A small ubiquitin binding domain inhibits ubiquitin-dependent protein recruitment to DNA repair foci

The rapid ubiquitination of chromatin surrounding DNA double-stranded breaks (DSB) drives the formation of large structures called ionizing radiation-induced foci (IRIF), comprising many DNA damage response (DDR) proteins. This process is regulated by RNF8 and RNF168 ubiquitin ligases and is thought to be necessary for DNA repair and activation of signaling pathways involved in regulating cell cycle checkpoints. Here we demonstrate that it is possible to interfere with ubiquitin-dependent recruitment of DDR factors by expressing proteins containing ubiquitin binding domains (UBDs) that bind to lysine 63-linked polyubiquitin chains. Expression of the E3 ubiquitin ligase RAD18 prevented chromatin spreading of 53BP1 at DSBs, and this phenomenon was dependent upon the integrity of the RAD18 UBD. An isolated RAD18 UBD interfered with 53BP1 chromatin spreading, as well as other important DDR mediators, including RAP80 and the BRCA1 tumor suppressor protein, consistent with the model that the RAD18 UBD is blocking access of proteins to ubiquitinated chromatin. Using the RAD18 UBD as a tool to impede localization of 53BP1 and BRCA1 to repair foci, we found that DDR signaling, DNA DSB repair, and radiosensitivity were unaffected. We did find that activated ATM (S1981P) and phosphorylated SMC1 (a specific target of ATM) were not detectable in DNA repair foci, in addition to upregulated homologous recombination repair, revealing 2 DDR responses that are dependent upon chromatin spreading of certain DDR factors at DSBs. These data demonstrate that select UBDs containing targeting motifs may be useful probes in determining the biological significance of protein–ubiquitin interactions.     

An RNF168 fragment defective for focal accumulation at DNA damage is proficient for inhibition of homologous recombination in BRCA1 deficient cells

The E3 ubiquitin ligase RNF168 is a DNA damage response (DDR) factor that promotes monoubiquitination of H2A/H2AX at K13/15, facilitates recruitment of other DDR factors (e.g. 53BP1) to DNA damage, and inhibits homologous recombination (HR) in cells deficient in the tumor suppressor BRCA1. We have examined the domains of RNF168 important for these DDR events, including chromosomal HR that is induced by several nucleases (I-SceI, CAS9-WT and CAS9-D10A), since the inducing nuclease affects the relative frequency of distinct repair outcomes. We found that an N-terminal fragment of RNF168 (1-220/N221*) efficiently inhibits HR induced by each of these nucleases in BRCA1 depleted cells, and promotes recruitment of 53BP1 to DNA damage and H2AX monoubiquitination at K13/15. Each of these DDR events requires a charged residue in RNF168 (R57). Notably, RNF168-N221* fails to self-accumulate into ionizing radiation induced foci (IRIF). Furthermore, expression of RNF168 WT and N221* can significantly bypass the role of another E3 ubiquitin ligase, RNF8, for inhibition of HR in BRCA1 depleted cells, and for promotion of 53BP1 IRIF. We suggest that the ability for RNF168 to promote H2A/H2AX monoubiquitination and 53BP1 IRIF, but not RNF168 self-accumulation into IRIF, is important for inhibition of HR in BRCA1 deficient cells.     

Tandem protein interaction modules organize the ubiquitin-dependent response to DNA double-strand breaks

The response to DNA double-strand breaks (DSBs) entails the hierarchical recruitment of proteins orchestrated by ATM-dependent phosphorylation and RNF8-mediated chromatin ubiquitylation. As in most ubiquitin-dependent processes, the ordered accumulation of DNA repair factors at the break site relies on ubiquitin-binding domains (UBDs). However, how UBDs select their ligands is poorly understood, and therefore we sought to uncover the basis for selectivity in the ubiquitin-dependent DSB response. We show that RNF168, its paralog RNF169, RAD18, and the BRCA1-interacting RAP80 protein accumulate at DSB sites through the use of bipartite modules composed of UBDs juxtaposed to peptide motifs that provide specificity. These sequences, named LR motifs (LRMs), are transferable, and we show that the RNF169 LRM2 binds to nucleosomes, the substrates of RNF168. The LRM-based selection of ligands is a parsimonious means to build a highly discrete ubiquitin-based signaling pathway such as the DNA damage response.     

