Structural and Mechanistic Insights Into the Artemis Endonuclease and Strategies for Its Inhibition Yuliana Yosaatmadja1,†, Hannah T
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9310–9326 Nucleic Acids Research, 2021, Vol. 49, No. 16 Published online 13 August 2021 https://doi.org/10.1093/nar/gkab693 Structural and mechanistic insights into the Artemis endonuclease and strategies for its inhibition Yuliana Yosaatmadja1,†, Hannah T. Baddock2,†, Joseph A. Newman1, Marcin Bielinski3, Angeline E. Gavard1, Shubhashish M.M. Mukhopadhyay1, Adam A. Dannerfjord1, Christopher J. Schofield3, Peter J. McHugh 2,* and Opher Gileadi 1,* 1Centre for Medicines Discovery, University of Oxford, ORCRB, Roosevelt Drive, Oxford OX3 7DQ, UK, 2Department 3 of Oncology, MRC-Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK and The Downloaded from https://academic.oup.com/nar/article/49/16/9310/6350767 by guest on 29 September 2021 Department of Chemistry and the Ineos Oxford Institute for Antimicrobial Research, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK Received December 22, 2020; Revised July 20, 2021; Editorial Decision July 25, 2021; Accepted August 11, 2021 ABSTRACT INTRODUCTION Artemis (SNM1C/DCLRE1C) is an endonuclease that Nucleases catalyse the hydrolysis of the phosphodiester plays a key role in development of B- and T- bonds in nucleic acids and are broadly classified as lymphocytes and in dsDNA break repair by non- exonucleases or endonucleases. Exonucleases are often homologous end-joining (NHEJ). Artemis is phos- non sequence-specific, while endonucleases can be fur- phorylated by DNA-PKcs and acts to open DNA hair- ther grouped into sequence-specific endonucleases, such as restriction enzymes, and structure-selective endonucleases pin intermediates generated during V(D)J and class- (1). Artemis (SNM1C or DCLRE1C), along with SNM1A switch recombination. Artemis deficiency leads to (DCLRE1A) and SNM1B (Apollo or DCLRE1B), are hu- congenital radiosensitive severe acquired immune man nucleases that are members of the extended structural deficiency (RS-SCID). Artemis belongs to a super- family of metallo--lactamase (MBL) fold enzymes (2,3). family of nucleases containing metallo--lactamase The N-terminal region of Artemis has a core MBL fold (MBL) and -CASP (CPSF-Artemis-SNM1-Pso2) do- (aa 1–170, 319–361) with an inserted -CASP (CPSF73, mains. We present crystal structures of the catalytic Artemis, SNM1 and PSO2) domain (aa 170–318) (4). - domain of wildtype and variant forms of Artemis, in- CASP domains are present within the larger family of eu- cluding one causing RS-SCID Omenn syndrome. The karyotic nucleic acid processing MBLs and confer both / catalytic domain of the Artemis has similar endonu- DNA RNA binding and nuclease activity (2,5). The C- clease activity to the phosphorylated full-length pro- terminal region of Artemis (aa 362–692) mediates protein- protein interactions, contains post translational modifica- tein. Our structures help explain the predominantly tion (PTM) sites, directs subcellular localization, and may endonucleolytic activity of Artemis, which contrasts modulate catalysis (6–9). with the predominantly exonuclease activity of the Although SNM1A, SNM1B and Artemis have similar closely related SNM1A and SNM1B MBL fold nucle- structures of their core catalytic domains, each has distinct ases. The structures reveal a second metal binding functions and selectivities. While SNM1A and SNM1B are site in its -CASP domain unique to Artemis, which exclusively 5 to 3 exonucleases, Artemis is an endonucle- is amenable to inhibition by compounds including ase (7,9), although a minor 5 to 3 exonuclease activity ebselen. By combining our structural data with that has been reported (10). Human SNM1A localizes to sites from a recently reported Artemis structure, we were of DNA damage, can digest past DNA damage lesions in able model the interaction of Artemis with DNA sub- vitro, and is involved in inter-strand crosslink (ICL) repair strates. The structures, including one of Artemis with (11–13). SNM1B is a shelterin-associated protein required for resection at newly-replicated leading-strand telomeres the cephalosporin ceftriaxone, will help enable the to generate the 3 -overhang necessary for telomere loop (t- rational development of selective SNM1 nuclease in- loop) formation and telomere protection (14–16). SNM1A hibitors. and SNM1B prefer ssDNA substrates in vitro, with an abso- lute requirement for a free 5-phosphate (3,17). By contrast, *To whom correspondence should be addressed. Tel: +44 7880553524; Email: [email protected] Correspondence may also be addressed to Peter J. McHugh. Email: [email protected] †The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors. C The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2021, Vol. 49, No. 16 9311 Artemis preferentially cleaves