The repair of lesions and gaps in DNA follows different pathways, each mediated by specific proteins and complexes. Post-translational modifications in many of these proteins govern their activities and interactions, ultimately determining whether a particular pathway is followed. Prominent among these modifications are the addition of phosphate or ubiquitin (and ubiquitin-like) moieties that confer new binding surfaces and conformational states on the modified proteins. The present review summarizes some of consequences of ubiquitin and ubiquitin-like modifications and interactions that regulate nucleotide excision repair, translesion synthesis, double-strand break repair and interstrand cross-link repair, with the discussion of relevant examples in each pathway.

Introduction

Cells have developed elaborate mechanisms for the rapid and sensitive sensing and repair of damaged DNA, collectively designated DDR (DNA­-damage response) [1]. In general, different types of DNA lesion result in different signals and present different structural problems that are dealt with by specialized proteins and molecular complexes dedicated to the recognition and repair of a particular type of lesion. A number of proteins and complexes participate in the resolution of more than one type of DNA lesion, and therefore represent more ubiquitous modulators of damage signalling, the recruitment of additional repair factors or the activation of checkpoints that switch the cell from a replication mode to a repair mode.

From the moment that broken or non-physiological DNA molecules are recognized within the cell, mechanisms are set in motion to attract to the site of the lesion proteins and complexes that amplify the initial damage-recognition signal, suppress ongoing DNA replication and transcription, initiate the repair of the lesions and the resolution of aberrant DNA structures, recognize the completion of the repair process and eventually signal the resumption of physiological DNA transactions. Many of these actions are initiated by the addition or removal of post-translational marks that modulate the structure, interactions, localization or stability of proteins with key roles in these hierarchically regulated steps. The most prominent post-translational marks directly involved in DDR are phosphorylation, modification by ubiquitin and UBL (ubiquitin-like) molecules and poly(ADP-ribosyl)ation. Other modifications such as acetylation and methylation also play important roles in DDR, although, with a few known exceptions, these modifications appear to involve a more general modulation of chromatin structure and dynamics in response to DNA damage, rather than specific recruitment of signalling and repair complexes to the sites of lesion.

The PIKKs [PI3K (phosphoinositide 3-kinase)-related kinases] ATM (ataxia telangiectasia mutated), ATR (ATM- and Rad3-related) and DNA-PK (DNA-dependent protein kinase), as well as Chk (checkpoint kinase) 1, Chk2 and protein kinase CK2 are responsible for most of the key phosphorylation events regulating DDR [2]. PP (protein phosphatase) 2A and other phosphatases reverse these reactions. In the nucleus, where the majority of the DDR transactions take place, many of the serine or threonine residues located within specific amino acid motifs that are phosphorylated by the PIKK enzymes are recognized by FHA (forkhead-associated) or BRCT [BRCA1 (breast-cancer early onset 1) C-terminal] domains borne by proteins containing additional functional domains, whereas residues phosphorylated by Chk1, Chk2 and CK2 are recognized by the 14-3-3 protein and also by phosphopeptide-recognition subunits within complex ubiquitin ligases and other multisubunit protein complexes [3]. These phosphorylation events can lead to multiple consequences: they can activate or inactivate the functions of the target proteins; they can tether to the site of the lesion other proteins that amplify and transduce the damage-recognition signal to activate downstream checkpoint events; or they can assist in substrate recognition by ubiquitin ligases that can lead to proteasome-dependent degradation of the modified protein.

One advantage for cells afforded by using phosphorylation reactions in DDR signalling is the short response time of these enzymatic reactions, a virtue necessary to rapidly prevent undue replication or cell death in the aftermath of significant DNA damage. However, phosphorylation reactions can be equally rapidly countered by potent phosphatases, which can prematurely terminate the phosphorylation-initiated actions unless other mechanisms take over when a sustained response is required. Thus cells have devised additional layers of regulation aimed at maintaining, potentiating and fine-tuning the DDR until the damage has been repaired. Foremost among this second tier of DDR regulation are modifications by ubiquitin and UBL moieties [4], in some cases exquisitely orchestrated along with phosphorylation to provide robust responses to DNA damage. Mono-ubiquitylation and polyubiquitylation reactions require the participation of at least two classes of enzymes, ubiquitin-conjugating enzymes (E2) and ubiquitin ligases (E3), of which several are directly involved in DDR. E2s interact with E3s in combinatorial fashion, and polyubiqutin chains can be synthesized using any of the seven lysine residues on ubiquitin or forming head-to-tail linear chains [4,5]. Mono-ubiquitin and the different classes of polyubiquitin chains are recognized by different UBDs (ubiquitin-binding domains) present on proteins that function in DDR and other processes [6]. Different UBDs may have preferences for mono-ubiquitin or for different types of polyubiquitin chains. Mono- and poly-ubiquitylation are antagonized by DUBs (deubiquitylating enzymes) that cleave the isopeptide bond at the C-terminus of ubiquitin and are grouped into six distinct structural and functional families that differ in their activities towards mono-ubiquitin or to different classes of polyubiquitin chains. It is clear that these combinations generate a large diversity of signals that can be used to co-ordinate the assembly and disassembly of many types of protein complex, allowing for multiple combinations between subunits that may participate in more than one complex at different times during the DDR. The enormous energy that cells spend to engage and execute these processes, and the consequences for the fate of the cell of entering these programmes, explain the existence of stringent regulations to keep a low signal background under physiological conditions.

SUMO (small ubiqutin-related modifier) in BER (base excision repair)

Small DNA lesions that result from oxidation, alkylation or deamination, which modify individual bases without large effects on the structure of the DNA double helix, are recognized and repaired by the BER pathways [7]. Individual base lesions are recognized by distinct DNA glycosylases that catalyse the cleavage of an N-glycosidic bond, removing the damaged base and creating an AP (apurinic/apyrimidinic) site [8]. The DNA backbone is cleaved by either a DNA AP endonuclease or a DNA AP lyase, an activity present in some glycosylases. An APE (AP endonuclease) activity creates an ssDNA (single-stranded DNA) nick 5′ to the AP site, and an AP lyase activity creates a 3′ nick. This single-nucleotide gap now contains a 3′-hydroxy and a 5′-phosphate group, substrates compatible with the downstream enzymatic reactions in BER. A DNA polymerase fills in the gap with the correct nucleotide. Finally, a DNA ligase completes the repair process and restores the integrity of the helix by sealing the nick.

One of the DNA glycosidases in BER is TDG (thymine-DNA glycosylase) that hydrolyses the N-glycosidic bonds of thymine or uracil that are mismatched with guanine. Modification of TDG by SUMO-1 or SUMO-3 causes the dissociation of the enzyme from the AP site, resulting in turnover of the enzyme [9], increasing the efficiency of the reaction. Controlled dissociation of TDG from the substrate allows BER to couple faithfully with APE activity, reducing the exposure of the potentially hazardous AP site. The mechanism of this SUMO-mediated dissociation has been determined with the resolution of the crystal structure of the central domain of the enzyme conjugated to SUMO that shows that conjugated SUMO interacts with a SUMO-binding element in the enzyme, in a intramolecular interaction that results in conformational changes that would release the enzyme from its product DNA [10].

Regulation by ubiquitin of NER (nucleotide excision repair)

Bulky DNA lesions that cause distortions in its structure are not processed by BER pathways, but by NER [11]. NER is critically important in the repair of UV-induced lesions. Short-wavelength UV light can cause the formation of covalent bonds between two adjacent pyrimidines on the same DNA strand. There are two main UV-induced lesions: CPDs (cyclobutane pyrimidine dimers) and (6-4)PPs [pyrimidine(6-4)pyrimidone photoproducts]. NER also deals with bulky DNA adducts formed by chemicals that bind DNA bases and cross-linking agents. Because NER requires an intact strand to serve as a template to repair the damaged strand, the repair of ICLs (interstrand cross-links) requires a combination of NER with translesion synthesis or homologous recombination. ICLs are treated as bulky adducts and can also be processed by NER.

