Iduna is a poly(ADP-ribose) (PAR)-dependent E3 ubiquitin ligase that regulates DNA damage
Ho Chul KangYun-Il LeeJoo‐Ho ShinShaida A. AndrabiZhikai ChiJean-Philippe GagnéYunjong LeeHan Seok KoByoung Dae LeeGuy G. PoirierValina L. DawsonTed M. Dawson
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Ubiquitin mediated protein degradation is crucial for regulation of cell signaling and protein quality control. Poly(ADP-ribose) (PAR) is a cell-signaling molecule that mediates changes in protein function through binding at PAR binding sites. Here we characterize the PAR binding protein, Iduna, and show that it is a PAR-dependent ubiquitin E3 ligase. Iduna’s E3 ligase activity requires PAR binding because point mutations at Y156A and R157A eliminate Iduna’s PAR binding and Iduna’s E3 ligase activity. Iduna’s E3 ligase activity also requires an intact really interesting new gene (RING) domain because Iduna possessing point mutations at either H54A or C60A is devoid of ubiquitination activity. Tandem affinity purification reveals that Iduna binds to a number of proteins that are either PARsylated or bind PAR including PAR polymerase-1, 2 (PARP1, 2), nucleolin, DNA ligase III, KU70, KU86, XRCC1, and histones. PAR binding to Iduna activates its E3 ligase function, and PAR binding is required for Iduna ubiquitination of PARP1, XRCC1, DNA ligase III, and KU70. Iduna’s PAR-dependent ubiquitination of PARP1 targets it for proteasomal degradation. Via PAR binding and ubiquitin E3 ligase activity, Iduna protects against cell death induced by the DNA damaging agent N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and rescues cells from G1 arrest and promotes cell survival after γ-irradiation. Moreover, Iduna facilitates DNA repair by reducing apurinic/apyrimidinic (AP) sites after MNNG exposure and facilitates DNA repair following γ-irradiation as assessed by the comet assay. These results define Iduna as a PAR-dependent E3 ligase that regulates cell survival and DNA repair.Keywords:
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The Brca1 A complex contains Brca1/Bard1, Abraxas, Rap80, and Brcc36; however, with the exception of the Brca1–Abraxas interaction, how the A complex is assembled is not known. The A complex is localized to sites of DNA damage through the UIM domains of RAP80, which bind K63-linked polyubiquitin chains. In this study, we identified an FHA domain RING finger E3 ubiquitin ligase, RNF8, and an E2-conjugating enzyme known to form K63–polyubiquitin chains, Ubc13, each of which is required to recruit the Brca1 A complex to sites of DNA damage. Rnf8 localizes to sites of DNA damage through an FHA-domain-containing region. We found that Rap80 contains an Abraxas interaction domain [AIR (Abraxas-interacting region)], required for association of Rap80 with Abraxas, Brca1, and Brcc36. Abraxas and Brcc36 associate through coiled-coil domains on each protein. These data suggest a model through which Ubc13 and Rnf8 are recruited to sites of DNA damage through DNA-damage-induced phosphorylation of a chromatin-associated protein and generate polyubiquitin chains that then recruit Rap80 and the entire Brca1 A complex to DNA-damage foci. This sequential E3 ubiquitin ligase recruitment constitutes a ubiquitin ligase cascade required for DNA repair and checkpoint signaling.
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AbstractThe ubiquitination of PCNA is an essential event in the operation of the DNA Damage Tolerance (DDT) pathway that is activated after DNA damage caused by UV or chemical agents during S-phase. This pathway allows the bypass of DNA damage by translesion synthesis that would otherwise cause replication fork stalling. PCNA is mono-ubiquitinated by Rad18-Rad6, and polyubiquitinated by Rad5-Ubc13/Uev1 in the DDT pathway. Mono-and polyubiquitination of PCNA are key processes in the translesion bypass and template switching sub-pathways of the DDT. DNA damage by IR causes DSBs, which trigger the DNA Damage Response (DDR). The ubiquitin ligase RNF8 has a critical role in the assembly of BRCA1 complexes at the DSBs in the DDR. We show that RNF8 readily mono-ubiquitinates PCNA in the presence of UbcH5c, and polyubiquitinates PCNA in the added presence of Ubc13/Uev1a. These reactions are the same as those performed by Rad18-Rad6 and Rad5-Ubc13. RNF8 depletion suppressed both UV and MNNG-stimulated mono-ubiquitination of PCNA, revealing that an RNF8-dependent pathway for PCNA ubiquitination is operative in vivo. These findings provide evidence that RNF8, a key E3 ligase in the DDR, may also play a role in the DDT.