Nucleosome Acidic Patch Promotes RNF168- and RING1B/BMI1-Dependent H2AX and H2A Ubiquitination and DNA Damage Signaling

Histone ubiquitinations are critical for the activation of the DNA damage response (DDR). In particular, RNF168 and RING1B/BMI1 function in the DDR by ubiquitinating H2A/H2AX on Lys-13/15 and Lys-118/119, respectively. However, it remains to be defined how the ubiquitin pathway engages chromatin to provide regulation of ubiquitin targeting of specific histone residues. Here we identify the nucleosome acid patch as a critical chromatin mediator of H2A/H2AX ubiquitination (ub). The acidic patch is required for RNF168- and RING1B/BMI1-dependent H2A/H2AXub in vivo. The acidic patch functions within the nucleosome as nucleosomes containing a mutated acidic patch exhibit defective H2A/H2AXub by RNF168 and RING1B/BMI1 in vitro. Furthermore, direct perturbation of the nucleosome acidic patch in vivo by the expression of an engineered acidic patch interacting viral peptide, LANA, results in defective H2AXub and RNF168-dependent DNA damage responses including 53BP1 and BRCA1 recruitment to DNA damage. The acidic patch therefore is a critical nucleosome feature that may serve as a scaffold to integrate multiple ubiquitin signals on chromatin to compose selective ubiquitinations on histones for DNA damage signaling.     

RNF126 Quenches RNF168 Function in the DNA Damage Response

DNA damage response (DDR) is essential for maintaining genome stability and protecting cells from tumorigenesis. Ubiquitin and ubiquitin-like modifications play an important role in DDR, from signaling DNA damage to mediating DNA repair. In this report, we found that the E3 ligase ring finger protein 126 (RNF126) was recruited to UV laser micro-irradiation-induced stripes in a RNF8-dependent manner. RNF126 directly interacted with and ubiquitinated another E3 ligase, RNF168. Overexpression of wild type RNF126, but not catalytically-inactive mutant RNF126 (CC229/232AA), diminished ubiquitination of H2A histone family member X (H2AX), and subsequent bleomycin-induced focus formation of total ubiquitin FK2, TP53-binding protein 1 (53BP1), and receptor-associated protein 80 (RAP80). Interestingly, both RNF126 overexpression and RNF126 downregulation compromised homologous recombination (HR)-mediated repair of DNA double-strand breaks (DSBs). Taken together, our findings demonstrate that RNF126 negatively regulates RNF168 function in DDR and its appropriate cellular expression levels are essential for HR-mediated DSB repair.     

USP14 regulates DNA damage repair by targeting RNF168-dependent ubiquitination

Recent reports have made important revelations, uncovering direct regulation of DNA damage response (DDR)-associated proteins and chromatin ubiquitination (Ubn) by macroautophagy/autophagy. Here, we report a previously unexplored connection between autophagy and DDR, via a deubiquitnase (DUB), USP14. Loss of autophagy in prostate cancer cells led to unrepaired DNA double-strand breaks (DSBs) as indicated by persistent ionizing radiation (IR)-induced foci (IRIF) formation for γH2AFX, and decreased protein levels and IRIF formation for RNF168, an E3-ubiquitin ligase essential for chromatin Ubn and recruitment of critical DDR effector proteins in response to DSBs, including TP53BP1. Consistently, RNF168-associated Ubn signaling and TP53BP1 IRIF formation were reduced in autophagy-deficient cells. An activity assay identified several DUBs, including USP14, which showed higher activity in autophagy-deficient cells. Importantly, inhibiting USP14 could overcome DDR defects in autophagy-deficient cells. USP14 IRIF formation and protein stability were increased in autophagy-deficient cells. Co-immunoprecipitation and colocalization of USP14 with MAP1LC3B and the UBA-domain of SQSTM1 identified USP14 as a substrate of autophagy and SQSTM1. Additionally, USP14 directly interacted with RNF168, which depended on the MIU1 domain of RNF168. These findings identify USP14 as a novel substrate of autophagy and regulation of RNF168-dependent Ubn and TP53BP1 recruitment by USP14 as a critical link between DDR and autophagy. Given the role of Ubn signaling in non-homologous end joining (NHEJ), the major pathway for repair of IR-induced DNA damage, these findings provide unique insights into the link between autophagy, DDR-associated Ubn signaling and NHEJ DNA repair.