NER can occur in two major pathways. TCR (transcription-coupled repair)/TC-NER (transcription-coupled NER) ensures that the transcribed strand of active genes is repaired in preference to the rest of the genome, probably by using RNAPII (RNA polymerase II) as a lesion sensor. GGR (global genome repair)/GG-NER (global genome NER) refers to NER of non-coding parts of the genome and non-transcribed strands of active genes. Mutations in NER underlie the extreme photosensitivity and predisposition to skin cancer exhibited in patients with CS (Cockayne's syndrome) and XP (xeroderma pigmentosum), although CS patients display developmental and neurological phenotypes not observed with XP patients. Mutations in the CSA and CSB proteins lead to specific defects in NER of the transcribed strand in transcriptionally active genes.

CUL4A–DDB1 ubiquitin ligases in NER

Lesions that cause DNA distortion can be recognized by the DDB (damaged DNA-binding) complex [12], a heterodimer consisting of DDB1 and DDB2 that has high affinity for (6-4)PPs and CPDs. DDB1 participates in at least two cullin-dependent E3 ubiquitin ligase complexes, one composed of CUL4A (scaffold), RBX1/ROC1/Hrt1 (E2 recruitment through its RING finger domain) and DDB2 (substrate recognition) [13], and a second ligase in which CSA is substituted for DDB2. Both DDB2 and CSA are WD40 repeat-harbouring proteins that confer substrate specificity on the ligase.

Once localized in the damage site, the CUL4A–RBX1–DDB1–DDB2 complex promotes the polyubiquitylation of XPC [14], which is subsequently deubiquitylated, without causing its degradation, but rather enhancing its affinity for DNA [14]. The type of polyubiquitin chain linkage that modifies XPC, whether Lys63-linked, Lys48-linked or otherwise, is not known. The activation of this E3 ligase also promotes the polyubiquitylation followed by degradation of its subunit DDB2, with consequent disassembly of the complex and down-regulation of its ligase activity (Figures 1A–1C). This autoregulatory mechanism ensures the transient nature of the activation of this NER subpathway [14].

Model for ubiquitin transactions in GG-NER

Figure 1
Model for ubiquitin transactions in GG-NER

(A) UV light induces CPDs and (6-4)PPs. Other agents induce local conformational changes in DNA. These structures are recognized by DDB2, in complex with DDB1. (B) This complex is recognized by CUL4A and ubiquitylated by an E2 recruited to CUL4A by RBX1, which leads to proteasome-dependent degradation of DDB2. (C) The CUL4A complex E3 ubiquitin ligase promotes the polyubiquitylation of XPC (Rad4 in S. cerevisiae), a modification that does not lead to its degradation, but rather to its enhanced recognition of DNA lesions. The chain-specificity of the polyubiquitin modifying XPC is not known. Lesion recognition by polyubiquitylated XPC is assisted by interactions with HR23B (Rad23 in S. cerevisiae) and CENP2 (Cdc31 in S. cerevisiae). A denaturation bubble is opened by the XPB and XPD helicases of TFIIH, whose activity is stimulated by XPC–HR23B. (D) XPA (Rad14 in S. cerevisiae) is recruited to this pre-excision complex and may co-operate with XPC in lesion recognition and TFIIH activation, and RPA associates with ssDNA. The XPF–ERCC1 and XPG endonucleases create incisions at the 5′ and 3′ ends of the lesion respectively. DNA-bound RPA recruits ATRIP and ATR kinase (Mec1 in S. cerevisiae) which phosphorylates H2AX, recruiting MDC1 by binding to its FHA domain. ATR also phosphorylates MDC1, recruiting the FHA domain E3 ubiquitin ligase RNF8. (E) The excised oligonucleotide. containing the lesion is released, and the gap is filled in by Polδ or Polε, in association with the PCNA processivity clamp. RNF8 promotes the ubiquitylation of H2AX and H2A, initiating the recruitment to extensive chromatin domains around the lesion of additional repair and checkpoint factors (see Figure 4).

Figure 1
Model for ubiquitin transactions in GG-NER

(A) UV light induces CPDs and (6-4)PPs. Other agents induce local conformational changes in DNA. These structures are recognized by DDB2, in complex with DDB1. (B) This complex is recognized by CUL4A and ubiquitylated by an E2 recruited to CUL4A by RBX1, which leads to proteasome-dependent degradation of DDB2. (C) The CUL4A complex E3 ubiquitin ligase promotes the polyubiquitylation of XPC (Rad4 in S. cerevisiae), a modification that does not lead to its degradation, but rather to its enhanced recognition of DNA lesions. The chain-specificity of the polyubiquitin modifying XPC is not known. Lesion recognition by polyubiquitylated XPC is assisted by interactions with HR23B (Rad23 in S. cerevisiae) and CENP2 (Cdc31 in S. cerevisiae). A denaturation bubble is opened by the XPB and XPD helicases of TFIIH, whose activity is stimulated by XPC–HR23B. (D) XPA (Rad14 in S. cerevisiae) is recruited to this pre-excision complex and may co-operate with XPC in lesion recognition and TFIIH activation, and RPA associates with ssDNA. The XPF–ERCC1 and XPG endonucleases create incisions at the 5′ and 3′ ends of the lesion respectively. DNA-bound RPA recruits ATRIP and ATR kinase (Mec1 in S. cerevisiae) which phosphorylates H2AX, recruiting MDC1 by binding to its FHA domain. ATR also phosphorylates MDC1, recruiting the FHA domain E3 ubiquitin ligase RNF8. (E) The excised oligonucleotide. containing the lesion is released, and the gap is filled in by Polδ or Polε, in association with the PCNA processivity clamp. RNF8 promotes the ubiquitylation of H2AX and H2A, initiating the recruitment to extensive chromatin domains around the lesion of additional repair and checkpoint factors (see Figure 4).

On the other hand, the CUL4A–RBX1–DDB1–CSA complex polyubiquitylates CSB, leading to its proteasome-dependent degradation. CSA and CSB specifically participate in the NER pathway associated with transcriptionally active regions, or TCR-NER.

CUL4A–DDB1 ubiquitin ligases associate with many other WD40 repeat-containing proteins that extend its range of substrates well beyond those directly involved in NER [15]. For example, the CUL4A–DDB1–Cdt2 complex promotes the ubiquitylation and proteasome-dependent degradation of the DNA replication-licensing factor Cdt1 and also the cyclin-dependent kinase regulator p21. Binding between DCAF2/Cdt2 and its target protein Cdt1 is not direct and it also requires PCNA (proliferating-cell nuclear antigen) and chromatin for complex formation [16]. Interestingly, skin-specific Cul4a-knockout mice accumulated DDB2, XPC and p21, and showed enhanced GG-NER, but not TC-NER, and resistance to UV-induced skin cancer [17]. This observation suggests that the CUL4A ubiquitin ligases not only are activators of XPC, but also determine its stability, highlighting the dynamic nature of these relationships. Additional CUL4A–DDB1 substrates include histones H2A, H3 and H4 around the site of damage [18,19], whose ubiquitylation antagonizes nucleosome binding in vitro, providing a pathway to assemble the NER complex in the otherwise inaccessible chromatin environment.

Like other cullin-ring ubiquitin ligases, CUL4–DDB1 complexes are regulated by the CAND1–NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) cycle [20]. Covalent attachment of the UBL protein NEDD8 enhances CUL4 ubiquitin ligase activity, in part by preventing inhibitory CAND1/TIP120 association with CUL4 [21]. The COP9 signalosome cleaves NEDD8 from CUL4, inhibiting its ubiquitin ligase activity [12]. Chromatin localization leads to the release of COP9 [12,22], activating CUL4A–DDB1.

HR23B/Rad23 in NER

Although DDB2 very specifically recognizes the distortions caused by photolesions, but has limited capacity to bind to other lesions, XPC, in conjunction with HR23B and CENP2 (Rad4, Rad23 and Cdc31 respectively in Saccharomyces cerevisiae) can recognize a wider range of duplex-destabilizing lesions.

