J. Radiat. Res., 51, 503–509 (2010) Review

Functions and Regulation of Artemis: A Goddess in the Maintenance of Genome Integrity

Aya KUROSAWA1* and Noritaka ADACHI1,2†

Artemis/DNA double-strand break/Non-homologous end-joining. Artemis is a structure-specific endonuclease when associated with and phosphorylated by DNA- dependent kinase catalytic subunit. This structure-specific endonuclease is responsible for the resolution of hairpin coding ends in V(D)J recombination. In DNA double-strand break repair, Artemis is implicated in the end-processing step of the non-homologous end-joining (NHEJ) pathway. Recently, we have demonstrated that the involvement of Artemis in NHEJ depends on the type of DNA damage. Inter- estingly, recent evidence suggests that the end-processing activity is not the only function of Artemis. Indeed, Artemis is rapidly phosphorylated by ataxia telangiectasia mutated in response to DNA damage, and such phosphorylation of Artemis appears to be involved in the regulation of cell cycle checkpoints. These findings suggest that Artemis is a multifunctional protein participating in the maintenance of genome integrity at two distinct levels; one at the end processing step of NHEJ, and the other at the signaling pathway of cell cycle regulation. Therefore, understanding Artemis function may give us profound insights into the DNA repair network. In this review, we summarize the functions and regulation of Artemis.

ing radiation (IR) than are HR-deficient cells. Instead, as HR DNA DOUBLE-STRAND BREAK REPAIR plays a major role at the replication fork, HR-deficient cells exhibit increased sensitivity to replication-associated, one- DNA double-strand breaks (DSBs), which can be caused ended DSBs that arise from replication fork collapse. It by a variety of exogenous and endogenous agents, pose a should also be noted that NHEJ is also important for V(D)J major threat to genome integrity and may cause cell death if recombination, which generates the diversity of left unrepaired.1,2) Eukaryotic cells have evolved two major and T cell receptor molecules.14) The NHEJ reaction can be pathways for repairing DSBs: homologous recombination divided into three steps; (1) end binding, (2) end processing, (HR) and non-homologous DNA end-joining (NHEJ).2–5) and (3) ligation. In the end-binding step, the Ku complex DNA repair by the HR pathway is restricted to the late S and (the heterodimer of Ku70 and Ku80) immediately binds to G2 phases of the cell cycle, while the NHEJ pathway is not DSB ends. After the Ku complex binding to DSB ends, restricted to a particular phase in the cell cycle and hence DNA-dependent protein kinase catalytic subunit (DNA- DSBs can be repaired via NHEJ throughout the cell cycle.6) PKcs) is recruited to DSB ends. The Ku/DNA-PKcs com- Consistent with the roles of the HR and NHEJ pathways for plex is thought to participate in end bridging during NHEJ DSB repair, cells deficient in HR or NHEJ show to protect the ends from nucleases as well as to facilitate end increased sensitivity to DSB-causing agents.6–13) In higher processing reactions.15,16) The end processing step is partic- eukaryotes, DSBs are mainly repaired by NHEJ,14) and thus ularly important when DSBs contain unligatable ends, such NHEJ-deficient cells are in general more sensitive to ioniz- as incompatible ends and chemically modified ends, because all DNA ligases, including DNA ligase IV, catalyze the for- mation of a phosphodiester bond between 5’-phosphate and *Corresponding author: Phone: +81-45-787-2228, 17) Fax: +81-45-787-2228, 3’-hydroxyl termini. The end processing step relies on E-mail: [email protected] several enzymes, including nuclease and polynucleotide †Corresponding author: Phone: +81-45-787-2228, kinase, to generate DNA ends suitable for ligation reac- Fax: +81-45-787-2228, tion.18,19) Finally, the ligatable ends are rejoined by DNA E-mail: [email protected] 1 ligase IV. Although higher eukaryotes have three different Graduate School of Nanobioscience, Yokohama City University, LIG1 LIG3 LIG4 Yokohama, Japan; 2Advanced Medical Research Center, Yokohama City that code for DNA ligase ( , , and ), the University, Yokohama, Japan. LIG4 product DNA ligase IV is the only DNA ligase doi:10.1269/jrr.10017 that can ligate DSB ends in the NHEJ reaction, and other 504 A. Kurosawa and N. Adachi

