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Dominant Mutations of the TREX1 Exonuclease Gene in Lupus and Aicardi-Goutières Syndrome

2011; Elsevier BV; Volume: 286; Issue: 37 Linguagem: Inglês

10.1074/jbc.m111.276287

ISSN

1083-351X

Autores

Jason M. Fye, Clinton D. Orebaugh, Stephanie R. Coffin, Thomas Hollis, Fred W. Perrino,

Tópico(s)

Inflammasome and immune disorders

Resumo

TREX1 is a potent 3′→5′ exonuclease that degrades single- and double-stranded DNA (ssDNA and dsDNA). TREX1 mutations at amino acid positions Asp-18 and Asp-200 in familial chilblain lupus and Aicardi-Goutières syndrome elicit dominant immune dysfunction phenotypes. Failure to appropriately disassemble genomic DNA during normal cell death processes could lead to persistent DNA signals that trigger the innate immune response and autoimmunity. We tested this concept using dsDNA plasmid and chromatin and show that the TREX1 exonuclease locates 3′ termini generated by endonucleases and degrades the nicked DNA polynucleotide. A competition assay was designed using TREX1 dominant mutants and variants to demonstrate that an intact DNA binding process, coupled with dysfunctional chemistry in the active sites, explains the dominant phenotypes in TREX1 D18N, D200N, and D200H alleles. The TREX1 residues Arg-174 and Lys-175 positioned adjacent to the active sites act with the Arg-128 residues positioned in the catalytic cores to facilitate melting of dsDNA and generate ssDNA for entry into the active sites. Metal-dependent ssDNA binding in the active sites of the catalytically inactive dominant TREX1 mutants contributes to DNA retention and precludes access to DNA 3′ termini by active TREX1 enzyme. Thus, the dominant disease genetics exhibited by the TREX1 D18N, D200N, and D200H alleles parallel precisely the biochemical properties of these TREX1 dimers during dsDNA degradation of plasmid and chromatin DNA in vitro. These results support the concept that failure to degrade genomic dsDNA is a principal pathway of immune activation in TREX1-mediated autoimmune disease. TREX1 is a potent 3′→5′ exonuclease that degrades single- and double-stranded DNA (ssDNA and dsDNA). TREX1 mutations at amino acid positions Asp-18 and Asp-200 in familial chilblain lupus and Aicardi-Goutières syndrome elicit dominant immune dysfunction phenotypes. Failure to appropriately disassemble genomic DNA during normal cell death processes could lead to persistent DNA signals that trigger the innate immune response and autoimmunity. We tested this concept using dsDNA plasmid and chromatin and show that the TREX1 exonuclease locates 3′ termini generated by endonucleases and degrades the nicked DNA polynucleotide. A competition assay was designed using TREX1 dominant mutants and variants to demonstrate that an intact DNA binding process, coupled with dysfunctional chemistry in the active sites, explains the dominant phenotypes in TREX1 D18N, D200N, and D200H alleles. The TREX1 residues Arg-174 and Lys-175 positioned adjacent to the active sites act with the Arg-128 residues positioned in the catalytic cores to facilitate melting of dsDNA and generate ssDNA for entry into the active sites. Metal-dependent ssDNA binding in the active sites of the catalytically inactive dominant TREX1 mutants contributes to DNA retention and precludes access to DNA 3′ termini by active TREX1 enzyme. Thus, the dominant disease genetics exhibited by the TREX1 D18N, D200N, and D200H alleles parallel precisely the biochemical properties of these TREX1 dimers during dsDNA degradation of plasmid and chromatin DNA in vitro. These results support the concept that failure to degrade genomic dsDNA is a principal pathway of immune activation in TREX1-mediated autoimmune disease. IntroductionDeoxyribonucleases are essential enzymes acting to degrade DNA polynucleotides in the orchestrated processes of dismantling dying cells and in defense from invading pathogens. Failure to efficiently degrade superfluous DNA macromolecules can result in persistent nucleic acids that activate the mammalian immune system (1Crow Y.J. Rehwinkel J. Hum. Mol. Genet. 