HR23B/Rad23 contains an N-terminal UBL domain and two ubiquitin-binding domains, a central UBA (ubiquitin-associated) 1 domain, and a C-terminal UBA2 domain [23,24]. Both domains bind mono- and poly-ubiquitin with distinct specificities [2527]. The UBA1 domain shows a preference for Lys63-linked polyubiquitin chains, whereas UBA2 preferentially recognizes Lys48-linked chains. Deletion of the UBA2 domain does not have a strong effect on the binding of polyubiquitylated proteins, whereas removal of the UBA1 domain has a major impact on the ubiquitin-binding properties of Rad23, suggesting that the UBA1 domain is responsible for the bulk of ubiquitin binding [23,25,26,28,29]. HR23B/Rad23 binds to XPC/Rad4 through a region located between its two UBA domains, stabilizing XPC and thus enhancing NER [3032]. Rad23 may play a second role in NER that does not require binding and stabilization of Rad4/XPC [31]. In addition, the ubiquitin-recognition function of Rad23 may be partially dispensable for NER, since yeast expressing Rad23 lacking the UBA domains display normal UV sensitivity [33], although, on the other hand, cells with UBA2 mutants are sensitive to multiple UV exposure, which could be due to the fact that Rad23 is unstable under these conditions [34].

The UBL domain or Rad23 interacts with a number of proteins and also participates in intramolecular interactions with its UBA domains [35]. Rad23 associates with the proteasome through the interaction of its UBL domain with the Rpn1 and Rpn2 subunits at the base of the 19S regulatory particle [36] and modulates the delivery of polyubiquitylated substrates to the proteasome [37,38]. The human orthologues hHR23A and hHR23B interact with S5a [39], a ubiquitin-binding protein associated with the proteasome, whereas yeast Rad23 does not interact with Rpn10, the yeast orthologue of S5a [40]. The UBL-dependent association of Rad23 with the 19S regulatory particle stimulates NER independently of the proteolytic activity of the proteasome [41]. However, disruption of the proteasome alleviates the defects in NER of Rad23 mutants [42]. The latter observations may be related to indirect effects of the proteasome on one or more components of the NER, for example ubiquitin-dependent degradation. Rad23 participates in additional pathways, such as ERAD (endoplasmic reticulum-associated degradation) [43], promoting protein turnover in response to stress, including UV and endoplasmic reticulum stress.

Downstream ubiquitin signalling in NER

After lesion recognition, a denaturation bubble is formed around the lesion, a task performed by the multisubunit TFIIH (transcription factor IIH). One of its subunits, XPB, is a helicase that facilitates clearance of RNAPII from the promoter. A subcomplex of three other subunits, cyclin H, Cdk7 and MAT1 (the CAK complex), phosphorylates the large subunit of RNAPII, switching it to elongation mode [44]. Another TFIIH subunit, the helicase subunit XPD, together with the ATPase function of XPB, opens a bubble of denaturation around the lesion (Figures 1C and 1D). XPB, XPD and five other subunits [p62, p52, p44, p34 and TTD-A (trichothiodystrophy A)] form a ring-shaped structure, to which the CAK complex is attached like a panhandle [45]. The yeast orthologue of p44 (Ssl1) possesses an E3 ubiquitin ligase activity, required for a proper transcriptional response to DNA damage [46]. The mammalian p44 also contains a RING finger motif, but it is not known whether it is indeed an E3 ligase.

The opening of a denaturation bubble around the site of lesion involves the exposure of ssDNA molecules that are rapidly protected by the heterotrimeric complex RPA (replication protein A) in association with the NER protein XPA, displacing the XPC complex. XPA recruits the nucleases XPG and ERCC1 (excision repair cross-complementing 1)/XPF that incise the damaged strand at the 3′ and the 5′ ends of the lesion respectively, resulting in the excision of a 25–30 nt fragment. An oligonucleotide spanning the lesion is displaced and the resulting gap is filled by DNA Pol (polymerase) δ or Polε, probably associated with PCNA. The nick is sealed by ligase III, with a minor contribution of ligase I in replicating cells (Figures 1D and 1E). In TC-NER, RNAPII is stalled by lesions in the transcribed strand of active genes and attracts NER enzymes. In TCR, lesion detection is performed by RNAPII, in the course of transcribing an active gene. This pathway proceeds as in GG-NER, although additional enzymes (CSA, CSB and XAB2) are required.

As discussed below, RPA also recruits and activates the ATR kinase through the ATRIP (ATR-interacting protein) adaptor, initiating a checkpoint response that includes the activation of Chk1 [47]. This results in the phosphorylation of H2AX, with subsequent deposition of the scaffold protein MDC1 (mediator of DNA-damage checkpoint protein 1) and also the ubiquitin ligase RNF (RING finger protein) 8, both of which recognize phosphopeptides through FHA domains [48] (Figures 1D and 1E). RNF8, in association with the ubiquitin-conjugating enzyme Ubc13, promotes the stable modification of H2A with Lys63-linkage polyubiquitin chains, necessary for the accretion at the lesion site of 53BP1 (p53-binding protein 1) and BRCA1 [48], and possibly additional repair and checkpoint mediators. These are late events in NER that are shared with processes involved in the recognition and repair of other types of lesions, notably double-strand breaks. It is not known whether these chromatin modifications are co-ordinated with those induced by the CUL4A ligase complexes.

Sensing stalled replication: the 9–1–1 checkpoint clamp

The presence of gaps or bulky DNA adducts in actively replicating chains causes DNA polymerases to stall, but the MCM (minichromosome maintenance) helicases continue to unwind DNA ahead of the replication fork. This leads to the generation of ssDNA, which is promptly coated by RPA [49]. The ssDNA–RPA complex recruits multiple proteins [50], including (i) ATRIP, that in turn tethers the ATR kinase and the BRCT domain protein TopBP1 (topoisomerase II-binding protein 1) to the damage site [47], (ii) the Rad17 clamp loader which then loads the heterotrimeric 9–1–1 (Rad9–Rad1–Hus1) complex on to DNA [51], and (iii) the Rad18–Rad6 ubiquitin ligase and conjugating enzyme complex [52,53].

The 9–1–1 complex forms a ring structure [5456] similar to that of the PCNA homotrimer [54,56,57], which is specifically loaded at the junction between duplex DNA and ssDNA coated by multiple copies of RPA [58,59]. Loading of 9–1–1 is facilitated by Rad17 (Rad24 in S. cerevisiae), a specialized form of the RFC (replication factor C) clamp loader [60,61]. In addition to detecting stalled replication through its interaction with RPA, the 9–1–1 checkpoint clamp physically associates with several factors required for BER [6267], and, like the structurally related PCNA, it interacts with translesion DNA polymerases [6870]. These interactions suggest that the checkpoint clamp may play additional roles in damage recognition and repair.

As a consequence of the recruiting events initiated by RPA at DNA replication blocks and damage sites, 9–1–1 and Rad17 are phosphorylated by ATR [71,72], and Rad17 is mono-ubiquitylated by Rad6 [53]. Phosphorylated Rad9 is recognized by TopBP1 [71], enhancing further the activity of ATR and its downstream activation of Chk1 [71,73]. Mono-ubiquitylation of Rad17 by Rad6 appears to be required for downstream signalling through Rad53 (Chk1 orthologue) [53], directly linking the Rad18–Rad6 ubiquitin ligase to checkpoint activation. This modification of Rad17 was also shown to be required for a co-ordinated transcriptional response to DNA damage in yeast that has been proposed to be analogous to the bacterial SOS response [53]. How Rad17 mono-ubiquitylation regulates this response is still unknown.

Damage bypass: PCNA as a switchboard for ubiquitin and SUMO signals

Stalled DNA polymerases at single-strand lesions promote modifications in the DNA processivity clamp PCNA that allow it to enter either a TLS (translesion synthesis) mode or a recombination-based error-free repair mode [74]. TLS is catalysed by error-prone translesion polymerases which make this pathway potentially mutagenic. The error-free mode uses the undamaged sister chromatid as a template, thus avoiding the propagation of mutations. The switch between different modes is dictated by ubiquitin or SUMO modifications of a single residue on PCNA, Lys164 [75].