DNA ligases cannot substitute for the DNA ligase IV func- ARTEMIS SNM1 PSO2). This domain is highly conserved tion.7) in other metallo-β-lactamases that specifically act on nucleic ARTEMIS (named after the Hellenic goddess of the hunt acids (Fig. 2).31) Recently, de Villartay et al. have reported who aids in childbirth) was identified as the gene responsible that a histidine residue within theβ-CASP domain (His254) for radiosensitive-severe combined immunodeficiency (RS- is critical for full activation of Artemis (Fig. 2).32) Although SCID) or Athabascan SCID (SCIDA).20–23) In vitro and in the precise role of His254 is currently unclear, it is suggested vivo studies have revealed that Artemis is the nuclease that His254 is involved in zinc binding.32) Pannicke et al. required for the resolution of hairpin coding ends during have reported that aspartic acid residue 37 and histidine res- V(D)J recombination.24,25) As mentioned above, Artemis- idues 33, 35, 38, 115, and 319 directly coordinate two pro- deficient cells display increased IR sensitivity, suggesting posed sites of metal (most likely Mg2+) binding (Fig. 2).33) that Artemis is also required for the NHEJ pathway of DSB While the N-terminus of Artemis is important for an enzy- repair.20) Interestingly, recent work suggests that Artemis matic role, the C-terminus of Artemis appears to be a region may be involved in the regulation of the cell cycle check- involved in the interaction with DNA-PKcs.34,35) Indeed, points as a downstream factor of ataxia telangiectasia Artemis is phosphorylated by DNA-PKcs only in the C- mutated (ATM) and/or the ATM- and Rad3-related kinase terminal domain.35) Because the C-terminal domain is (ATR).26–30) It is therefore possible that Artemis is a multi- dispensable for hairpin opening activity in V(D)J recombi- functional protein in the maintenance of genome integrity nation in vivo,35,36) the phosphorylation of the C-terminus and an important protein for understanding the mechanism may cause a conformational change, resulting in an activated of DSB repair. In this review, we summarize the recent form of Artemis.35) On the other hand, Goodarzi et al. progress on biological functions of Artemis in DSB repair reported that autophosphorylated DNA-PKcs recruits and DNA damage response. Artemis to the sites of DSBs.37) As autophosphorylation of

BIOCHEMICAL AND STRUCTURAL PROPERTIES OF ARTEMIS

Artemis has a ssDNA-specific 5’ to 3’ exonuclease act- ivity and acquires an endonuclease activity when associated with and phosphorylated by DNA-PKcs.25) The Artemis/ DNA-PKcs complex specifically cleaves boundary of ssDNA and dsDNA and hairpin DNA, generating blunt or 3’ overhang DNA ends (Fig. 1).25) Although the three-dimen- sional structure has not been determined yet, two domains in the N-terminus of Artemis are shown to be important for enzymatic activity.31) One of the domains is called the metallo-β-lactamase domain, amino acids 1–155 of human Artemis, which is commonly observed in members of the Fig. 1. Endonucleolytic properties of the Artemis/DNA-PKcs metallo-β-lactamase superfamily (Fig. 2).31) Another domain, complex. Shown are chematic structures of a hairpin end and DNA amino acids 156–385 of human Artemis, is called the β- ends with a 3’- or 5’-overhang. Arrows mark the major cleavage CASP domain (metallo-β-lactamases-associated CPSF sites.

