RING domain is required for E2 protein interactions. found in damage-induced foci [4,7]. It has been previously pro- Yeast two hybrid experiments using full-length FANCL have iden- posed that a chromatin receptor for the monoubiquitylated ID tified two E2 proteins capable of interacting with FANCL, UBE2T complex might be present at the chromatin [44,52,53], although and UBE2W [42]. The C-terminal RING domain is both necessary such a receptor has not yet been identified. In the light of FAN1 and sufficient for FANCL binding of UBE2T [43]. The importance binding to the chromatin bound monoubiquitylated ID complex of the RING domain is reflected in the evolutionary conservation (see below), existence of such a receptor is less likely, although of this domain in FANCL homologs [41]. UBE2T is considered to not impossible. If such a receptor does exist, understanding of be the most functionally important E2 enzyme required for the re-modeling of the ID complex architecture from interacting FANCL-mediated ubiquitylation of FANCD2 in vivo and is required with a receptor via monoubiquitin to interacting with FAN1 and for cellular resistance to ICLs [43]. In vitro data differ from in vivo- other effector proteins at chromatin will be very important. derived data as to the minimal requirements for FANCD2 monoub- Fanconi anemia proteins are regulated by DNA damage signal- iquitylation, since E1, UBE2T, and FANCL are shown to be sufficient ing involving ATM and ATR kinases. ATM is activated in response for FANCD2 monoubiquitylation in an in vitro context [42]. How- to double-strand breaks and in a manner that involves DNA resec- ever, this reaction was quite inefficient and ubiquitylation tion while ATR signals replicative stress and is activated by binding occurred only on FANCD2, even when FANCI was also present in of 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 including sensitivity to crosslinking agents [67,68]. This implies that the cy- FANCA, FANCG, FANCM, FANCD1 and both constituents of the ID cling of the ID complex between ubiquitylated and non-ubiquity- complex [4,5,10,45,56–62]. The importance of many of the phos- lated forms is essential for the activity of the pathway. USP1 is phorylation events is not entirely clear but some of them are the deubiquitylating enzyme that interacts with FANCD2 in chro- important for crosslink resistance. In particular, phosphorylation matin and removes its monoubiquitin [69] (Fig. 2). USP1 is regu- of FANCI on SQ/TQ sites close to the monoubiquitylation site of lated on many levels. USP1 levels are tightly regulated at a FANCI is necessary for the monoubiquitylation and localization of transcriptional level to peak during S-phase, presumably when both FANCI and FANCD2 to damage-induced foci [45]. FANCI carry- its activity is most required. It is also regulated by polyubiquityla- ing phosphomimic mutations of these sites showed constitutive tion and proteasome-dependent degradation at the end of S phase activation of the pathway even in the absence of damage. Based once it is no longer needed. USP1 levels during S phase however on these findings, it has been proposed that FANCI phosphorylation are fairly constant [69]. This implies that there will be as of yet functions as a molecular switch to turn on the FA pathway. unidentified regulatory steps in the pathway, possibly at the chro- Although it is unclear how this switch my work, one possibility matin level, which will allow deubiquitylation of FANCD2 and is that phosphorylation of FANCI drives an interaction of the ID resetting of the pathway. Interestingly, upon damage USP1 complex with a protein that interacts with the core complex or transcription is shut off with a subsequent rapid decline of USP1 with a protein in the core complex itself. It will be important to protein [70]. It is not known what happens to the chromatin- understand how phosphorylation of FANCI triggers monoubiquity- associated USP1 pool after DNA damage and it is this fraction of lation of FANCD2. USP1 that is of particular interest. It is unlikely that it disappears; rather its activity is probably regulated in some way to allow for 5. Ubiquitin mediated interaction of the ID complex and FAN1 de-ubiquitylation of the ID complex only after the damage is re- paired. USP1 activity is greatly enhanced by its partner, a WD40 The chromatin bound monoubiquitylated ID complex facilitates domain containing protein, USP1 Associated Factor 1 (UAF1) [70]. subsequent steps of ICL repair in ways that are still being eluci- Future work should concentrate on regulation of USP1 since our dated. The ID complex and monoubiquitylation of FANCD2 is re- appreciation of the mechanisms of deactivation, lags behind our quired for ICL unhooking and translesion DNA synthesis to