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Published in final edited form as: Bioessays. 2021 Feb 23;43(5):e2100011. doi: 10.1002/bies.202100011

Chaperones for dancing on chromatin: Role of post-translational modifications in dynamic damage detection hand-offs during nucleotide excision repair

Bennett Van Houten 1,2,3,4, Brittani Schnable 1,3, Namrata Kumar 3,4
PMCID: PMC9756857  NIHMSID: NIHMS1856031  PMID: 33620094

Abstract

We highlight a recent study exploring the hand-off of UV damage to several key nucleotide excision repair (NER) proteins in the cascade: UV-DDB, XPC and TFIIH. The delicate dance of DNA repair proteins is choreographed by the dynamic hand-off of DNA damage from one recognition complex to another damage verification protein or set of proteins. These DNA transactions on chromatin are strictly chaperoned by post-translational modifications (PTM). This new study examines the role that ubiquitylation and subsequent DDB2 degradation has during this process. In total, this study suggests an intricate cellular timer mechanism that under normal conditions DDB2 helps recruit and ubiquitylate XPC, stabilizing XPC at damaged sites. If DDB2 persists at damaged sites too long, it is turned over by auto-ubiquitylation and removed from DNA by the action of VCP/p97 for degradation in the 26S proteosome.

Keywords: DNA damage, global genome repair, nucleotide excision repair, post-translational modifications, ubiquitylation

INTRODUCTION

Damage to genomic DNA is ubiquitous: as many as 70,000 lesions occur daily in human cells.[1] Life has evolved a series of complex pathways to repair these potentially harmful lesions.[2] One pathway that removes a wide array of chemically diverse lesions caused by UV-light, chemical carcinogens and the chemotherapeutic drug, Cisplatin, is nucleotide excision repair (NER), which can be described in three major steps: recognition, verification, and repair.[3] Depending on the recognition/initiation events, NER consists of two sub-pathways: global genome (GG-NER) and transcription-coupled (TC-NER). The former is initiated by UV-damaged DNA binding protein (UV-DDB), a heterodimer of DDB1 and DDB2, which recognizes a lesion in the context of chromatin, flipping out two bases into a recognition pocket. The lesion is then passed to XPC-RAD23B, which inserts a beta-hairpin at the damaged site to bend and open the helix. During TC-NER, repair is initiated by RNA polymerase II stalling at a lesion.[4] Both sub-pathways converge after the damage recognition step, at which point transcription factor IIH (TFIIH) is recruited. TFIIH is a complex consisting of seven core proteins that are arranged in a donut shape.[5,6] These subunits include: p8, p34, p44, p52, p62, XPB, and XPD. XPD, a DNA helicase, works along with the ATPase activity of XPB, a DNA translocase, to help create a bubble in the DNA to verify the location of the lesion on the damaged strand. XPA along with RPA help to stabilize the recognition bubble and form the pre-excision complex to recruit XPF/ERCC1 heterodimer, and XPG. XPF creates an incision 5′ of the lesion. DNA polymerases (Pol δ, Pol κ or Pol ɛ) are recruited and gap filling DNA synthesis begins, which triggers XPG nuclease activity to incise the strand 3′ of the lesion, allowing the lesion to be excised as a 22–25 base oligonucleotide. NER is completed with DNA ligase I or III sealing the nick, Figure 1. This essay briefly reviews the role of post-translational modifications in NER and discusses a recent, exciting study demonstrating that timely departure of UV-DDB through autoubiquitylation is required for stable XPC and TFIIH recruitment.

FIGURE 1.

FIGURE 1

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Global nucleotide excision repair. The recognition step of GG-NER is initiated by UV-DDB scanning DNA for lesions (step 1). UV-DDB binding to a lesion (step 2) serves as a signal for XPC-RAD23B to bind (step 3). During the verification steps, UV-DDB dissociates from the lesion and TFIIH, consisting of XPB and XPD, is recruited (step 4). The helicase activity of XPD, along with the translocase activity of XPB, opens the double helix around the lesion. XPA together with RPA stabilize the recognition bubble, verify the existence of lesions, and recruit XPF-ERCC1 and XPG to complete the pre-excision complex (step 6). During the repair steps, XPF makes an incision 5′ of the lesion and PCNA ring and Pol δ/κ/ɛ are recruited to begin gap filling DNA synthesis (step 7). This triggers XPG to cut 3′ of the lesion to remove a 22–25 region (not to scale) (step 8). NER is completed by Ligase I or III sealing the nick