PCNA forms a homotrimeric clamp that requires the pentameric complex RFC for loading on DNA templates [60]. The loading proceeds in an orderly sequence that requires ATP and directs the stepwise assembly by RFC of circular PCNA structures that encircle double helical DNA and then release the loader. Lys164 resides on the outer edge of the loaded processivity clamp, away from the encircled DNA. In the yeast, PCNA is SUMOylated on Lys164 during the S-phase of the cell cycle, in a reaction catalysed by Ubc9 and enhanced by the SUMO ligase Siz1 {PIAS1 [protein inhibitor of activated STAT (signal transducer and activator of transcription) 1] in mammals}. This creates a high-affinity binding site for Srs2 [76], a helicase that suppresses recombination by virtue of its capacity to displace the Rad51 recombinase from the replication fork, preventing unscheduled recombination during normal replication (Figure 2). A second lysine residue at position 127, at the interdomain loop of PCNA, can be also SUMOylated directly by Ubc9 without the participation of a ligase, and both the modification and the binding of Ubc9 dislodge proteins that use that region for interaction with PCNA [77], among which is Eco1, responsible for sister chromatid cohesion during S-phase [78]. SUMOylation at both sites is greatly enhanced by the loading of the PCNA clamp on to DNA [79]. The SUMO hydrolase Ulp1 catalyses the removal of SUMO from these sites.

Model for ubiquitin and SUMO transactions associated with PCNA in S. cerevisiae

Figure 2
Model for ubiquitin and SUMO transactions associated with PCNA in S. cerevisiae

During the S-phase, PCNA is SUMOylated on Lys164, in a reaction catalysed by Ubc9 and enhanced by the SUMO ligase Siz1. This creates a binding site for Srs2 that antagonizes the Rad51 recombinase and prevent unscheduled recombination during normal replication. In response to DNA damage in S-phase or stalled replication forks, ssDNA intermediates are generated and coated with RPA. This recruits Rad18–Rad6, catalysing the mono-ubiquitylation of PCNA at Lys164 (K164) and creating a surface for tethering to PCNA low-fidelity polymerases of the Y-family in preference to replicative polymerases. This allows DNA synthesis to proceed even in the presence of lesions. In the event of unsuccessful TLS or in the continued presence of lesions and stalled forks at the damaged site, the mono-ubiquitin on PCNA serves as the acceptor ubiquitin for Lys63 (K63)-based polyubiquitylation catalysed by Ubc13–MMS2. Ubc13–MMS2 is recruited to the site of lesion by the ubiquitin ligase Rad5 with in turn is recruited to the stalled fork by Rad18. Presumably, the addition of this chain dislodges TLS polymerases and promotes template switching from the leading strand to the nascent lagging strand. A DNA polymerase extends the 3′ end of the leading nascent strand by copying from the nascent lagging strand, and the back-migration of the four-way junction completes error-free replication through the DNA damage.

Figure 2
Model for ubiquitin and SUMO transactions associated with PCNA in S. cerevisiae

During the S-phase, PCNA is SUMOylated on Lys164, in a reaction catalysed by Ubc9 and enhanced by the SUMO ligase Siz1. This creates a binding site for Srs2 that antagonizes the Rad51 recombinase and prevent unscheduled recombination during normal replication. In response to DNA damage in S-phase or stalled replication forks, ssDNA intermediates are generated and coated with RPA. This recruits Rad18–Rad6, catalysing the mono-ubiquitylation of PCNA at Lys164 (K164) and creating a surface for tethering to PCNA low-fidelity polymerases of the Y-family in preference to replicative polymerases. This allows DNA synthesis to proceed even in the presence of lesions. In the event of unsuccessful TLS or in the continued presence of lesions and stalled forks at the damaged site, the mono-ubiquitin on PCNA serves as the acceptor ubiquitin for Lys63 (K63)-based polyubiquitylation catalysed by Ubc13–MMS2. Ubc13–MMS2 is recruited to the site of lesion by the ubiquitin ligase Rad5 with in turn is recruited to the stalled fork by Rad18. Presumably, the addition of this chain dislodges TLS polymerases and promotes template switching from the leading strand to the nascent lagging strand. A DNA polymerase extends the 3′ end of the leading nascent strand by copying from the nascent lagging strand, and the back-migration of the four-way junction completes error-free replication through the DNA damage.

In response to DNA damage in S-phase or stalled replication forks, ssDNA intermediates are generated and coated with RPA. This recruits Rad18–Rad6 to the site of the stalled fork, catalysing the mono-ubiquitylation of PCNA at Lys164 [75], presumably outcompeting SUMOylation at the same site by Siz1 (Figure 2). This creates a new surface for the recognition by low-fidelity polymerases of the Y-family [80] that harbour ubiquitin-binding domains such as UBZ (Polη and Polκ), or UBM (Polζ and Polι) [6,8183] (Figure 2). These polymerases use their UBDs together with N-terminal sequences known as PIP (PCNA-interacting protein) boxes [8285] to recognize mono-ubiquitylated PCNA in preference to the replicative Polδ [81]. The Y-family polymerases lack the high specificity for Watson–Crick base pairs of Polδ and other replicative polymerases, and can accommodate bulky DNA adducts in their active sites [80]. This allows DNA synthesis to proceed even in the presence of lesions, a process with intrinsic mutagenic potential. After polymerization is resumed, the ubiquitin on PCNA can be removed by the DUB Usp1, reverting to replicative mode with the re-entry of Polδ in exchange for TLS polymerases [81] (Figure 2).

In the event of unsuccessful TLS or in the continued presence of lesions and stalled forks at the damaged site, the mono-ubiquitin on PCNA is not removed and rather serves as the acceptor ubiquitin for Lys63-based polyubiquitylation catalysed by the Ubc13–MMS2 heterodimeric ubiquitin-conjugating enzyme [75] (Figure 2). Ubc13–MMS2 is recruited to the site of lesion through the RING finger domain of the ubiquitin ligase Rad5 and Rad5 is recruited to the stalled fork through interaction with Rad18 [75,86]. In yeast, Ubc13–MMS2 localizes preferentially in the cytoplasm and translocate to the nucleus only after DNA damage, by unknown mechanisms [75]. The enzymatic activity of the Ubc13–MMS2 dimer now localized in close proximity to Lys164 on PCNA is responsible for the synthesis of the Lys63-linked polyubiquitin chain, primed by the ubiquitin modification at that residue [87]. Presumably, the addition of this chain dislodges TLS polymerases and, instead promotes template switching from the leading strand to the nascent lagging strand. Although the biochemical details of this process are not well understood, it requires the helicase activity of Rad5 that mediates replication fork regression [88]. One model proposes that Rad5 unwinds both the lagging and leading nascent DNA strands, followed by annealing them as well as annealing the template strands. By this fork regression activity, a four-way junction intermediate called a ‘chicken foot’ is formed (Figure 2). Following that, a DNA polymerase extends the 3′ end of the leading nascent strand by copying from the nascent lagging strand. Finally, the back-migration of the four-way junction completes error-free replication through the DNA damage [88]. Questions remain as to whether Lys63 polyubiquitin chains on PCNA recruit specific factors that enable template switching and error-free repair or how the Ubc13–MMS2–Rad5 pathway is turned off to return to normal replicative modes. Although most of the mechanistic details of these pathways have been elucidated in S. cerevisiae, most of the components have clear orthologues in mammals, including the human Rad5 homologues SHPRH (SNF2 histone linker PHD RING helicase) and HLTF (helicase-like transcription factor) [89,90], and thus a reasonable prediction is that many of the processes and mechanisms described here will be conserved from yeast to mammals.

The very different functional modes adopted by PCNA through mutually exclusive SUMO and ubiquitin modifications on a single amino acid have become a paradigm of the exquisitely selective engagement in alternative pathways conferred by each of these modifications.