understanding of the activation of the Fanconi anemia pathway. facilitate bypass of the lesion [63,64]. It is likely that monoubiqui- Interestingly, USP1 is also a de-ubiquitylating enzyme for PCNA tylation of the ID complex forms a platform for at least some of (Fig. 2), suggesting that regulation of de-ubiquitylation of PCNA these events and that the protein components involved in making and the ID complex might be co-regulated. Clearly there will also the incisions and translesion synthesis may harbor ubiquitin-bind- be separate regulatory networks, for example ELG1 interacts with ing motifs themselves (Fig. 1C). The newly identified nuclease re- USP1-UAF1 and affects PCNA deubiquitylation, but not FANCD2 quired for ICL repair, FAN1, is one such protein. FAN1 has a UBZ ubiquitylation status [71]. domain 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 localizing 7. The Ubiquitin binding motifs of FANCP/SLX4 to damage made by laser irradiation or by crosslinking damage [13–16]. The ID complex was also required for foci formation by FANCP/SLX4 represents the newest member of the FA pathway FAN1. Moreover, cells with endogenous FANCD2 replaced with to date and constitutes the 14th Fanconi anemia complementation the K561R FANCD2 mutant lost FAN1 localization. Since FANCD2 group FA-P [26,27]. The SLX4 protein is a large scaffold with multi- and FAN1 were able to interact in immunoprecipitation experi- ple recognizable domains. At the N terminus, it has two UBZ do- ments, monoubiquitylated FANCD2 is considered a platform for mains followed by an MLR (MUS312/MEI9 interaction like FAN1 binding through the UBZ domain. Other regions of FAN1 pro- region), BTB domain, SAP domain, and finally a helix-turn-helix tein might also be required for interaction with FANCD2 analogous motif at the C terminus [28–30]. Human SLX4 interacts with multi- to REV1 interacting not only with the monoubiquitin on PCNA but ple proteins including XPF-ERCC1, MUS81-EME1, SLX1, MSH2, also with PCNA itself through the BRCT motifs [66]. Since FAN1 dis- MSH3, TRF2, RAP1, and PLK1 among others. It appears that most plays both endonuclease and exonuclease activity this ubiquitin- if not all of the cellular pool of MUS81 and SLX1 interact with mediated recruitment focuses the nuclease activity of FAN1 to SLX4 while only a small portion of the cellular XPF pool interacts the site of the ICL. Thus far, in vitro experiments have been per- with SLX4 [30]. In vitro SLX4 is able to enhance the activity of formed on non-crosslinked substrates so it is unclear at which step the associated nucleases. Cells derived from FA-P patients display of repair FAN1 acts. It may work in concert with the MUS81-EME1 normal monoubiquitylation of FANCD2 following DNA damage and XPF-ERCC1 nuclease that have been implicated in crosslink but genomic instability and sensitivity to inter-strand crosslinking unhooking [31,32]. It might also act at the later stages of excision agents characteristic of an FA phenotype. The early steps of ICL re- of the unhooked crosslink or even later during preparation for pair that requires the core components of the FA pathway are unaf- homologous recombination events. The concept of ubiquitin- fected by a loss of SLX4 suggesting that it is a downstream effector mediated recruitment via the ID complex will undoubtedly be an of the pathway [26,27]. Obvious candidates for the essential com- important aspect of understanding the downstream processes of ponents of SLX4 in the setting of FA are XPF and MUS81 but the ex- FA pathway-mediated DNA repair. It will be interesting to uncover act role of SLX4 in FA pathway still needs to be determined. Patient how the ID complex co-ordinates the downstream effectors of the mutations suggest an important role for the UBZ domains. Two of pathway, some of which may yet to be identified. the four families with SLX4 mutations have a very interesting in- frame internal deletion of amino acids 317–387, corresponding to half of the first UBZ domain and the whole second part of UBZ 6. Deubiquitylation and FA pathway regulation domain. Although the level of expression of this allele was lower than wild type, it was able to interact with XPF and MUS81. Ubiquitylation of the ID complex is an activating event neces- The UBZ domains of SLX4 interact efficiently with K63-linked sary for the repair of ICLs. Equally important is the event of turning ubiquitin chains in vitro in a manner that requires intact UBZ that signal off once the repair is completed. Failure to de-ubiquity- domains [26]. Mutation of the two cysteines in both of the UBZ late the ID complex results, somewhat counter intuitively, in domains resulted in abrogation of ubiquitin chain binding. These E. Garner, A. Smogorzewska / FEBS Letters 585 (2011) 2853–2860 2857