POST-TRANSLATIONAL MODIFICATIONS PLAY A CRITICAL ROLE DURING NER

Ubiquitylation and deubiquitylation influence NER

Some of the first evidence that ubiquitylation regulated NER was the work of several labs showing that the budding yeast protein, RAD23, helped couple ubiquitylation to proteosome degradation.[7–11] Ubiquitin is a small protein (8.6 kDa) that is conjugated to proteins through the series of steps involving activation (E1), conjugation (E2) and ultimately attachment to the target protein through the action of E3-ubiquitin ligases. Work by Nakatani and coworkers demonstrated that UV-DDB was recognized to work with CUL4A and RBX/ROC1 as an E3-ligase.[12] One puzzling feature of UV-DDB was that while it was apparently required for global NER of UV-induced photoproducts, various types of DNA damage triggered rapid degradation (within 3–4 h) of DDB2/p48, the protein defective in the human sun sensitive syndrome, xeroderma pigmentosum, complementation group E. However, DDB1 levels were unchanged after DNA damage.[13] In the same study the authors reported that DDB2 was induced by p53 after damage, perhaps to replenish the cell of the degraded subunit of UV-DDB. In two key studies published in 2005 by the Tanaka and Sugasawa laboratories, it was shown that DDB2 was important for ubiquitylation of XPC.[14,15] The following year it was shown that UV-DDB-CUL4A/4B-RBX works to ubiquitylate H2A, H3, and H4.[16–18] Wani and colleagues demonstrated that DDB1 works with CUL4A to ubiquitylate DDB2 leading to its degradation through proteolysis, allowing proper XPC recruitment at sites of damage.[19,20] Later Lan et al., showed that monoubiquitylation of H2A Lys119 and Lys120 were necessary and sufficient to destabilize the nucleosome containing a UV-induced photoproduct.[21] Structural work by Thoma’s laboratory has provided elegant structures of UV-DDB bound to nucleosomes[22] and as part of the CUL4A-RBX complex,[23] Figure 2. These studies provide an amazing snapshot of how UV-DDB binds to lesions in the context of chromatin.

FIGURE 2.

FIGURE 2

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Structural model of UV-DDB-CUL4A-RBX bound to a nucleosome containing aUV-photoproduct. (A) Model of human UV-DDB-CUL4A-RBX bound to a 6–4 photoproduct in the context of a nucleosome, built from PDB codes: 4A0K and 6R8Y [22,23]; (B) cyclobutane pyrimidine dimer (CPD), PDB code: 1N4E; and (C) 6–4 pyrimidine-pyrimidone product, (6–4PP), PDB code: 3EI1. The damaged strand is red and the non-damaged strand is black

The Naegeli laboratory revealed that ubiquitylation of XPC by UV-DDB-CUL4A-RBX helped to stabilize XPC at sites of damage in chromatin.[24] Finally, in 2012, it was proved that the deubiquitinating protein USP24 worked to help stabilize DDB2 through a direct interaction.[25] All these studies suggest that proper hand-off of UV induced photoproducts to XPC-RAD23 required ubiquitylation of XPC, and that repair could not proceed properly without removal of DDB2 from the chromatin through polyubiquitylation and subsequent degradation. One question that remained was what protein helped to promote the removal of ubiquitylated DDB2 from sites of DNA damage. A key piece of the puzzling role of ubiquitylation in NER came with the finding that the p97 segregase complex was essential for the faithful removal of pyrimidine dimers. Loss of this protein led to long-lived DDB2 protein and increased genome instability after UV damage.[26] This p97 segregase is ubiquitin-selective and is also known as valosin containing protein (VCP), or Cdc48 in yeast, and had been shown to allow extraction of key proteins during mitosis and helps to remodel stalled replication or transcription complexes, reviewed in.[27]