Focus on DSBs (double-strand breaks): ubiquitin-mediated recruitment of repair complexes

DSBs are seriously damaging lesions that can trigger cell death or genomic instability unless promptly repaired. They can be produced by ionizing radiation through production of ROS (reactive oxygen species), or UV light and radiomimetic drugs that block replication. They can also be caused by excess ROS accumulated as metabolic by-products, and during programmed genome rearrangements such as yeast mating-type, meiosis, V(D)J recombination or class-switch recombination. Defects in components of DSB repair mechanisms can lead to immune deficiency and cancer syndromes.

Two major pathways are used to repair DSBs, NHEJ (non-homologous end-joining) and HDR (homology-directed repair). NHEJ joins the two ends of a DSB without resorting to homologous sequences and thus can be mutagenic, and can function throughout the cell cycle, although it is the most important mode of DSB repair in G1-phase. This repair mechanism is prevalent in vertebrates, but not in yeast, which rely almost exclusively on HDR to repair DSBs. HDR uses the homologous regions in sister chromatids as templates for repair, and thus is considerably less error-prone than NHEJ, except when repair templates are not sister chromatids and therefore not perfectly homologous. In vertebrates, HDR is the prevalent mode of repair of DSBs in the S- and G2-phases of the cell cycle, when templates from sister chromatids are available.

In NHEJ, exposed DNA ends are bound by Ku (Ku70–Ku80), a heterodimer that forms a loop structure that encircles DNA. Binding to DNA induces conformational changes in Ku that promote its recruitment of proteins to the site of damage, including DNA-PK, the X4-L4 [XRCC4 (X-ray repair complementing defective repair in Chinese-hamster cells 4)–ligase IV] complex, XLF, and the X-family DNA polymerases Polμ and Polλ. In lymphocytes, also TdT (terminal deoxyribonucleotidyltransferase) interacts with DNA-bound Ku. The large DNA-PK protein recruits and activates the 5′→3′ exonuclease Artemis, critical for the processing of broken ends in preparation for ligation. DNA-PK phosphorylates many proteins, including itself. Autophosphorylation of DNA-PK inhibits its kinase activity and causes its dissociation from DNA, thereby facilitating access to damaged sites of additional factors that are critical for repair after the initial activation of the pathway. Ku-bound XRCC4 is phosphorylated by CK2, creating phosphosites for binding by the FHA domains of the DNA end-processing enzymes PNK (polynucleotide kinase) (with 3′ phosphatase and 5′ kinase activities) and APLF (aprataxin PNK-like factor) (with endo- and exo-nuclease activities). Additional phosphosites on XRCC4 are recognized by BRCT domains on the gap-filling polymerases Polμ and Polλ. The ligation of processed DNA ends is performed by ligase 4. Thus Ku-bound XRCC4 functions as a scaffold that brings together the machinery necessary for DNA end-processing, gap filling and ligation. XRCC4 is SUMOylated on Lys210 and this modification is necessary for its localization to DSBs and for V(D)J repair by NHEJ [91] (Figure 3).

Major pathways in DSB repair

Figure 3
Major pathways in DSB repair

In NHEJ, DNA ends are encircled by Ku which recruits proteins such as DNA-PK, X4-L4, XLF, Polμ and Polλ. DNA-PK recruits and activates the 5′→3′ exonuclease Artemis. Autophosphorylation of DNA-PK causes its dissociation from DNA. XRCC4 is phosphorylated by CK2, creating phosphosites for binding by the FHA domains of nucleases PNK and APLF. Additional phosphosites on XRCC4 are recognized by BRCT domains on Polμ and Polλ. The ligation of processed DNA ends is performed by ligase 4. HDR is initiated by binding of the broken ends by the MRN complex that, along with Sae2/CtIP, EXO1, Sgs1/BLM and Dna2, produces 3′ overhangs that are coated by RPA. The MRN/MRX complex also recruits and activates ATM, which phosphorylates numerous substrates, including RPA and H2AX. Phosphorylated RPA recruits Rad51 and ATR. With the assistance of BRCA2, Rad51 displaces RPA and is loaded on to ssDNA, forming nucleoprotein filaments that scan for sequences homologous to the bound ssDNA, producing complementary-paired duplex structures known as D-loops. Repair polymerases extend DNA complementary to parental strands, and the resulting cross-over structures are resolved by the RecQ-homologous helicase BLM. IR, ionizing radiation.

Figure 3
Major pathways in DSB repair

In NHEJ, DNA ends are encircled by Ku which recruits proteins such as DNA-PK, X4-L4, XLF, Polμ and Polλ. DNA-PK recruits and activates the 5′→3′ exonuclease Artemis. Autophosphorylation of DNA-PK causes its dissociation from DNA. XRCC4 is phosphorylated by CK2, creating phosphosites for binding by the FHA domains of nucleases PNK and APLF. Additional phosphosites on XRCC4 are recognized by BRCT domains on Polμ and Polλ. The ligation of processed DNA ends is performed by ligase 4. HDR is initiated by binding of the broken ends by the MRN complex that, along with Sae2/CtIP, EXO1, Sgs1/BLM and Dna2, produces 3′ overhangs that are coated by RPA. The MRN/MRX complex also recruits and activates ATM, which phosphorylates numerous substrates, including RPA and H2AX. Phosphorylated RPA recruits Rad51 and ATR. With the assistance of BRCA2, Rad51 displaces RPA and is loaded on to ssDNA, forming nucleoprotein filaments that scan for sequences homologous to the bound ssDNA, producing complementary-paired duplex structures known as D-loops. Repair polymerases extend DNA complementary to parental strands, and the resulting cross-over structures are resolved by the RecQ-homologous helicase BLM. IR, ionizing radiation.

If a DSB is not repaired by NHEJ, it is eventually processed by 5′→3′ resection, which commits the repair process to HDR. HDR is initiated by binding of the broken ends by the MRN (Mre11–Rad50–NSB1) complex [MRX (Mre1–Rad50–Xrs2) in S. cerevisiae] [92]. The MRN/MRX complex holds together the broken DNA ends, and, along with Sae2 [CtIP (C-terminal-binding protein-interacting protein) in vertebrates], Exo1 (EXO1), Sgs1 [BLM (Bloom's syndrome protein)] and Dna2, extends the 5′→3′ trimming to produce 3′ overhangs that are then coated by RPA [9395]. Sgs1/BLM is also critical for the resolution of double Holliday junctions in a later step in HDR [96,97]. The MRN/MRX complex also recruits and activates the ATM kinase (Tel1 in S. cerevisiae), which phosphorylates numerous substrates, including RPA (with phosphorylation sites mostly on its 32 kDa subunit) and H2AX (H2A in S. cerevisiae). Hyperphosphorylation of RPA enhances its recruitment of additional proteins, including Rad51 and the kinase ATR (Mec1 in S. cerevisiae) [98]. With the assistance of BRCA2 [also known as FANC (Fanconi's anaemia complementation group) D1], Rad51 displaces RPA and is loaded on to ssDNA, forming nucleoprotein filaments that scan for sequences homologous with the bound ssDNA, producing complementary-paired duplex structures known as D-loops (Figure 3). Repair polymerases, primed by the 3′ ends of invading strands, extend DNA complementary to parental strands, and the resulting cross-over structures are resolved by the RecQ-homologous helicase BLM and possibly additional complexes.

A concert of phosphorylation and ubiquitin-mediated events at DSB sites

Phosphorylation of H2AX triggers a second set of recruiting events to the damage sites, orchestrated by phosphorylation and ubiquitin modifications and recognition [99]. This modification can be produced by any of the PIKK family kinases, ATM, ATR and DNA-PK, and therefore can associate not only with DSBs, but also with other types of DNA damage in which kinases other than ATM are preferentially activated [100]. A combination of advanced single-cell imaging techniques, gene-silencing tools and protein–protein interaction analyses has unveiled complex cascades of events and interactions that are set in motion by the appearance of this chromatin modification. Remarkably, and unlike other DDR mechanisms, most of these findings come from functional studies in mammalian cells, rather than yeast models, largely because many of the factors involved are absent from S. cerevisiae.