2009; 18: R130-R136Crossref PubMed Scopus (229) Google Scholar). TREX1 is a 314-amino acid polypeptide containing a robust 3′-exonuclease that degrades ssDNA and dsDNA and is expressed ubiquitously in mammalian cells (2Höss M. Robins P. Naven T.J. Pappin D.J. Sgouros J. Lindahl T. EMBO J. 1999; 18: 3868-3875Crossref PubMed Scopus (148) Google Scholar, 3Mazur D.J. Perrino F.W. J. Biol. Chem. 1999; 274: 19655-19660Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 4Mazur D.J. Perrino F.W. J. Biol. Chem. 2001; 276: 17022-17029Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The catalytic core of TREX1 is contained in the N-terminal 242 amino acids, and the C-terminal 72 amino acids contain a hydrophobic region that localizes TREX1 to the endoplasmic reticulum in the perinuclear space of cells (5Chowdhury D. Beresford P.J. Zhu P. Zhang D. Sung J.S. Demple B. Perrino F.W. Lieberman J. Mol. Cell. 2006; 23: 133-142Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Located in the cytosol, TREX1 prevents the initiation of a cell-intrinsic autoimmune pathway by degrading ssDNA derived from endogenous retroelements (6Stetson D.B. Ko J.S. Heidmann T. Medzhitov R. Cell. 2008; 134: 587-598Abstract Full Text Full Text PDF PubMed Scopus (894) Google Scholar). TREX1 also degrades HIV DNA generated during HIV-1 infection, preventing activation of intrinsic DNA sensors (7Yan N. Regalado-Magdos A.D. Stiggelbout B. Lee-Kirsch M.A. Lieberman J. Nat. Immunol. 2010; 11: 1005-1013Crossref PubMed Scopus (395) Google Scholar). Upon activation of a cell death pathway and treatment of cells with DNA-damaging agents, TREX1 relocates to the nucleus, where it acts on DNA 3′ termini (5Chowdhury D. Beresford P.J. Zhu P. Zhang D. Sung J.S. Demple B. Perrino F.W. Lieberman J. Mol. Cell. 2006; 23: 133-142Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 8Chowdhury D. Lieberman J. Annu. Rev. Immunol. 2008; 26: 389-420Crossref PubMed Scopus (419) Google Scholar, 9Yang Y.G. Lindahl T. Barnes D.E. Cell. 2007; 131: 873-886Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar).The TREX1 gene is a single open reading frame located on chromosome 3p21.31 (10Mazur D.J. Perrino F.W. J. Biol. Chem. 2001; 276: 14718-14727Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Mutations in the human TREX1 gene have been linked to a spectrum of autoimmune diseases including the severe neurological brain disease Aicardi-Goutières syndrome (AGS) 2The abbreviations used are: AGSAicardi-Goutières syndromeFCLfamilial chilblain lupusMBPmaltose-binding proteinMUTmutant. (11Crow Y.J. Hayward B.E. Parmar R. Robins P. Leitch A. Ali M. Black D.N. van Bokhoven H. Brunner H.G. Hamel B.C. Corry P.C. Cowan F.M. Frints S.G. Klepper J. Livingston J.H. Lynch S.A. Massey R.F. Meritet J.F. Michaud J.L. Ponsot G. Voit T. Lebon P. Bonthron D.T. Jackson A.P. Barnes D.E. Lindahl T. Nat. Genet. 2006; 38: 917-920Crossref PubMed Scopus (637) Google Scholar), to a monogenic form of cutaneous lupus erythematosus named "familial chilblain lupus" (FCL) (12Lee-Kirsch M.A. Chowdhury D. Harvey S. Gong M. Senenko L. Engel K. Pfeiffer C. Hollis T. Gahr M. Perrino F.W. Lieberman J. Hubner N. J. Mol. Med. 2007; 85: 531-537Crossref PubMed Scopus (169) Google Scholar, 13Lee-Kirsch M.A. Gong M. Schulz H. Rüschendorf F. Stein A. Pfeiffer C. Ballarini A. Gahr M. Hubner N. Linné M. Am. J. Hum. Genet. 2006; 79: 731-737Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 14Rice G. Newman W.G. Dean J. Patrick T. Parmar R. Flintoff K. Robins P. Harvey S. Hollis T. O'Hara A. Herrick A.L. Bowden A.P. Perrino F.W. Lindahl T. Barnes D.E. Crow Y.J. Am. J. Hum. Genet. 