SUMOylation and PARylation impact protein transactions during NER

In addition to ubiquitylation, several groups have demonstrated that NER proteins are SUMOylated or PARylated. Building on work in yeast illustrating that SUMO played an important role in NER,[28,29] Wani and co-workers showed that XPC could be SUMOylated after UV-damage.[30,31] They further demonstrated that levels of XPC were transiently reduced after UV-damage and that DDB1 and CUL4A were necessary to protect from XPC degradation. This is consistent with an important role of UV-DDB-CUL4A-RBX helping to stabilize XPC at damage sites as mentioned in the previous section. In 2013, a group reported that DDB2 is SUMOylated by a SUMO E3 ligase, PIASy. PIASy belongs to a class of SUMO E3 ligases identified as protein inhibitor of activated STAT1 (PIAS) with four members in mammals: 1,2, x and y. They found that PIASy and XPC interact, and knockdown of PIASy caused cyclobutane pyrimidine dimers (CPD), Figure 2, but not 6–4 photoproducts, to be repaired more slowly. Sugasawa and co-workers mapped SUMO sites in XPC to K81, K89, K183, and K655.[32] Mutating these first three Lys residues to Arg in XPC lead to reduced NER, which could be abrogated by knocking down DDB2. These results lead to a model in that SUMOylation of XPC was required for efficient interaction with DDB2. Mutating these critical Lys residues in XPC blocked efficient ubiquitylation by DDB2 causing a defect in proper lesion hand-off from UV-DDB to XPC. It is interesting to note that the Marteijn laboratory discovered that RNF111, a SUMO-targeted ubiquitin ligase, promoted K63-linked ubiquitylation of SUMOylated XPC only after DNA damage.[33] They further showed that RNF111 was required for efficient XPC release, and subsequent binding of XPG to damaged sites. These results were confirmed by Naegli and coworkers in a separate study and led to a model that UV-DDB-CUL4A ubiquitylation, PIAS SUMOylation, and subsequent RNF111 ubiquitylation were necessary for stabilization of XPC and release of XPC during NER.[34] Finally, Wani and co-workers showed that SUMOylation of DDB2 on K309 was necessary for efficiently recruiting XPC to sites of damage.[35]

Simultaneous with these SUMOylation studies, it was reported that DDB2 is also PARylated, which apparently stabilized UV-DDB at damaged sites and increased DDB2 stability after UV damage.[36] Shah and colleagues went on to reveal that PARP inhibition blocked DDB2 interaction with PARP1 or XPC.[33] These two studies would suggest that PARP1 can stabilize DDB2 after UV treatment and help mediate productive hand-off of UV-damage from UV-DDB to XPC. Finally, this model was extended to suggest that XPC-RAD23B interacts directly with PARP1 in solution and is recruited to PARylated DDB2 at sites of damage.[37] Together these studies suggest that there is a complex dance between UV-DDB and XPC that is carefully chaperoned by ubiquitylation, SUMOylation and PARylation, summarized in Figure 3. Despite these results, one significant limitation in our understanding of NER was why DDB2 is ubiquitylated and destroyed after initial recognition. Another important question is how the initial detection and recruitment of UV-DDB to damage and subsequent hand-off to TFIIH is fully coordinated. The following section details a new and exciting study that helps answer these questions and provides molecular insights into these well-choreographed events.[38]

FIGURE 3.

FIGURE 3

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Post translational modification sites on DDB2 and XPC. (A) Schematic model of DDB2 showing sites of ubiquitylation (green) and PARylation (black). The N-terminal tail (K5, K11, K22, K35, K40) is targeted by CRL4 for ubiquitylation after UV-induced damage and necessary for degradation of DDB2. PARylation or SUMOylation of K309 is necessary for the recruitment of XPC. (B) Model of human DDB2 bound to DNA containing an abasic lesion. The five lysine residues shown in green are sites for ubiquitylation and the PARylation site is shown in black (PDB code 4E54). (C) Schematic model XPC showing sites of SUMOylation (purple) and ubiquitylation (green). SUMOylation of K81, K89, K183, and K655 allow XPC to interact with UV-DDB. Height of the bar is not proportional to the degree of modification

HOW DOES UBIQUITYLATION CONTROL THE HAND-OFF OF UV-INDUCED DNA DAMAGE FROM UV-DDB TO XPC AND TFIIH?

Downstream NER proteins affect DDB2 and XPC, differently

Despite our knowledge of ubiquitylation in NER, a clear mechanistic understanding was lacking. In this recent paper from Ribeiro-Silva et al., at Erasmus University,[38] the authors used state-of-the-art CRIPSR/Cas9 knockout and knock-in approaches to follow tagged proteins that are expressed at endogenous levels. This is a key scientific advance as dysregulated levels of repair proteins can adversely affect specific steps in the repair cascade. In this landmark study the authors first used fluorescence recovery after photobleaching (FRAP) to follow how UV-DDB and XPC tagged with GFP are immobilized after UV-damage.[39,40] They demonstrated that UV-DDB remained longer at UV damaged sites after depletion of the p62 subunit (GTF2H1) of TFIIH. Furthermore, XPG knockdown (KD) caused retention of UV-DDB albeit somewhat reduced to p62 depletion. This is in marked contrast to XPC binding to damage sites. XPG depletion significantly increased XPC binding, whereas p62 depletion greatly reduced XPC binding to DNA damage. These data suggested that TFIIH damage verification strengthened XPC interaction with damaged sites and helps to decrease UV-DDB binding.