Very shortly after damage induction, H2AX is phosphorylated on Ser139 (also designated γH2AX), a modification that is detectable with specific antibodies. This phosphosite is recognized by the BRCT motifs on the large adaptor protein MDC1 [101,102] that protects it from dephosphorylation by PP2A or PP4 [103]. MDC1 bound to chromatin promotes further the recruitment of the MRN complex to DSBs through interaction with BRCT motifs on NBS1 to sites on MDC1 phosphorylated by CK2 [104107], which in turn promotes additional recruitment and autoactivation of ATM. In addition to BRCT motifs, MDC1 harbours an FHA domain at its N-terminus, through which it binds phosphorylated ATM and Chk2 [102]. This progressively stabilizes and amplifies the γH2AX mark, reaching a maximal intensity by 30 min after the insult, and also greatly propagates the marked chromatin domains, up to 100 kb in yeasts and 1 Mb in vertebrate cells beyond the site of lesion. Such amplified signals are readily visible as IRIFs (ionizing-radiation-induced foci) in immunoassays with antibodies specific for γH2AX, frequently used as an indirect indicator of the extent of DNA damage [108].

The association of MDC1 with ATM results in the phosphorylation of threonine residues within tandem TQXF (Thr-Gln-Xaa-Phe) motifs on MDC1 that are subsequently recognized by the FHA domain of RNF8 [109111]. This ubiquitin ligase recruits the ubiquitin-conjugating enzyme Ubc13 [112] for the catalysis of Lys63-linked di-ubiquitylation of H2AX on Lys119 and also H2A [110,111] and H2B [113]. The H2AX and H2A ubiquitin modifications are recognized by the MIU (motif interacting with ubiquitin) domains on a second RING finger ubiquitin ligase, RNF168 [114,115]. Again in conjunction with Ubc13, RNF168 promotes the elongation of Lys63-linked polyubiquitin chains on H2AX and H2A, primed by the ubiquitin moieties conjugated previously by RNF8–Ubc13 [114,115]. The longer Lys63-linked polyubiquitin chains now formed on the chromatin associated with the DSBs are recognized by the adaptor protein RAP80 (receptor-associated protein 80) through its tandem UIMs (ubiquitin-interacting motifs) [114,115] which show a preferential binding for this class of polyubiquitin chains over Lys48-linked chains [116118]. It has not been resolved whether the RNF8-directed di-ubiquitin modification of histones could suffice to recruit RAP80 to DSB sites, although RNF168 clearly provides a greatly amplified signal and more robust recruitment. RAP80 forms a complex with the adaptor protein Abraxas (also called CCDC98 or ABRA1) that recruits BRCA1 (mutated in familial breast cancer) by virtue of the recognition of specific phosphoserine residues by the BRCT domain at the C-terminus of BRCA1 [116,117,119123].

The RAP80–Abraxas complex also hosts the JAMM (JAB1/MPN/MOV34 metalloenzyme)-domain deubiquitylating enzyme BRCC36 (BRCA1/BRCA2-containing complex 36) [117,119,124126] that antagonizes the formation of Lys63-linked polyubiquitin chains [124,127] formed by RNF8–Ubc13 and RNF168–Ubc13 at DSB sites, dismantling at least part of the complex [124]. The activity of BRCC36 might serve functions other than simply down-regulating the DSB signalling and repair complex, because its depletion also attenuates BRCA1-containing IRIFs and increases sensitivity of cells to ionizing radiation [125,128,129], suggesting that dynamic Lys63-linked polyubiquitin chain turnover plays a role in the maintenance of these complexes. There is evidence that other DUBs, such as USP3, may also play roles in the dynamics of histone ubiquitylation and of DSB-associated checkpoint and repair complexes [130].

Additional components of the RAP80–Abraxas–BRCC36 complex are BRCC45 and MERIT4 (C19orf62, NBA1) [128,129,131]. These five proteins form stoichiometric associations and are mutually interdependent in their interactions and functions, such that cells lacking any component are impaired in the recruitment of BRCA1 to IRIFs, are sensitive to ionizing radiation and show a compromised G2 checkpoint activation [128,129,131]. Through mediation by PALB2 (FANCN), BRCA1 recruits the unrelated protein BRCA2 [132], whose mutations are also associated with gynaecological cancers. BRCA2, like its yeast homologue Rad52, plays critical roles in the recruitment and recombinogenic activity of Rad51 [133,134], the key factor in homology-directed repair. These events are summarized in Figure 4.

The recruitment of repair and checkpoint proteins to DSB foci requires highly concerted ubiquitylation and phosphorylation reactions

Figure 4
The recruitment of repair and checkpoint proteins to DSB foci requires highly concerted ubiquitylation and phosphorylation reactions

(A) Ionizing irradiation (IR), radiomimetic chemicals or programmed DNA rearrangements induce DSBs. (B) Immediately after DNA ends are exposed, they are bound by the MRN complex that recruits and activates ATM that phosphorylates H2AX (γH2AX). This phosphosite is recognized by the BRCT motifs on the MDC1 that promotes further the recruitment of the MRN complex to DSBs, which in turn promotes additional recruitment and autoactivation of ATM. This results in the phosphorylation of TQXF motifs on MDC1, subsequently recognized by the FHA domain of RNF8. (C) RNF8–Ubc13 catalyses Lys63-linked di-ubiquitylation of H2AX, H2A and H2B. These di-ubiquitins are recognized by a second RING finger ubiquitin ligase, RNF168 that, with Ubc13, promotes the elongation of Lys63-linked polyubiquitin chains on H2AX and H2A. These Lys63-linked polyubiquitin chains are recognized by RAP80 through its tandem UIMs. RAP80 forms a complex with Abraxas, BRCC36, BRCC45 and MERIT4, all of which are necessary for the recruitment of BRCA1. BRCA1 recruits BRCA2 by means of their mutual interaction with PALB2 (FANCN). 53BP1 is also recruited to IRIFs via the RNF8- and RNF168-dependent pathway by unknown mechanisms that make H3K79me and H4K20me accessible for interaction with the Tudor domains of 53BP1. MIU, motif interacting with ubiquitin; Ub, ubiquitin.

Figure 4
The recruitment of repair and checkpoint proteins to DSB foci requires highly concerted ubiquitylation and phosphorylation reactions

(A) Ionizing irradiation (IR), radiomimetic chemicals or programmed DNA rearrangements induce DSBs. (B) Immediately after DNA ends are exposed, they are bound by the MRN complex that recruits and activates ATM that phosphorylates H2AX (γH2AX). This phosphosite is recognized by the BRCT motifs on the MDC1 that promotes further the recruitment of the MRN complex to DSBs, which in turn promotes additional recruitment and autoactivation of ATM. This results in the phosphorylation of TQXF motifs on MDC1, subsequently recognized by the FHA domain of RNF8. (C) RNF8–Ubc13 catalyses Lys63-linked di-ubiquitylation of H2AX, H2A and H2B. These di-ubiquitins are recognized by a second RING finger ubiquitin ligase, RNF168 that, with Ubc13, promotes the elongation of Lys63-linked polyubiquitin chains on H2AX and H2A. These Lys63-linked polyubiquitin chains are recognized by RAP80 through its tandem UIMs. RAP80 forms a complex with Abraxas, BRCC36, BRCC45 and MERIT4, all of which are necessary for the recruitment of BRCA1. BRCA1 recruits BRCA2 by means of their mutual interaction with PALB2 (FANCN). 53BP1 is also recruited to IRIFs via the RNF8- and RNF168-dependent pathway by unknown mechanisms that make H3K79me and H4K20me accessible for interaction with the Tudor domains of 53BP1. MIU, motif interacting with ubiquitin; Ub, ubiquitin.