? Ub Ub UAF1 I D2 P ID2 P Ub Ub P ID2D2 P

Ub Ub USP1 ID complex proteasome Ub

Ub

Ub Ub UAF1 Ub

ELG1 Ub Ub Ub Ub USP1 Ub PCNA

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 repair has 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 subject to regulation on both a transcriptional and post-transcriptional level. Importantly, USP1 is polyubiquitylated as DNA replication concludes. Polyubiquitylated USP1 is targeted for proteasomal degradation.

data led to a proposal that FANCP might localize to sites of damage ICL is an exciting topic. Many others polymerases might also be in- via ubiquitin mediated interactions. The substrates of such interac- volved in ICL repair including POLg, POLj, POLi, POLh, and POLm. tions are unknown at this time (Fig. 1C). It is still possible that SLX4 The involvement of these polymerase was recently extensively re- interacts with monoubiquitylated ID complex as suggested for the viewed in [79]. Of particular interest to the FA pathway is POLm chicken FANCP in DT40 cells [72]. However, the presence of tan- (POLN), which is an A type DNA polymerase. Depletion of POLN dem UBZ domains would be consistent with SLX4 binding to a tar- in human cells results in DNA crosslink sensitivity with defects get that is endowed with ubiquitin chains. Identity of ubiquitylated in homologous recombination [80]. POLN was found to interact substrates is a hot subject and identification of these in the context with monoubiquitylated FANCD2 on chromatin after mitomycin of the FA pathway will be truly exciting. It is appealing to think C treatment, although it is unclear if monoubiquitylation of that different nucleases will arrive at the sites of a crosslink via dif- FANCD2 is required for the interaction, Furthermore, the POLN ferent, albeit ubiquitin-dependent interactions. FAN1 would arrive dependent requirements for this interaction are not yet known. via monoubiquitylated ID complex, SLX4/XPF/MUS81 complex, via a different, unknown interactor. This would give an opportunity to 9. RAD18 and FA pathway activation regulate these nucleases separately and make sure that the inci- sions are made only at the correct sites and at the right time. Although RAD18 does not seem to be involved in REV1 localiza- tion to ICLs, lack of RAD18 has been reported by several laborato- 8. Translesion polymerases in the ICL repair ries to be involved in activation of the FA pathway since depletion or lack of RAD18 led to slower kinetics of FANCD2 and FANCI Translesion synthesis (TLS) polymerases are specialized en- monoubiquitylation [81–84]. The mechanism of how RAD18 might zymes that are able to insert a nucleotide across a damaged site affect ID complex monoubiquitylation is not clear at this time since in DNA. Multiple translesion polymerases have been implicated the published data from different groups do not agree with each in the FA pathway. Knockout of REV1, and components of POLf other. RAD18 is an E3 ligase and together with RAD6 it has been (REV3, REV7) in chicken DT40 cells leads to exquisite sensitivity shown to catalyze monoubiquitylation of PCNA on lysine 164 to crosslinking agents [73–77]. Moreover REV1 and REV3 are epi- and this modification promotes error-prone translesion bypass static with FANCC for crosslink sensitivity suggesting that they [85,86]. According to two papers [82,83], PCNA ubiquitination on are in the same pathway [73,75]. The current model for ICL repair lysine 164 is required for activation of the FA pathway upon cis- based on the repair of a crosslinked plasmid in Xenopus egg extract platin or benzo[a]pyrenedihydrodiol epoxide (BPDE) damage and proposes that translesion polymerases are essential for the bypass that ubiquitylated PCNA recruits the core complex which in turn of the crosslink that was freed, commonly referred to as ‘‘un- ubiquitylates the ID complex. Findings of Williams et al. [81] con- hooked’’, by endonucleolytic incisions [63,64]. It has been pro- tradict those conclusions. They show that RAD18 co-immunopre- posed that REV1 inserts one nucleotide against the unhooked cipitates with FANCD2 and that the RING domain of RAD18 is crosslink, which is further extended by the REV3 catalytic subunit necessary for this interaction. However, they show that FANCD2 of POLf [63,64]. Depletion of FANCD2 affected the translesion step ubiquitylation is normal after mitomycin C treatment of cells con- as well as the ‘‘unhooking’’ step. It is not yet known whether both taining an ubiquitylation-resistant form of PCNA, and that the of these events are direct consequences of FANCD2 depletion. It is chromatin loading of FA core complex proteins appears normal possible that both of these activities are co-regulated by the ID in RAD18-knockout cells. The authors suggest that RAD18 could complex or that the ID complex regulates only one of them and be responsible for modifying the core complex itself or other that the second depends upon the initiation (or even completion) unidentified DNA repair proteins upstream of the ID complex. of the first. Interestingly, REV1 focus formation due to ICLs is These discrepancies need to be addressed by the labs that reported dependent on the functional FA core complex but does not seem these findings. There were some obvious differences in cell lines to involve FANCD2 or RAD18-dependent PCNA monoubiquityla- and in damaging agents used between all the reports, including tion [78] (Fig. 1D). How the translesion polymerases get to the the 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 in also adoption of MUS81 into the SLX4 complex, offering an oppor- the report that did not. Regardless of the mechanism, RAD18 is cer- tunity for coordination of multiple nuclease activities through the tainly a factor that regulates the FA pathway. SLX4 protein.