Abbreviations: ATG7: autophagy related 7; CQ: chloroquine; DDR: DNA damage response; DUB: deubiquitinase; HR: homologous recombination; IR: ionizing radiation; IRIF: ionizing radiation-induced foci; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MIU1: motif interacting with ubiquitin; NHEJ: non homologous end-joining; PCa: prostate cancer; TP53BP1/53BP1: tumor protein p53 binding protein 1; RNF168: ring finger protein 168; SQSTM1/p62 sequestosome 1; γH2AFX/γH2AX: H2A histone family member X: phosphorylated, UBA: ubiquitin-associated; Ub: ubiquitin; Ubn: ubiquitination; USP14: ubiquitin specific peptidase 14.     

RNF8 and RNF168 but not HERC2 are required for DNA damage-induced ubiquitylation in chicken DT40 cells

The ubiquitylation cascade plays an important role in the recruitment of repair factors at DNA double-strand breaks. The involvement of a growing number of ubiquitin E3 ligases adds to the complexity of the DNA damage-induced ubiquitin signaling. Here we use the genetically tractable avian cell line DT40 to investigate the role of HERC2, RNF8 and RNF168 in the DNA damage-induced ubiquitylation pathway. We show that formation of ubiquitin foci as well as cell survival after DNA damage depends on both RNF8 and RNF168. However, we find that RNF8 and RNF168 knockout cell lines respond differently to treatment with camptothecin indicating that they do not function in a strictly linear manner. Surprisingly, we show that HERC2 is required neither for survival nor for ubiquitin foci formation after DNA damage in DT40. Moreover, the E3 ubiquitin ligase activity of HERC2 is not redundant to that of RNF8 or RNF168.     

Crystal structure of the ubiquitin binding domains of rabex-5 reveals two modes of interaction with ubiquitin

The interaction between ubiquitinated proteins and intracellular proteins harboring ubiquitin binding domains (UBDs) is critical to a multitude of cellular processes. Here, we report that Rabex-5, a guanine nucleotide exchange factor for Rab5, binds to Ub through two independent UBDs. These UBDs determine a number of properties of Rabex-5, including its coupled monoubiquitination and interaction in vivo with ubiquitinated EGFRs. Structural and biochemical characterization of the UBDs of Rabex-5 revealed that one of them (MIU, motif interacting with ubiquitin) binds to Ub with modes superimposable to those of the UIM (ubiquitin-interacting motif):Ub interaction, although in the opposite orientation. The other UBD, RUZ (Rabex-5 ubiquitin binding zinc finger) binds to a surface of Ub centered on Asp58(Ub) and distinct from the "canonical" Ile44(Ub)-based surface. The two binding surfaces allow Ub to interact simultaneously with different UBDs, thus opening new perspectives in Ub-mediated signaling.     

Give me a break,but not in mitosis

DNA double-strand breaks (DSBs) are extremely cytotoxic with a single unrepaired DSB being sufficient to induce cell death. A complex signalling cascade, termed the DNA damage response (DDR), is in place to deal with such DNA lesions and maintain genome stability. Recent work by us and others has found that the signalling cascade activated by DSBs in mitosis is truncated, displaying apical, but not downstream, components of the DDR. The E3 Ubiquitin ligases RNF8, RNF168 and BRCA1, along with the DDR mediator 53BP1, are not recruited to DSB sites in mitosis, and activation of downstream checkpoint kinases is also impaired. Here, we show that RNF8 and RNF168 are recruited to DNA damage foci in late mitosis, presumably to prime sites for 53BP1 recruitment in early G1. Interestingly, we show that, although RNF8, RNF168 and 53BP1 are excluded from DSB sites during most of mitosis, they associate with mitotic structures such as the kinetochore, suggesting roles for these DDR factors during mitotic cell division. We discuss these and other recent findings and suggest how these novel data collectively contribute to our understanding of mitosis and how cells deal with DNA damage during this crucial cell cycle stage.     