2007; 80: 811-815Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar), to systemic lupus erythematosus (15Lee-Kirsch M.A. Gong M. Chowdhury D. Senenko L. Engel K. Lee Y.A. de Silva U. Bailey S.L. Witte T. Vyse T.J. Kere J. Pfeiffer C. Harvey S. Wong A. Koskenmies S. Hummel O. Rohde K. Schmidt R.E. Dominiczak A.F. Gahr M. Hollis T. Perrino F.W. Lieberman J. Hübner N. Nat. Genet. 2007; 39: 1065-1067Crossref PubMed Scopus (511) Google Scholar, 16Namjou B. Kothari P.H. Kelly J.A. Glenn S.B. Ojwang J.O. Adler A. Alarcón-Riquelme M.E. Gallant C.J. Boackle S.A. Criswell L.A. Kimberly R.P. Brown E. Edberg J. Stevens A.M. Jacob C.O. Tsao B.P. Gilkeson G.S. Kamen D.L. Merrill J.T. Petri M. Goldman R.R. Vila L.M. Anaya J.M. Niewold T.B. Martin J. Pons-Estel B.A. Sabio J.M. Callejas J.L. Vyse T.J. Bae S.C. Perrino F.W. Freedman B.I. Scofield R.H. Moser K.L. Gaffney P.M. James J.A. Langefeld C.D. Kaufman K.M. Harley J.B. Atkinson J.P. Genes Immun. 2011; 12: 270-279Crossref PubMed Scopus (203) Google Scholar), and to retinal vasculopathy and cerebral leukodystrophy (17Richards A. van den Maagdenberg A.M. Jen J.C. Kavanagh D. Bertram P. Spitzer D. Liszewski M.K. Barilla-Labarca M.L. Terwindt G.M. Kasai Y. McLellan M. Grand M.G. Vanmolkot K.R. de, Vries B. Wan J. Kane M.J. Mamsa H. Schäfer R. Stam A.H. Haan J. de Jong P.T. Storimans C.W. van Schooneveld M.J. Oosterhuis J.A. Gschwendter A. Dichgans M. Kotschet K.E. Hodgkinson S. Hardy T.A. Delatycki M.B. Hajj-Ali R.A. Kothari P.H. Nelson S.F. Frants R.R. Baloh R.W. Ferrari M.D. Atkinson J.P. Nat. Genet. 2007; 39: 1068-1070Crossref PubMed Scopus (306) Google Scholar). Approximately 40 TREX1 disease-causing missense and frameshift mutations have been identified, mapping to positions located throughout the gene (18Kavanagh D. Spitzer D. Kothari P.H. Shaikh A. Liszewski M.K. Richards A. Atkinson J.P. Cell Cycle. 2008; 7: 1718-1725Crossref PubMed Scopus (105) Google Scholar, 19Ramantani G. Kohlhase J. Hertzberg C. Innes A.M. Engel K. Hunger S. Borozdin W. Mah J.K. Ungerath K. Walkenhorst H. Richardt H.H. Buckard J. Bevot A. Siegel C. von Stülpnagel C. Ikonomidou C. Thomas K. Proud V. Niemann F. Wieczorek D. Häusler M. Niggemann P. Baltaci V. Conrad K. Lebon P. Lee-Kirsch M.A. Arthritis Rheum. 2010; 62: 1469-1477Crossref PubMed Scopus (150) Google Scholar). Furthermore, mice lacking TREX1 develop inflammatory myocarditis consistent with autoimmune disease (20Morita M. Stamp G. Robins P. Dulic A. Rosewell I. Hrivnak G. Daly G. Lindahl T. Barnes D.E. Mol. Cell. Biol. 2004; 24: 6719-6727Crossref PubMed Scopus (276) Google Scholar).Multiple mechanisms of dysfunction underlie the observed clinical phenotypes in patients carrying TREX1 mutations, reflecting the position of the mutation and the stable dimeric structure. Our structural studies of the TREX1 catalytic domain with bound DNA reveal the protein-polynucleotide interactions that explain the requirement for ssDNA in the active site and highlight the extensive interface contacts in the remarkably stable dimeric enzyme (21de Silva U. Choudhury S. Bailey S.L. Harvey S. Perrino F.W. Hollis T. J. Biol. Chem. 2007; 282: 10537-10543Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The TREX1 AGS-causing mutations locate predominantly to the catalytic core region with a few notable exceptions positioned in the C-terminal hydrophobic region (22Rice G. Patrick T. Parmar R. Taylor C.F. Aeby A. Aicardi J. Artuch R. Montalto S.A. Bacino C.A. Barroso B. Baxter P. Benko W.S. Bergmann C. Bertini E. Biancheri R. Blair E.M. Blau N. Bonthron D.T. Briggs T. Brueton L. A Brunner. H.G. Burke C.J. Carr I.M. Carvalho D.R. Chandler K.E. Christen H.J. Corry P.C. Cowan F.M. Cox H. D'Arrigo S. Dean J. De Laet