Next, the authors used UV-irradiation through filters to produce local UV damage (LUD) and then followed recruitment of DDB2 and XPC by immunofluorescence. They show that inhibition of later stages of NER by suppressing the incision steps through the removal of one of the endonucleases, XPF, leads to longer lived DDB2 and XPC at damaged sites. However, this long-lived DDB2 results in an increase of DDB2 ubiquitylation. Knockdown of p62 of TFIIH further increased accumulation of DDB2 at damaged sites in XPF knockout cells (KO) cells, but also led to increased ubiquitylation and degradation of DDB2. Importantly, the authors demonstrated that cellular degradation of DDB2 was not observed in WT cells treated with LUD. However, this degradation of UV-DDB was triggered when cells lacked either XPF or p62. These results indicated to the authors that DDB2 degradation prevents DDB2 rebinding to lesions, which could alter XPC and TFIIH recruitment. DDB2 immunoprecipitation (IP) followed by western blot analysis for ubiquitin indicated that absence of p62 greatly increased DDB2 ubiquitylation. Together, these data indicate that p62 as part of TFIIH is important for DDB2 dissociation. Finally, our own single-molecule observations demonstrate that UV-DDB can dimerize at sites of damage,[41] sometimes binding two DNA molecules, creating long-lived persistent binding events.[42] Collectively, these data suggest that proper control of transient UV-DDB complexes is essential for coordinated NER.

VCP/p97 displaces ubiquitylated UV-DDB from damage site facilitating a DNA damage handover

The authors further support their working model that DDB2 must be ubiquitylated to be removed from damaged sites through inhibition of VCP/p97, which normally helps extract ubiquitylated DDB2 from chromatin, see “Ubiquitylation and deubiquitylation influence NER”.[26] Addition of the inhibitor caused delayed XPC and XPB accumulation at locally damaged sites. KO of DDB2 also caused slower XPC accumulation and the important control of adding VCPi under these conditions had no further effect. In the final and most important experiment of this study, the authors used a third fluorescence technique, inverse fluorescence recovery after photobleaching (iFRAP). In this approach a 266 nm UV-C laser is first used to create foci of damage to induce the accumulation of GFP-tagged DDB2. Once steady-state levels of recruitment are observed the entire nuclear region is photobleached, except for the damage site and a control non-damaged region. The decay of the fluorescence signal in the damaged site is a direct measure of the DDB2 residence time at UV-induced photoproducts. To test the role of DDB2 ubiquitylation the authors expressed two constructs in a DDB2 KO cell line: an N-terminal 40 amino acid truncated DDB2, and the same truncation construct carrying five additional Lys to Arg variants to block ubiquitylation. These two constructs were shown to ubiquitylate XPC, but because it could not auto-ubiquitylate, DDB2 remained on DNA 30% longer and thus decreased the recruitment of XPC. It is important to point out that inhibition of the 26S proteosome by MG132, while preventing DDB2 degradation, did not prevent VCP removal of ubiquitinylated UV-DDB from chromatin.

In total, these exciting data suggest an intricate cellular timer mechanism that under normal conditions UV-DDB is rapidly recruited to damage sites and helps recruit XPC, then subsequent UV-DDB dissociation is facilitated by TFIIH arrival, allowing UV-DDB to dissociate and move to another damaged site to recruit XPC. Under these conditions DDB2, due to its relatively short-lived time at damage sites, is not highly ubiquitylated and not degraded. This study clearly demonstrates that ubiquitylated XPC is stabilized at damaged sites; if DDB2 persists at damaged sites, either due to high levels of protein causing rebinding or higher affinity for specific types of DNA damage, it is thus turned over by auto-ubiquitylation and degradation. If UV-DDB does not release in a timely manner, TFIIH cannot be properly recruited to damaged sites, Figure 4.

FIGURE 4.

FIGURE 4

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Handover of repair proteins in GG-NER. Upon UV-DDB binding to a lesion and recruiting XPC, CUL4A ubiquitylates DDB2 and XPC. The p62 subunit of TFIIH interacts with and stabilizes XPC, while also displacing DDB2. Ubiquitin-proteasome system (UPS) factors such as VCP and the 26s proteasome further mediates the extraction and degradation of DDB2. The degradation of DDB2 facilitates stable binding of XPC, which allows for the recruitment of the next NER proteins (TFIIH) to complete the pre-incision complex. Figure adapted from Riveiro-Silva, et al., 2020

DO PTMS OF UV-DDB PLAY A ROLE IN BASE EXCISION REPAIR?