RNF4 interacts with both SUMO and nucleosomes to promote the DNA damage response

The post‐translational modification of DNA repair and checkpoint proteins by ubiquitin and small ubiquitin‐like modifier (SUMO) critically orchestrates the DNA damage response (DDR). The ubiquitin ligase RNF4 integrates signaling by SUMO and ubiquitin, through its selective recognition and ubiquitination of SUMO‐modified proteins. Here, we define a key new determinant for target discrimination by RNF4, in addition to interaction with SUMO. We identify a nucleosome‐targeting motif within the RNF4 RING domain that can bind DNA and thereby enables RNF4 to selectively ubiquitinate nucleosomal histones. Furthermore, RNF4 nucleosome‐targeting is crucially required for the repair of TRF2‐depleted dysfunctional telomeres by 53BP1‐mediated non‐homologous end joining.     

Structural basis for role of ring finger protein RNF168 RING domain

Ubiquitin adducts surrounding DNA double-strand breaks (DSBs) have emerged as molecular platforms important for the assembly of DNA damage mediator and repair proteins. Central to these chromatin modifications lies the E2 UBC13, which has been implicated in a bipartite role in priming and amplifying lys63-linked ubiquitin chains on histone molecules through coupling with the E3 RNF8 and RNF168. However, unlike the RNF8-UBC13 holoenyzme, exactly how RNF168 work in concert with UBC13 remains obscure. To provide a structural perspective for the RNF168-UBC13 complex, we solved the crystal structure of the RNF168 RING domain. Interestingly, while the RNF168 RING adopts a typical RING finger fold with two zinc ions coordinated by several conserved cystine and histine residues arranged in a C3HC4 “cross-brace” manner, structural superimposition of RNF168 RING with other UBC13-binding E3 ubiquitin ligases revealed substantial differences at its corresponding UBC13-binding interface. Consistently, and in stark contrast to that between RNF8 and UBC13, RNF168 did not stably associate with UBC13 in vitro or in vivo. Moreover, domain-swapping experiments indicated that the RNF8 and RNF168 RING domains are not functionally interchangeable. We propose that RNF8 and RNF168 operate in different modes with their cognate E2 UBC13 at DSBs.     

Give me a break,but not in mitosis: The mitotic DNA damage response marks DNA double strand breaks with early signaling events

DNA double-strand breaks (DSBs) are extremely cytotoxic lesions with a single unrepaired DSB being sufficient to induce cell death. A complex signaling cascade, termed the DNA damage response (DDR), is in place to deal with such DNA lesions and maintain genome stability. Recent work by us and others has found that the signaling cascade activated by DSBs in mitosis is truncated, displaying apical, but not downstream, components of the DDR. The E3 Ubiquitin ligases RNF8, RNF168 and BRCA1, along with the DDR mediator 53BP1, are not recruited to DSB sites in mitosis, and activation of downstream checkpoint kinases is also impaired. Here, we show that RNF8 and RNF168 are recruited to DNA damage foci in late mitosis, presumably to prime sites for 53BP1 recruitment in early G₁. Interestingly, we show that, although RNF8, RNF168 and 53BP1 are excluded from DSB sites during most of mitosis, they associate with mitotic structures such as the kinetochore, suggesting roles for these DDR factors during mitotic cell division. We discuss these and other recent findings and suggest how these novel data collectively contribute to our understanding of mitosis and how cells deal with DNA damage during this crucial cell cycle stage.Key words: mitosis, DNA damage response, DNA double-strand breaks, signaling cascade, chromatin     

Close encounters of the RNF8th kind: when chromatin meets DNA repair

Cells counteract the adverse effects of chromosome breakage by activating the DNA damage response (DDR), which entails a coordinated series of events that regulate cell cycle progression and repair of DNA lesions. The packaging of genomic DNA into condensed, often inaccessible chromatin severely complicates efficient DNA damage repair in living cells. Recent studies implicate a large number of chromatin-modifying enzymes in the DDR, suggesting a stepwise model in which chromatin is continually reconfigured to accommodate the association and action of repair factors during the different stages of the DDR. Emerging evidence suggests that the histone ubiquitin ligases RNF8/RNF168 act in concert with ATP-dependent chromatin remodelling enzymes to orchestrate the signalling and repair of DNA lesions in specific chromatin topologies.     