Fig. 2. Schematic representation of human Artemis. Human Artemis consists of 692 amino acids. The metallo-β-lactamase/β-CASP domain contains the active site of the enzyme. The C-terminus of this protein is thought to be a regulatory domain. The positions of amino acid residues directly involved in metal ion- binding (Asp37, His33, His35, His38, His115, and His319), playing a key role in the Artemis activity (His254), and phosphorylated in response to DNA damage (Ser516 and Ser645) are indicated. Functions and Regulation of Artemis 505

Fig. 4. Artemis is important for repairing low-dose X-ray- induced DSBs in human lymphocytes. Sensitivities of Nalm-6 wild-type, ARTEMIS–/– cells and LIG4–/– cells to X-rays were deter- mined by clonogenic survival assays. Shown are the mean ± SD of Fig. 3. Regulation of Artemis in NHEJ. When Artemis is associ- three independent experiments. Where absent, error bars fall within ated with and phosphorylated by DNA-PKcs, a conformational symbols. change occurs in Artemis, resulting in an activated form of Artemis. The activated form of Artemis can create DNA ends suit- able for ligation by DNA ligase IV. formed targeted disruption of the ARTEMIS gene in the human pre- line Nalm-6, and found that ARTEMIS–/– cells showed increased sensitivity to low-dose IR (Fig. 4), DNA-PKcs is also suggested to cause a conformational but not to cisplatin.42) These findings are consistent with the change in DNA-PKcs, the conformational change of DNA- notion that Artemis is not involved in ICL repair, but is PKcs rather than Artemis itself may be important for involved in DSB repair by the NHEJ pathway. Indeed, Artemis activity. Taken together, Artemis protein is divided human ARTEMIS–/– cells displayed increased sensitivity to into two functional domains, the N-terminal catalytic etoposide, a potent topoisomerase II inhibitor that causes domain and the C-terminal regulatory domain, and the reg- DSBs. Importantly, however, the extent of etoposide sensi- ulation of endonuclease activity of Artemis may contribute tivity of Artemis-deficient cells was much smaller than that to preventing unnecessary DNA degradation (Fig. 3).35) of cells lacking DNA ligase IV,42,43) suggesting a limited role for Artemis in DSB repair by NHEJ. Intriguingly, Riballo et ARTEMIS AND ITS RELATED PROTEINS al. have reported that Artemis is only required for the repair of a subset (15%) of IR-induced DSBs, specifically those Artemis is a member of the SNM1 family, which is con- repaired with slow kinetics and those located at regions of stituted by gene products homologous to yeast SNM1, and .27,44) These findings provide important clues thus Artemis is often referred to as SNM1C. Yeast SNM1 to Artemis function, as it is suggested that the Artemis- possesses a 5’ to 3’ exonuclease activity, depending on its dependent DSB repair is ATM dependent (see below). catalytic domain.38) Genetic analysis has shown that SNM1 Another member of the SNM1 family, SNM1B, also pos- participates in the repair of interstrand cross-links (ICLs) sess a 5’ to 3’ exonuclease activity.45,46) SNM1B is referred and that exonuclease activity of SNM1 is important for the to as Apollo, as SNM1B is closely related to Artemis; Apollo ICL repair.38) Interestingly, however, Artemis exonuclease is the twin brother of Artemis in Hellenic mythology.45,46) activity is independent of its catalytic domain,33) implying Despite structural similarities between Apollo and Artemis, that exonuclease activities of Artemis and SNM1 are func- it has been reported that Snm1b-deficient DT40 cells, unlike tionally unrelated. Consistent with this, fibroblast cell lines Snm1c-deficient cells, display increased sensitivity to ICL- established from RS-SCID/SCIDA patients do not exhibit generating agents, but not to IR, suggesting that Apollo is increased sensitivity to ICL-causing agents, though these involved in ICL repair and not in DSB repair.41) It is shown cells are hypersensitive to IR.39,40) Similarly, in chicken in HEK293 cells that shRNA-mediated knockdown of DT40 cells, SNM1 is involved in ICL repair and not in DSB Apollo results in increased sensitivity to ICL-generating repair, while Snm1c-deficient cells are sensitive to IR but not agents.47) Interestingly, however, exonuclease activity of to the ICL-causing agent cisplatin.41) Recently, we per- Apollo appears to be dispensable for repairing ICLs. Since 506 A. Kurosawa and N. Adachi