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Protein modifications by ubiquitin and small ubiquitin-like modifier (SUMO) play key roles in cellular signaling pathways. SUMO-targeted ubiquitin ligases (STUbLs) directly couple these modifications by selectively recognizing SUMOylated target proteins through SUMO-interacting motifs (SIMs), promoting their K48-linked ubiquitylation and degradation. Only a single mammalian STUbL, RNF4, has been identified. We show that human RNF111/Arkadia is a new STUbL, which used three adjacent SIMs for specific recognition of poly-SUMO2/3 chains, and used Ubc13–Mms2 as a cognate E2 enzyme to promote nonproteolytic, K63-linked ubiquitylation of SUMOylated target proteins. We demonstrate that RNF111 promoted ubiquitylation of SUMOylated XPC (xeroderma pigmentosum C) protein, a central DNA damage recognition factor in nucleotide excision repair (NER) extensively regulated by ultraviolet (UV)-induced SUMOylation and ubiquitylation. Moreover, we show that RNF111 facilitated NER by regulating the recruitment of XPC to UV-damaged DNA. Our findings establish RNF111 as a new STUbL that directly links nonproteolytic ubiquitylation and SUMOylation in the DNA damage response.
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Macroautophagy is a bulk degradation system in which double membrane-bound structures called autophagosomes to deliver cytosolic materials to lysosomes. Autophagy promotes cellular homeostasis by selectively recognizing and sequestering specific targets, such as damaged organelles, protein aggregates, and invading bacteria, termed selective autophagy. We previously reported a type of selective autophagy, lysophagy, which helps clear damaged lysosomes. Damaged lysosomes become ubiquitinated and recruit autophagic machinery. Proteomic studies using transfection reagent-coated beads and further evaluations reveal that a CUL4A-DDB1-WDFY1 E3 ubiquitin ligase complex is essential to initiate lysophagy and clear damaged lysosomes. Moreover, we show that LAMP2 is ubiquitinated by the CUL4A E3 ligase complex as a substrate on damaged lysosomes. These results reveal how cells selectively tag damaged lysosomes to initiate autophagy for the clearance of lysosomes.
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8-Oxoguanine DNA glycosylase (OGG1) is the major cellular enzyme required for the excision of 8-oxoguanine DNA base lesions in DNA through the base excision repair (BER) pathway, and therefore plays a major role in suppressing mutagenesis and in controlling genome stability. However, the mechanism of regulation of cellular OGG1 protein, particularly in response to oxidative stress, is unclear. We have purified the major E3 ubiquitin ligase responsible for OGG1 ubiquitylation from human cell extracts, and identify this as E3 ubiquitin-protein ligase NEDD4-like (NEDD4L). We demonstrate that recombinant NEDD4L stimulates ubiquitylation of OGG1 in vitro, particularly on lysine 341, and that NEDD4L and OGG1 interact in U2OS cells. Depletion of NEDD4L in U2OS cells has no impact on the stability and steady-state protein levels of OGG1, however, OGG1 stability is enhanced in response to oxidative stress induced by ionizing radiation. Furthermore, ubiquitylation of OGG1 by NEDD4L in vitro inhibits its DNA glycosylase/lyase activity. As a consequence of prolonged OGG1 stability and increased excision activity in the absence of NEDD4L, cells display increased DNA repair capacity but conversely that this decreases cell survival post-irradiation. This effect can be reproduced following OGG1 overexpression, suggesting that dysregulation of OGG1 increases the formation of lethal intermediate DNA lesions. Our study therefore highlights the importance of balancing OGG1 protein levels and BER capacity in maintaining genome stability.
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The chemotherapeutic cisplatin is widely used to treat various tumors. By inducing crosslinking of DNA, signaling and repair pathways are activated which are referred to as the DNA damage response (DDR). However, the cellular and molecular mechanisms of cisplatin treatment are incompletely understood. We set up a study to find new regulators in the DDR to cisplatin. Since ubiquitination plays a major role in the DDR, we applied a high-content siRNA screen targeting 327 human ubiquitin ligases and 92 deubiquitinating enzymes in U2OS cells. We detected phosphorylation of the histone variant H2AX (yielding γH2AX), a marker for DNA damage. Knockdown of one of the candidates, the ubiquitin ligase G2E3, led to decrease in γH2AX levels. G2E3 had previously been proposed to play a role in the DDR and in cell survival. However, little was known about the underlying mechanisms. In the work presented here, we show that G2E3 is a DNA damage-responsive, cell cycle-dependent survival factor.
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UV-damaged-DNA-binding protein (UV-DDB) is a heterodimer comprised of DDB1 and DDB2 and integrated in a complex that includes a ubiquitin ligase component, cullin 4A, and Roc1. Here we show that the ubiquitin ligase activity of the DDB2 complex is required for efficient global genome nucleotide excision repair (GG-NER) in chromatin. Mutant DDB2 proteins derived from xeroderma pigmentosum group E patients are not able to mediate ubiquitylation around damaged sites in chromatin. We also found that CSN, a negative regulator of cullin-based ubiquitin ligases, dissociates from the DDB2 complex when the complex binds to damaged DNA and that XPC and Ku oppositely regulate the ubiquitin ligase activity, especially around damaged sites. Furthermore, the DDB2 complex-mediated ubiquitylation plays a role in recruiting XPA to damaged sites. These findings shed some light on the early stages of GG-NER.
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