10. Ubiquitin binding motif of SNM1A 12. Proteolysis and DNA damage signaling

Saccharomyces cerevisiae SNM1 (sensitivity to nitrogen mus- Ubiquitylation clearly plays pan-cellular roles that are critical for tard), also known as PSO2 (sensitivity to psoralen), has been iden- signaling and, as discussed here, crucial for FA pathway regulation tified in screens for selective sensitivity to ICLs [87,88]. The 50 to 30 and DNA repair. Monoubiquitylation and polyubiquitin chains exonuclease activity of Pso2p is thought to be important for pro- may be of primary importance in signaling events, as platforms for cessing of incisions made by nucleotide excision repair in yeast. complex interactions. However, the importance of proteolytic deg- In vertebrates, there are three orthologs of SNM1; SNM1A, radation in response to polyubiquitylation and proteasome target- SNM1B/Apollo, and SNM1C/Artemis (for review see [89]) SNM1A ing cannot be underestimated in terms of its importance for DNA and SNM1B both seem to participate in ICL repair, and the double repair. Many of the regulatory factors discussed here may be tar- knockout in DT40 cells is more sensitive to cisplatin than either of geted for proteasomal degradation as is the case for the FA regula- the single knockouts, suggesting that they function in separate tory deubiquitylating enzyme USP1 (discussed above, Fig. 2) and pathways or that they are redundant for cisplatin resistance [90]. FANCM, which is degraded during M phase, releasing the core com- SNM1A is also not epistatic in crosslink resistance with FANCC, plex from chromatin [102]. It has also been shown that proteasome RAD18 or XRCC3 (homologous recombination pathway) in DT40 function is in fact required for monoubiquitylation and foci forma- cells. SNMA1A, but not SNM1B was able to complement crosslink tion of FANCD2 [103]. The proteasome has also been linked to vari- sensitivity of the yeast Pso2 mutant [91] suggesting that SNM1 is ous aspects of DSB repair and homologous recombination [104,105]. a functional ortholog of Pso2. Recent work led to the identification Understanding how the proteasome balances the accumulation of of a PIP box motif in vertebrate SNM1 required for binding to PCNA repair factors with their degradation will involve a more thorough irrespective of DNA damage [71]. A newly identified UBZ domain in understanding of polyubiquitylation targets. In turn, how the pro- the N-terminus of SNM1A, which is conserved even in S. cerevisiae teasome is localized at sites of damage and its regulation is an Pso2, was required for damage induced foci formation by a GFP- important aspect of DNA damage signaling and repair. tagged SNM1 in human cells (Fig. 1C). SNM1A foci formation after DNA damage was dependent on RAD18 [71] though the functional 13. Perspectives consequences of these interactions are not yet clear. It remains to be determined whether SNM1B functions in crosslink repair in hu- There is a lot to be learned about the role of ubiquitylation in man cells. the Fanconi anemia pathway. Many of the remaining issues were noted in their respective sections above. Here we highlight a few, including the activation of ubiquitylation of the ID complex, role 11. Evolutionary conservation of proteins in the Fanconi of ubiquitin in regulation of nucleases, and a potential role of other, anemia pathway ubiquitin-like modifiers in the FA pathway.