Apollo interacts with the human telomeric protein TRF2 via complex in an ATM-dependent manner.28,29) Remarkably, its N-terminal domain, Apollo may act to protect telomeres Chen et al. revealed that serine residue 645 (Ser645) of from unwanted DNA repair.45,46) Thus, unlike Artemis, Artemis is phosphorylated in response to IR irradiation (Fig. SNM1 and Apollo are likely to be mainly involved in ICL 2).29) Several groups have investigated the relationship repair. between Artemis phosphorylation and cell cycle progres- sion. Jeggo and coworkers reported that Artemis-deficient BIOCHEMICAL ROLE OF ARTEMIS IN NHEJ cells had normal G2/M checkpoint and thus exhibited a pro- longed G2/M arrest after IR irradiation; by contrast, Leger- As mentioned above, genetic analysis indicates the ski and coworkers presented data showing that Artemis was involvement of Artemis in NHEJ.41,42) In vitro studies required for normal G2/M arrest after IR irradiation.28,60,61) showed that the Artemis/DNA-PKcs complex can generate A possible explanation for this discrepancy could be that either blunt ends or 3’ overhangs of 2–4 bases.25) Therefore, those studies employed different cell lines (primary fibro- Artemis is believed to be involved in the end processing step blasts derived from SCIDA patients versus transformed 293 of NHEJ. As DNA ligase IV can ligate incompatible DNA kidney cells depleted for Artemis). Alternatively, the dis- ends with 3’ overhangs in the presence of Ku,48) Artemis crepancy may simply be due to the difference in cell cycle may have a role in NHEJ only when end trimming is phases when cells were irradiated in those studies (asynchro- necessary prior to ligation. For example, the Artemis/DNA- nous cells versus S phase-enriched cells). PKcs complex may remove chemically modified termini. The Legerski group also showed that Artemis is phospho- Consistent with this idea, biochemical analysis revealed that the Artemis/DNA-PKcs complex can convert such chemically modified ends to a form suitable for ligation with minimal loss of terminal sequence.49,50) Such chemically modified DSBs are often induced by IR and other radiomimetic agents.51) It is known that IR and other free radicals induce DSBs with either 3’-phosphate or 3’- phosphoglycolate termini,52–55) while radiomimetic enediyne antibiotics, such as neocarzinostatin, induce 5’-aldehyde termini.54,55) Thus, the end processing step mediated by an endonuclease activity of the Artemis/DNA-PKcs complex may be required for removing chemically modified termini. Recently, Ma et al. have constructed a biochemically defined system for mammalian NHEJ, and carefully examined the joining of incompatible DNA ends.56) In that system, the core NHEJ components, Ku70, Ku80, Artemis, DNA-PKcs, DNA ligase IV, and XRCC4, were able to join incompatible DNA ends.56) Interestingly, the length of the reaction prod- ucts was affected by the presence of Artemis/DNA-PKcs, consistent with a nucleolytic role of this complex in end processing. Furthermore, recent studies have suggested that the C-terminus of Ku80 is important not only for repairing IR-induced DNA damage but also for Artemis-mediated processing of DNA ends.30) Although it is possible that other nucleases also participate in the processing reaction,57–59) the Artemis/DNA-PKcs complex is likely to be a central enzyme for the end processing step of NHEJ in higher eukaryotes.