The Tudor domain protein 53BP1 (Rad9 in S. cerevisiae; Crb2 in Schizosaccharomyces pombe) [135] is also recruited to IRIFs through the RNF8- and RNF168-dependent pathway in mammals [109111,114,119,136]. The Tudor domains on this protein recognize H3K79me (histone H3 methylated at Lys79) in S. cerevisiae and mammals [137], and H4K20me (histone H4 methylated at Lys20) in S. pombe and mammals [138140], but it does not have recognizable UBDs and is not known to bind ubiquitin, RNF8 or RNF168. It has been speculated that the dependence on RNF8, RNF168 and Ubc13 of the recruitment of 53BP1 to IRIFs is based on conformational changes in the chromatin induced by the ubiquitylation of histones catalysed by these ligases, that expose pre-existing H3K79me and H4K20me and make them accessible for interaction by the Tudor domains of 53BP1 [99,111,138]. The function of 53BP1/Rad9/Crb2 at DSB sites is associated with checkpoint signalling through activation of Chk2 and ATM [135].

BRCA1: essential DDR ubiquitin ligase scarce in ubiquitylation substrates

In addition to the central role played by ATM in most DDR pathways [141], many components of this cascade also have independent functions in DNA-damage signalling. Their importance is highlighted by the occurrence of pathologies causally associated with mutations in some of these factors, such as the RIDDLE (radiosensitivity, immunodeficiency, dysmorphic features and learning difficulties) syndrome, a rare disorder caused by mutations that inactivate RNF168 [115,136], or by the observation of genome instability and sensitivity to DNA-damaging agents in animal models [142,143]. Nevertheless, it may be argued that these actors could play relatively subsidiary roles in an elaborate and costly scheme mainly designed to lure BRCA1, another ubiquitin ligase, to DSB sites. There is evidence that pathways different from the one summarized above can also bring BRCA1 to such sites [144]. BRCA1 is the most prevalent tumour-suppressor gene mutated in familial breast and ovarian cancer [145147], and mutations are also strongly associated with specific types of sporadic breast cancer [148,149]. Cellular and animal models have shown that BRCA1 is required for genome stability and repair by HDR [150154] and also for chromosomal stability [155] (possibly reflecting functions separate from DNA-damage repair). As a consequence, mice with homozygous deletions of BRCA1 suffer from embryonic lethality [156158], indicating essential roles in development. Finally, mammary-specific deletion or functional inactivation of BRCA1 fosters the development of adenocarcinomas that mimic BRCA1-associated human breast cancer [159]. In spite of the unequivocal evidence of the importance of BRCA1 in DDR and cancer, knowledge of the molecular mechanisms by which it exerts its functions remains surprisingly limited.

BRCA1 is a large protein harbouring multiple domains that associates with numerous other proteins and protein complexes to perform many different functions in the cell [159]. Two important functional domains on BRCA1 containing sequence motifs that are conserved in other proteins are the C-terminal repeats that recognize phosphorylated serine residues in SXX motifs (the BRCT domains), critical for the recruitment of BRCA1 to IRIFs as discussed above, and the N-terminal RING finger domain. The main function of this domain is to engage in α-helical interactions with the RING finger domain of BARD1 (BRCA1-associated RING domain 1) [160], and it is the BRCA1–BARD1 heterodimer that forms a functional ubiquitin ligase in vitro [161,162]. On the basis of in vitro assays, numerous ubiquitylation substrates have been proposed for BRCA1–BARD1, including BRCA1 itself. Surprisingly, very few have been reported as substrates in vivo, and these include γ-tubulin [163], nucleophosmin [164], topoisomerase IIα [165] and CtIP [166]. Evidence for several of these putative in vivo BRCA1–BARD1 substrates is based on a single report and requires further confirmation. Of these proteins, only CtIP (Sae2 in S. cerevisiae) is directly involved in HDR [9395].

Indirect evidence in support of the importance of the ubiquitin ligase function of BRCA1–BARD1 in HDR is provided by the fact that many familial breast cancer clusters associate with mutations in the RING finger domain of BRCA1 or BARD1 that inactivate their ligase function [167170]. In contrast, evidence that the ubiquitin ligase of BRCA1–BARD1 does not significantly affect DSB repair by HDR is furnished by experiments in which mouse ES (embryonic stem) cells were knocked-in with a BRCA1 mutant (I26A) unable to recruit E2s, but that still partners with BARD1. These cells showed augmented chromosomal abnormalities upon damage, but normal levels of IRIFs and HDR [171]. The elaborate choreography that follows the establishment of DSBs and IRIFs culminates in the arrival to the site of damage of a protein with essential roles whose detailed molecular function continues to resist elucidation.

The FA (Fanconi's anaemia)–ubiquitin connection in ICL repair

Bivalent chemicals that react with DNA can cause ICLs, forming structures that represent a challenge for DNA-repair mechanisms, particularly when they occur in the vicinity of replication forks. These lesions result in stalled replication and generation of DSB intermediates and thus, in mammals, their repair requires a combination of NER, HDR and post-replicative repair/TLS.

FA is a rare genetic disease characterized by bone marrow failure, developmental abnormalities and a high incidence of cancer [172]. To date, 13 complementation groups have been characterized in FA, for each of which a responsible gene has been identified. Cells derived from FA patients exhibit high sensitivity and prolonged S-phase arrest in response to cross-linking agents such as diepoxybutane, mitomycin C or cisplatin. These cells display a high degree of chromosomal instability, as is evident from spontaneous and drug-induced chromosomal breakages and sister chromatid interchanges that produce ‘chromosome radials’, a hallmark of FA. Remarkably, each of the 13 proteins encoded by the genes of the FA complementation groups physically associates with at least one of the other proteins, and eight of them form an FA core complex with stoichiometric representations of the FA proteins A, B, C, E, F, G, L and M, together with the associated proteins FAAP (FA-associated protein) 24 and FAAP100 [173,174]. This complex can associate with the remaining FA proteins, FANCD2 and FANCI (the ID complex), and FANCD1 (BRCA2), FANCN (PALB) and FANCJ (BRIP1 or BACH1) (the BRCA complex), and also with other proteins, notably the Bloom's syndrome complex (BLM, BLAP75, BLAP250, RPA70, RPA34, RPA14, topoisomerase IIIα) [175]. Interestingly, FA proteins are not conserved in yeast, which suggests that they participate in unique mechanisms for ICL repair in higher eukaryotes.

The only enzymatic activity of the FA core complex demonstrated is to serve as a ubiquitin ligase for the ID complex. All eight proteins of the core complex are required for mono-ubiquitylation of both FANCD2 and FANCI, and these two proteins are dependent on each other for mono-ubiquitylation [176,177]. UBE2T has been identified as a major E2 ubiquitin-conjugating enzyme catalysing this reaction [178], being recruited to the FA core complex through interaction with FANCL, a subunit that harbours a PHD/RING finger domain and WD40 repeats [179]. Activation of ATR promotes the ubiquitin ligase activity of the FA complex on the ID complex [180], and this double mono-ubiquitylation promotes the association of the FA complex with chromatin [177]. These mono-ubiquitins are removed by the deubiquitylation enzyme USP1 [181], the same enzyme involved in removing the mono-ubiquitin on Lys164 of PCNA, and this turnover is important for efficient functioning of the FA complex in ICL repair [182].

ICLs are initially recognized by XPA that recruits to the lesion the structure-specific heterodimeric endonuclease ERCC1–XPF [183] which creates a nick at the 5′ side of the lesion [184]. Another endonuclease, XPG, nicks the 3′ side of the lesion, thus excising the damaged DNA [185]. End-trimming reactions expose 3′ overhangs that are coated by RPA, which then recruits ATRIP and ATR to the site of the lesion, with activation of the latter. Gap filling and repair can proceed through NER and/or TLS pathways. Lesion excision by ERCC1–XPF that occurs in the proximity of replication forks generates double-stranded ends and complex structures that are bound by the ATR-activated FA core complex containing mono-ubiquitylated FANCI and FANCD2 [186]. These ends are a class of DSBs and trigger the activation of pathways preferentially dedicated to DSB repair. Activated ATR phosphorylates H2AX and thus promotes the recruitment of BRCA1, which interacts with BRCA2–PALB2 (FANCD1–FANCN) and Rad51 [132134]. BRCA2 could also be recruited to the lesion through interaction with the ID complex, and this in turn recruits BRCA1. These interactions link ICL repair to homology-directed recombination, where Rad51 plays an essential role. In contrast with the recruitment of the ID complex, the recruitment of the BRCA complex to the lesion appears to require active replication and can be independent of the core and ID complexes [186], suggesting the existence of at least two separate branches of the FA pathway whose activation depend on replicative status.