Ring Finger Protein RNF169 Antagonizes the Ubiquitin-dependent Signaling Cascade at Sites of DNA Damage

Ubiquitin signals emanating from DNA double-strand breaks (DSBs) trigger the ordered assembly of DNA damage mediator and repair proteins. This highly orchestrated process is accomplished, in part, through the concerted action of the RNF8 and RNF168 E3 ligases, which have emerged as core signaling intermediates that promote DSB-associated ubiquitylation events. In this study, we report the identification of RNF169 as a negative regulator of the DNA damage signaling cascade. We found that RNF169 interacted with ubiquitin structures and relocalized to DSBs in an RNF8/RNF168-dependent manner. Moreover, ectopic expression of RNF169 attenuated ubiquitin signaling and compromised 53BP1 accumulation at DNA damage sites, suggesting that RNF169 antagonizes RNF168 functions at DSBs. Our study unveils RNF169 as a component in DNA damage signal transduction and adds to the complexity of regulatory ubiquitylation in genome stability maintenance.     

USP3 counteracts RNF168 via deubiquitinating H2A and γH2AX at lysine 13 and 15

Histone ubiquitination plays a vital role in DNA damage response (DDR), which is important for maintaining genomic integrity in eukaryotic cells. In DDR, ubiquitination of histone H2A and γH2AX by the concerted action of ubiquitin (Ub) ligases, RNF168 and RNF8, generates a cascade of ubiquitination signaling. However, little is known about deubiquitinating enzymes (DUBs) that may catalyze the removal of Ub from these histones. This study demonstrated that USP3, an apparent DUB for mono-ubiquitinated H2A, is indeed the enzyme for deubiquitinating Ub conjugates of γH2AX and H2A from lysine sites, where the ubiquitination is initiated by RNF168. Here, we showed that ectopic expression of USP3 led to the deubiquitination of both H2A and γH2AX in response to UV-induced DNA damage. Moreover, ectopic USP3 expression abrogated FK2 antibody-reactive Ub-conjugate foci, which co-localize with damage-induced γH2AX foci. In addition, USP3 overexpression impaired the accumulation of downstream repair factors BRCA1 and 53BP1 at the damage sites in response to both UV and γ-irradiation. We further identified that the USP3 removes Ub at lysine 13 and 15 of H2A and γH2AX, as well as lysine 118 and 119 of H2AX in response to DNA damage. Taken together, the results suggested that USP3 is a negative regulator of ubiquitination signaling, counteracting RNF168- and RNF8-mediated ubiquitination.     

DNA damage signaling in response to double-strand breaks during mitosis

The signaling cascade initiated in response to DNA double-strand breaks (DSBs) has been extensively investigated in interphase cells. Here, we show that mitotic cells treated with DSB-inducing agents activate a “primary” DNA damage response (DDR) comprised of early signaling events, including activation of the protein kinases ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK), histone H2AX phosphorylation together with recruitment of mediator of DNA damage checkpoint 1 (MDC1), and the Mre11–Rad50–Nbs1 (MRN) complex to damage sites. However, mitotic cells display no detectable recruitment of the E3 ubiquitin ligases RNF8 and RNF168, or accumulation of 53BP1 and BRCA1, at DSB sites. Accordingly, we found that DNA-damage signaling is attenuated in mitotic cells, with full DDR activation only ensuing when a DSB-containing mitotic cell enters G1. Finally, we present data suggesting that induction of a primary DDR in mitosis is important because transient inactivation of ATM and DNA-PK renders mitotic cells hypersensitive to DSB-inducing agents.