C. De Praeter C. Dery C. Ferrie C.D. Flintoff K. Frints S.G. Garcia-Cazorla A. Gener B. Goizet C. Goutieres F. Green A.J. Guet A. Hamel B.C. Hayward B.E. Heiberg A. Hennekam R.C. Husson M. Jackson A.P. Jayatunga R. Jiang Y.H. Kant S.G. Kao A. King M.D. Kingston H.M. Klepper J. van der Knaap M.S. Kornberg A.J. Kotzot D. Kratzer W. Lacombe D. Lagae L. Landrieu P.G. Lanzi G. Leitch A. Lim M.J. Livingston J.H. Lourenco C.M. Lyall E.G. Lynch S.A. Lyons M.J. Marom D. McClure J.P. McWilliam R. Melancon S.B. Mewasingh L.D. Moutard M.L. Nischal K.K. Ostergaard J.R. Prendiville J. Rasmussen M. Rogers R.C. Roland D. Rosser E.M. Rostasy K. Roubertie A. Sanchis A. Schiffmann R. Scholl-Burgi S. Seal S. Shalev S.A. Corcoles C.S. Sinha G.P. Soler D. Spiegel R. Stephenson J.B. Tacke U. Tan T.Y. Till M. Tolmie J.L. Tomlin P. Vagnarelli F. Valente E.M. Van Coster R.N. Van der Aa N. Vanderver A. Vles J.S. Voit T. Wassmer E. Weschke B. Whiteford M.L. Willemsen M.A. Zankl A. Zuberi S.M. Orcesi S. Fazzi E. Lebon P. Crow Y.J. Am. J. Hum. Genet. 2007; 81: 713-725Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). Some TREX1 AGS-causing mutants exhibit dramatically lower levels of catalytic function, whereas others show more modest effects on the ssDNA degradation activities, yet all yield similar human pathologies (21de Silva U. Choudhury S. Bailey S.L. Harvey S. Perrino F.W. Hollis T. J. Biol. Chem. 2007; 282: 10537-10543Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 23Lehtinen D.A. Harvey S. Mulcahy M.J. Hollis T. Perrino F.W. J. Biol. Chem. 2008; 283: 31649-31656Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The TREX1 systemic lupus erythematosus-associated mutations are located mostly in the C-terminal region with a few variants positioned in the catalytic core. The heterozygous TREX1 mutations that cause retinal vasculopathy and cerebral leukodystrophy are all frameshifts in the C-terminal region (17Richards A. van den Maagdenberg A.M. Jen J.C. Kavanagh D. Bertram P. Spitzer D. Liszewski M.K. Barilla-Labarca M.L. Terwindt G.M. Kasai Y. McLellan M. Grand M.G. Vanmolkot K.R. de, Vries B. Wan J. Kane M.J. Mamsa H. Schäfer R. Stam A.H. Haan J. de Jong P.T. Storimans C.W. van Schooneveld M.J. Oosterhuis J.A. Gschwendter A. Dichgans M. Kotschet K.E. Hodgkinson S. Hardy T.A. Delatycki M.B. Hajj-Ali R.A. Kothari P.H. Nelson S.F. Frants R.R. Baloh R.W. Ferrari M.D. Atkinson J.P. Nat. Genet. 2007; 39: 1068-1070Crossref PubMed Scopus (306) Google Scholar). The TREX1 enzymes containing C-terminal mutations retain full catalytic function but fail to localize to the perinuclear space in cells (15Lee-Kirsch M.A. Gong M. Chowdhury D. Senenko L. Engel K. Lee Y.A. de Silva U. Bailey S.L. Witte T. Vyse T.J. Kere J. Pfeiffer C. Harvey S. Wong A. Koskenmies S. Hummel O. Rohde K. Schmidt R.E. Dominiczak A.F. Gahr M. Hollis T. Perrino F.W. Lieberman J. Hübner N. Nat. Genet. 2007; 39: 1065-1067Crossref PubMed Scopus (511) Google Scholar, 17Richards A. van den Maagdenberg A.M. Jen J.C. Kavanagh D. Bertram P. Spitzer D. Liszewski M.K. Barilla-Labarca M.L. Terwindt G.M. Kasai Y. McLellan M. Grand M.G. Vanmolkot K.R. de, Vries B. Wan J. Kane M.J. Mamsa H. Schäfer R. Stam A.H. Haan J. de Jong P.T. Storimans C.W. van Schooneveld M.J. Oosterhuis J.A. Gschwendter A. Dichgans M. Kotschet K.E. Hodgkinson S. Hardy T.A. Delatycki M.B. Hajj-Ali R.A. Kothari P.H. Nelson S.F. Frants R.R. Baloh R.W. Ferrari M.D. Atkinson J.P. Nat. Genet. 