The most conserved of the FA proteins are FANCM, SLX4 and 13.1. Turning on ubiquitylation BRCA2, orthologs of which are present from yeast to human [28– 30,92–94]. Such an exquisite conservation; however, may reflect Phosphorylation of FANCI turns on the FA pathway in response functions of these proteins outside of ICL repair. Vertebrates, flies, to damage. Understanding how this takes place is an important frogs, worms, plants and even a slime mold D. discoideum, have rec- concept in how ubiquitylation events occur in response to chal- ognizable orthologs of some of the components of the core com- lenges such as DNA damage. Are ubiquitin ligase complexes consti- plex, importantly FANCL [41,95–98]. Conservation of the tutively active such that the recruitment of substrates to the ligase components of the core complex, other than FANCL, fluctuate complex is the regulated step? Are ubiquitin ligase complexes throughout evolution with some components lost or gained in dif- themselves activated by post-translational modification? In the ferent species (reviewed in [98]). All organisms that have FANCL case of damage-induced ubiquitylation of the ID complex it may also have FANCD2 and FANCI, which have been shown to be be that phosphorylation of both the core complex and the ID com- monoubiquitylated in some [64,95,97,98]. S. cerevisiae and Saccha- plex regulate the ‘activation’ of ID complex ubiquitylation. In this romyces pombe have no core complex nor ID complex components. respect phospho-binding motifs may be the chromatin ‘receptors’ However, S. pombe does have a FAN1 ortholog whose function is that localize core complex with substrate leading to ubiquitylation. unknown. S. pombe Fan1 has a well-conserved nuclease domain and a SAP domain but no recognizable ubiquitin binding domain 13.2. Targets of FANCL monoubiquitylation [16]. SAP (SAF-A/B, Acinus and PIAS) domains have been postulated to be modules involved in targeting proteins to chromatin [99].As Thus far only FANCD2 and FANCI have been identified as targets such, it is possible that the SAP domain in the S. pombe FAN1 might of FANCL monoubiquitylation. It is compelling to think that there be involved in the localization of FAN1 to sites of damage. may be alternative targets for FANCL monoubiquitylation. This Both S. cerevisiae and S. pombe have Slx4 orthologs. Indeed, Slx4 hypothesis is supported by the observation that the core complex and Slx1 were first identified in yeast in a screen for factors re- is not dispensable for crosslink repair even when FANCD2 quired for the viability of S. cerevisiae lacking RecQ helicase Sgs1 monoubiquitylation has been satisfied [44]. The missing monoub- [100]. There are clear similarities between the yeast and human iquitylated factor in that study could have been FANCI, however, orthologs of Slx4 in that they both interact with XPF-ERCC1 and FANCI monoubiquitylation appears not to be very important in SLX1 orthologs [101]. SLX4 from yeast to human also contains the DT40 cells. In light of these findings, there still might be some SAP domains and the C-terminal Helix-turn-helix domains. The missing substrates of the core complex. Experimentally, finding Slx4 orthologs in yeast do not have the UBZ domains, BTB domains alternative targets of FANCL monoubiquitylation may not be sim- nor do they interact with MUS81. These differences suggest that ple. Such a modification is not represented in abundance and there has been an evolutionary expansion of SLX4 function, but dissecting monoubiquitylated targets from polyubiquitylated E. Garner, A. Smogorzewska / FEBS Letters 585 (2011) 2853–2860 2859 targets may prove challenging. 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