Conclusions and perspectives

Recent evidence shows that DNA-damage repair mechanisms rely on elaborate interplays between phosphorylation, ubiquitin and UBL modifications coupled to their recognition by specialized protein motifs. The specificity provided by these induced interactions in dictating localizations, activities and pathways is elegantly exemplified by the consequences of alternative modifications on Lys164 of PCNA. The power and importance of a single ubiquitin modification in deciding to pursue a given repair pathway is evident in the role played by mono-ubiquitylation of the ID complex in ICL repair, for which a multisubunit ubiquitin ligase appears to be dedicated full-time.

It is also becoming clear that, although different pathways and strategies are employed by the cell to tackle different types of DNA lesions, composing specific phosphorylation–ubiquitylation choreographies for each pathway, several pathways can share the same or very similar interactions and complexes for the resolution of shared structural problems in the process of DNA repair. This overlap is most evident in ICL repair, in which TLS, NER and HDR pathways participate in concert. From this evidence, it is reasonable to expect that the prominent role played in DSB repair of ATM phosphorylation and Ubc13 Lys63-linked polyubiquitylation events may also become of importance in ICL repair, as well as other lesions that become marked by γH2AX [48]. Similarly, the finding that the DNA-bound RPA complex directly recruits Rad18–Rad6 should lead to the finding that this ubiquitin ligase plays important roles in most DNA-repair pathways [187].

Many questions remain to be answered with regards to the diverse roles played by ubiquitin and UBL modifications in DNA repair. For example, it is unclear why so many different ubiquitin ligases and reactions are necessary during the formation of IRIFs in response to DSBs. It may be that histone polyubiquitylation requires RNF168, and that the role of RNF8 is limited to priming substrates in preparation for RNF168's activity. Alternatively, histone di-ubiquitylation by RNF8 could be important in remodelling the chromatin around the DSB site, in preparation for homologous recombination and HDR [187]. It will be very interesting to verify this possibility with new cellular and animal models. In fact, that RNF8 plays more complex roles is suggested by the discovery of new interactors, such as the large protein HERC2, once again a putative ubiquitin ligase harbouring a HECT (homologous with E6-associated protein C-terminus) domain (V. Plans, M. Guerra-Rebollo and T. Thomson, unpublished work). A more long-standing question continues to be the role, if any, played by the ubiquitin ligase activity of the BRCA1–BARD1 heterodimer in DDR.

Ubiquitin–Proteasome System, Dynamics and Targeting: 4th Intracellular Proteolysis Meeting, a Biochemical Society Focused Meeting held at Institut d'Estudis Catalans, Casa de Convalescència, Barcelona, Spain, 27–29 May 2009. Organized and Edited by Bernat Crosas (Institute of Molecular Biology of Barcelona, Spain), Rosa Farràs (Centro de Investigación Príncipe Felipe, Valencia, Spain), Gemma Marfany (University of Barcelona, Spain), Manuel Rodríguez (CIC bioGUNE, Derio, Spain) and Timothy Thomson (Institute of Molecular Biology of Barcelona, Spain)

Abbreviations

     
  • 9–1–1

    Rad9–Rad1–Hus1

  •  
  • AP

    apurinic/apyrimidinic

  •  
  • APE

    AP endonuclease

  •  
  • APLF

    aprataxin polynucleotide kinase-like factor

  •  
  • ATM

    ataxia telangiectasia mutated

  •  
  • ATR

    ATM- and Rad3-related

  •  
  • ATRIP

    ATR-interacting protein

  •  
  • BER

    base excision repair

  •  
  • BLM

    Bloom's syndrome protein

  •  
  • 53BP1

    p53-binding protein 1

  •  
  • BRCA1

    breast-cancer early onset 1

  •  
  • BARD1

    BRCA1-associated RING domain 1

  •  
  • BRCC36

    BRCA1/BRCA2-containing complex 36

  •  
  • BRCT

    BRCA1 C-terminal

  •  
  • Chk

    checkpoint kinase

  •  
  • CPD

    cyclobutane pyrimidine dimer

  •  
  • CS

    Cockayne's syndrome

  •  
  • CtIP

    C-terminal-binding protein-interacting protein

  •  
  • DDB

    damaged DNA-binding

  •  
  • DDR

    DNA-damage response

  •  
  • DNA-PK

    DNA-dependent protein kinase

  •  
  • DSB

    double-strand break

  •  
  • DUB

    deubiquitylating enzyme

  •  
  • ERCC1

    excision repair cross-complementing 1

  •  
  • FA

    Fanconi's anaemia

  •  
  • FAAP

    FA-associated protein

  •  
  • FANC

    FA complementation group

  •  
  • FHA

    forkhead-associated

  •  
  • GG-NER

    global genome nucleotide excision repair

  •  
  • H3K79me

    histone H3 methylated at Lys79

  •  
  • H4K20me

    histone H4 methylated at Lys20

  •  
  • HDR

    homology-directed repair

  •  
  • ICL

    interstrand cross-link

  •  
  • IRIF

    ionizing-radiation-induced focus

  •  
  • Ku

    Ku70–Ku80

  •  
  • MDC1

    mediator of DNA-damage checkpoint protein 1

  •  
  • MRN

    Mre11–Rad50–NSB1

  •  
  • MRX

    Mre1–Rad50–Xrs2

  •  
  • NEDD8

    neural-precursor-cell-expressed developmentally down-regulated 8

  •  
  • NER

    nucleotide excision repair

  •  
  • NHEJ

    non-homologous end-joining

  •  
  • PCNA

    proliferating-cell nuclear antigen

  •  
  • PIKK

    PI3K (phosphoinositide 3-kinase)-related kinase

  •  
  • PNK

    polynucleotide kinase

  •  
  • Pol

    polymerase

  •  
  • PP

    protein phosphatase

  •  
  • (6-4)PP

    pyrimidine(6-4)pyrimidone photoproduct

  •  
  • RAP80

    receptor-associated protein 80

  •  
  • RFC

    replication factor C

  •  
  • RNAPII

    RNA polymerase II

  •  
  • RNF

    RING finger protein

  •  
  • ROS

    reactive oxygen species

  •  
  • RPA

    replication protein A

  •  
  • ssDNA

    single-stranded DNA

  •  
  • SUMO

    small ubiquitin-related modifier

  •  
  • TC-NER

    transcription-coupled nucleotide excision repair

  •  
  • TCR

    transcription-coupled repair

  •  
  • TDG

    thymine-DNA glycosylase

  •  
  • TFIIH

    transcription factor IIH

  •  
  • TLS

    translesion synthesis

  •  
  • TopBP1

    topoisomerase II-binding protein 1

  •  
  • UBA

    ubiquition-associated

  •  
  • UBD

    ubiquitin-binding domain

  •  
  • UBL

    ubiquitin-like

  •  
  • UIM

    ubiquitin-interacting motif

  •  
  • XP

    xeroderma pigmentosum

  •  
  • XRCC4

    X-ray repair complementing defective repair in Chinese-hamster cells 4

  •  
  • X4-L4

    XRCC4–ligase IV

We are grateful for the support shown by all the members of the Cell Signalling and Cancer Laboratory at the Instituto de Biología Molecular.

Funding

M.G-R. is funded by a fellowship of the Spanish Ministry of Science. This work was supported by Spanish Ministry of Science [grant number SAF2008-04136-C02-01] and institutional funds provided by the Centre de Referència en Biotecnologia (Generalitat de Catalunya).

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