hairpins and DNA junctions, cloning (LIC) (38)) into the baculovirus expression vector although it is able to process ssDNA substrates (18–20). pBF-6HZB (GenBank™ accession number KP233213.1), The key roles of Artemis and related DSB repair en- which combines an N-terminal His6 sequence,the Z-basic zymes in both programmed V(D)J recombination and non- tag and a TEV protease cleavage site for efficient purifica- programmed c-NHEJ DSB repair, make them attractive tion. Site-directed mutagenesis was carried out using an in- targets for treatment of cancer, either on their own or in verse PCR method whereby an entire plasmid is amplified combination with chemo- or radiotherapy. The Artemis using complementary mutagenic primers (oligonucleotides) endonuclease activity is responsible for hairpin opening with minimal cloning steps (39), using the Herculase II Fu- in variable (diversity) joining (V(D)J) recombination (21) sion DNA Polymerase (Agilent) for amplification and the and contributes to end-processing in the canonical non- KLD enzyme mix (NEB). homologous end joining (c-NHEJ) DNA repair path- way (22–25). V(D)J recombination is initiated by recog- nition and binding of recombination-activating gene pro- Expression and purification of WT and mutant Artemis with Downloaded from https://academic.oup.com/nar/article/49/16/9310/6350767 by guest on 29 September 2021 teins (RAG1 and RAG2) to recombination signal sequences IMAC (aa 1-362) (RSSs) adjacent to the V, D and J gene segments. Upon Baculovirus generation was performed as described (3). Re- binding, the RAG proteins induce double-strand breaks combinant proteins were produced by infecting Sf9 cells at (DSBs) and create a hairpin at the coding ends (26–28). The 2 × 106 cells/ ml with 1.5 ml of P2 virus for WT and 3 ml Ku heterodimer recognizes the DNA double-strand break of P2 virus for mutants respectively. Infected Sf9 cells were and recruits DNA-dependent protein kinase catalytic sub- harvested 70 h after infection by centrifugation (900 × g, unit (DNA-PKcs) and Artemis to mediate hairpin open- 20 min). The cell pellets were resuspended in 30 ml/lly- ing (19). Following hairpin opening, the NHEJ machin- sis buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 10 ery containing the XRCC4/XLF(PAXX)/DNA-Ligase IV mM imidazole, 5% (v/v) glycerol and 1 mM TCEP), snap complex is recruited to catalyse the processing and ligation frozen in liquid nitrogen, then stored at −80◦C for later reactions at the DNA ends (22,29,30). V(D)J recombination use. is an essential process in antibody maturation (18,31,32). Thawed cell aliquots were lysed by sonication. The lysates Mutations in the Artemis gene cause aberrant hairpin were centrifuged (40 000 × g, 30 min); the supernatant was opening, resulting in severe combined immune deficiency passed through a 0.80 m filter (Millipore), then loaded (RS-SCID), with sensitivity to ionizing radiation due to im- onto an immobilized metal affinity chromatography col- pairment of the predominant DSB repair pathway in mam- umn (IMAC) (Ni-NTA Superflow Cartridge, Qiagen) equi- malian cells, NHEJ (19,21,29), and another form of SCID librated in lysis buffer. The column was washed with lysis (Omenn syndrome) associated with hypomorphic Artemis buffer, then eluted using a linear gradient to elution buffer mutations (33,34). Artemis loss-of-function mutations of- (50 mM HEPES pH 7.5, 500 mM NaCl, 300 mM imidazole, ten comprise large deletions in the first four exons or non- 5% (v/v) glycerol and 1 mM TCEP). Protein-containing sense founder mutation, as found in Navajo and Apache fractions were pooled and passed through an ion exchange Native Americans (35). In addition, missense mutations and column (HiTrap® SP FF GE Healthcare Life Sciences) pre- in-frame deletions in the highly conserved residues such as equilibrated in the SP buffer A (25 mM HEPES pH 7.5, 300 H35, D165 and H228 can also abolish Artemis’ protein mM NaCl, 5% (v/v) glycerol and 1 mM TCEP). Protein was function (36). eluted using a linear gradient to SP buffer B (SP buffer A We present high-resolution crystal structures of the cat- with 1 M NaCl), and fractions containing the ZB-tagged alytic core of Artemis (aa 1–361) containing both MBL and Artemis were identified by electrophoresis. a -CASP domains. The structures reveal that Artemis pos- Artemis containing fractions were pooled and dialysed sesses a second metal binding site in its -CASP domain, overnight at 4◦C in SP buffer A, supplemented with re- not present in SNM1A and SNM1B, that resembles classi- combinant tobacco etch virus (TEV) protease to cleavage cal Cys2His2 zinc finger motifs. Based on our data andan- the His6-ZB tag. The protein was loaded into an ion ex- other recently reported Artemis structure (37), we present a change column (HiTrap® SP FF GE Healthcare Life Sci- model for Artemis DNA binding.