We have recently discovered that UV-DDB plays an important role in base excision repair of 8-oxoG moieties.[43] Biochemical studies indicated that UV-DDB binds to 8-oxoG and stimulated the turnover of OGG1 and APE1 at abasic sites some three and eight-fold, respectively. Single molecule studies helped confirm this turnover and revealed transient interactions of UV-DDB with these two BER proteins. Using a unique chemoptogenetic approach, which allows direct production of 8-oxoG in telomeric DNA, we demonstrated that UV-DDB arrival precedes and overlaps with OGG1 suggesting that initial recognition of 8-oxoG in chromatin is facilitated by UV-DDB, since 8-oxoG embedded in nucleosomes are poor substrates for OGG1. It remains to be determined whether UV-DDB is degraded under these conditions and whether UV-DDB-CUL4A-RBX-mediated histone ubiquitylation is important in this process. To this end it is important to point out that Rapic-Otrin found that while DDB2 is first degraded and induced after UV in a p53 dependent manner, DDB2 levels did not go down, but actually rose after ionizing radiation.[13] Finally, since PARP1 is activated after 8oxoG removal by OGG1 and subsequent action of APE1, it will be interesting to investigate whether PARP1 PARylates DDB2 under these conditions, potentially decreasing its own ubiquitylation and degradation.

CONCLUSIONS AND OUTLOOK

This outstanding study has given us important insights into the “reciprocal coordination” of damage detection by UV-DDB and XPC with damage verification by TFIIH. However, this work did not look at the role of XPC SUMOylation or whether SUMO-dependent RNF111 ubiquitylation of XPC was the necessary switch for XPC departure and subsequent XPG binding to TFIIH. Nor did it revisit whether PARP1 activity is important for the efficient hand-off from UV-DDB to XPC to TFIIH. Finally, the nature of how and where UV-DDB is degraded was not fully detailed. It is interesting to note that functional roles of the 26S proteosome in nuclear events including DNA repair, transcription, chromatin structure, and nuclear-quality control have been well documented.[44,45] The MG132 results suggested, but did not prove, that ubiquitylated DDB2 is degraded at the 26S proteosome. Recent work from Sugasawa and coworkers have further investigated the nature of DDB2 degradation,[46] and demonstrated that the proteasomal subunit, PSMD14, working with the ubiquitin-proteosome shuttle protein RAD23B can be recruited to sites of DNA damage, even in the absence of XPC. They further showed that PMSD14 KD causes a decrease in CPD, but not 6–4 photoproduct removal and that MG132 inhibition after UV damage causes proteasomal aggregation and immobilization of DDB2 at sites of nucleoli periphery, compromising further DNA repair. These results also suggest that RAD23B is not a silent partner in this well-choreographed DNA repair dance of damage hand-offs. Finally, one wonders what other chromatin modifications might guide DNA repair proteins to and from sites of damage. To this end Naegeli and co-workers, have found that three key histone methyltransferases, DOT1L, ASH1L, and NSD2, enabled recognition of damage within nucleosomes.[47]

ACKNOWLEDGMENTS

The authors thank Maria Beecher, Sripriya Raja, and Drs. Matt Schaich and Zhou Zhong for careful reading of, and commenting on, this manuscript. This work was supported by the National Institutes of Health, R01ES019566 to B.V.H., R35ES03163801 to B.V.H., T32GM088119 to B.S., and 2P30CA047904 to UPMC Hillman Cancer

Abbreviations:

6–4 PP

6–4 pyrimidine-pyrimidone product, a form of UV photoproduct

CPD

cyclobutane pyrimidine dimers, a form of UV photoproduct

FRAP

fluorescence recovery after photobleaching

GG-NER

global-genome nucleotide excision repair

iFRAP

inverse fluorescence recovery after photobleaching

LUD

local UV damage

MG132

an inhibitor of the 26S proteosome

NER

nucleotide excision repair

OGG1

8-oxoguanine glycosylase

PARP

poly (ADP-ribose) polymerase

PIAS

protein inhibitor of activated STAT1

PTM

post-translational modifications

TC-NER

ranscription-coupled nucleotide excision repair

TFIIH

transcription factor IIH, consisting of p8, p34, p44, p52, p62, XPB, and XPD. XPD

UV-DDB

UV-damaged DNA binding protein, DDB1/DDB2 heterodimer

VCP/p97

valosin containing protein, ubiquitin-selective segregase

XP

xeroderma pigmentosum, complementation groups A-G

XPC

a damage detection protein involved in NER

XPB

a DNA translocase

XPD

a DNA helicase

XPF

a damage specific 5′ endonuclease

XPG

a damage specific 3/ endonuclease

Footnotes

CONFLICT OF INTEREST

The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

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