Fig. 5. The Artemis/DNA-PKcs complex is required for generat- INVOLVEMENT OF ARTEMIS IN DNA ing ligatable DNA ends. IR and radiomimetic agents induce DSBs DAMAGE RESPONSE with unligatable ends, such as 3’ phosphoglycolate termini. After the Ku70/Ku80 complex immediately binds to DSB ends, the It has been shown that ATM and ATR as well as DNA-PKcs Artemis/DNA-PKcs complex generates ligatable DNA ends with 26–30,60) phosphorylate Artemis in response to DNA damage, minimal loss of nucleotides. Finally, the DNA ligase IV/XRCC4/ and the phosphorylated Artemis physically associates with XLF complex ligates the DSB ends. When NHEJ proteins leave the the Mre11/Rad50/Nijmegen breakage syndrome 1 (Nbs1) ligated DNA, NHEJ is completed. Functions and Regulation of Artemis 507 rylated at Ser516 and Ser645 by ATR in response to UV (Fig. 6). As ATM triggers apoptosis via phosphorylation of light (Fig. 2), and this phosphorylation is involved in the p53 at Ser15,66) it is possible that Artemis may be involved recovery from S-phase arrest.62) In the ATM/ATR signaling in the p53-dependent apoptosis signaling pathway. Interest- pathways, Chk1 and Chk2 kinases act as key downstream ingly, however, it was recently reported that Artemis may act regulators that phosphorylate various proteins involved in as a negative regulator of p53.67) Specifically, after oxidative cell cycle checkpoints after DNA damage.63) Intriguingly, stress, Artemis knockdown led to a spontaneous activation neither Chk1 nor Chk2 is involved in IR- and UV-induced of p53 via phosphorylation at Ser15 and Ser37, resulting in hyperphosphorylation of Artemis.28,29) These observations G1 arrest and subsequent apoptosis.67) Although the bio- suggest that Artemis is a direct downstream factor of the logical significance of such Artemis function is currently ATM (and presumably ATR) signaling pathway. Consistent unclear, this finding suggests the possibility that Artemis with this notion, epistasis analysis using human fibroblasts may signal to p53 under certain conditions. Further analysis derived from ataxia telangiectasia patients and from will clarify the exact relationship between Artemis and the Artemis-deficient RS-SCID patients showed that ATM and p53 pathway. Artemis function in a common DSB repair pathway.27,64) In this review, we have described the biochemical proper- Additionally, this pathway requires H2AX, 53BP1 and ties of Artemis and its biological functions in light of the DNA-PKcs, as well as Mre11 and Nbs1.27) Therefore, maintenance of genome integrity. It is conceivable that Ser645 phosphorylation may trigger a conformational Artemis acts at two distinct levels; one at the end processing change in Artemis that enables its physical interaction with step of NHEJ and the other at the signaling pathway of cell the Mre11/Rad50/Nbs1 complex. A genetic interaction cycle progression. In either case, such Artemis functions are between 53BP1 and Artemis has been shown in mammalian likely to be regulated through phosphorylation.27,28,35,60,66) cells, consistent with the observation that Artemis physically Since Artemis does not exist in yeast, it is reasonable to interacts with 53BP1,27,65) though in chicken DT40 cells speculate that Artemis contributes to efficient DSB repair in 53BP1 reported to play a role in a pathway distinct from the higher eukaryotes. For instance, the Artemis/DNA-PKcs Artemis-dependent ATM pathway.64) Together, these obser- complex may serve to create DNA ends that are preferentially vations strongly suggest that Artemis acts as a genome care- rejoined by DNA ligase IV, resulting in prompt DSB repair taker to suppress genome instability, by regulating cell cycle with minimal loss of nucleotides. Further analysis of progression in concert with ATM and its downstream factors Artemis function will provide detailed information about the mechanism that ensures the integrity of the genome in higher eukaryotes.

ACKNOWLEDGEMENTS

We thank the Editor-In-Chief of the Journal of Radiation Research, Dr. Yoshiya Furusawa, for giving us the oppor- tunity to write this review article.

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