2007; 39: 1068-1070Crossref PubMed Scopus (306) Google Scholar). Thus, disruptions in catalytic function and in cellular trafficking are mechanisms of TREX1 dysfunction in autoimmunity.TREX1-mediated autoimmune disease exhibits both dominant and recessive genetics dependent upon the nature of the mutation. Heterozygous de novo and inherited mutations in the highly conserved TREX1 Asp-18 and Asp-200 metal-binding residues exhibit dominant FCL and AGS (12Lee-Kirsch M.A. Chowdhury D. Harvey S. Gong M. Senenko L. Engel K. Pfeiffer C. Hollis T. Gahr M. Perrino F.W. Lieberman J. Hubner N. J. Mol. Med. 2007; 85: 531-537Crossref PubMed Scopus (169) Google Scholar, 14Rice G. Newman W.G. Dean J. Patrick T. Parmar R. Flintoff K. Robins P. Harvey S. Hollis T. O'Hara A. Herrick A.L. Bowden A.P. Perrino F.W. Lindahl T. Barnes D.E. Crow Y.J. Am. J. Hum. Genet. 2007; 80: 811-815Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 19Ramantani G. Kohlhase J. Hertzberg C. Innes A.M. Engel K. Hunger S. Borozdin W. Mah J.K. Ungerath K. Walkenhorst H. Richardt H.H. Buckard J. Bevot A. Siegel C. von Stülpnagel C. Ikonomidou C. Thomas K. Proud V. Niemann F. Wieczorek D. Häusler M. Niggemann P. Baltaci V. Conrad K. Lebon P. Lee-Kirsch M.A. Arthritis Rheum. 2010; 62: 1469-1477Crossref PubMed Scopus (150) Google Scholar, 24Haaxma C.A. Crow Y.J. van Steensel M.A. Lammens M.M. Rice G.I. Verbeek M.M. Willemsen M.A. Am. J. Med. Genet. A. 2010; 152A: 2612-2617Crossref PubMed Scopus (33) Google Scholar). The disease phenotypes in dominant FCL and AGS patients correlate best with the dsDNA degradation activities measured in the TREX1 D18N and D200N enzymes and not with the ssDNA degradation activities (23Lehtinen D.A. Harvey S. Mulcahy M.J. Hollis T. Perrino F.W. J. Biol. Chem. 2008; 283: 31649-31656Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). We have proposed that the inability to perform chemistry of phosphodiester bond cleavage resulting from the D18N or D200N mutation might trap the TREX1 mutant enzyme onto the dsDNA in a nonproductive enzyme-DNA complex at the site of the nick. These data support the proposal that TREX1 degrades dsDNA by acting at 3′ termini generated by the NM23-H1 endonuclease during cell death (5Chowdhury D. Beresford P.J. Zhu P. Zhang D. Sung J.S. Demple B. Perrino F.W. Lieberman J. Mol. Cell. 2006; 23: 133-142Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). The studies presented here show that the dsDNA degradation activities of TREX1 enzymes containing the FCL and AGS dominant D18N, D200N, and D200H mutations are defective and that these mutants inhibit the dsDNA degradation activity of TREX1WT enzyme, likely present in cells of these patients. The dominant effect of these TREX1 mutant enzymes is dependent upon the functional DNA binding by residues Arg-174 and Lys-175 positioned adjacent to the active sites and the Arg-128 positioned in the catalytic cores (see Fig. 1). In addition, metal-dependent DNA binding in the active sites of the catalytically inactive dominant TREX1 mutants contributes to DNA retention and precludes access to the DNA 3′ termini of nicked dsDNA by the TREX1WT enzyme.RESULTS AND DISCUSSIONThe TREX1 exonuclease degrades ssDNA and dsDNA polynucleotide substrates containing available 3′ termini. Structural studies of the TREX1-DNA complex provide direct evidence for ssDNA binding of at least four nucleotides in length in the active sites (Fig. 1) (21de Silva U. Choudhury S. Bailey S.L. Harvey S. Perrino F.W. Hollis T. J. Biol. Chem. 2007; 282: 10537-10543Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Amino acid residues Asp-18 and Asp-200 are two of the divalent metal ion Mg2+-coordinating aspartates in the TREX1 active site that contribute to DNA binding and are required for catalysis. Residues Arg-174 and Lys-175 located on a flexible loop adjacent to the active sites and Arg-128 in the catalytic core are positioned appropriately to function in DNA binding to locate available 3′ termini and generate ssDNA for entry into the active sites.The TREX1 ssDNA Exonuclease Activities of Dominant MutantsThe dominant TREX1 D18N, D200N, and D200H de novo and inherited mutations have been identified in FCL and AGS (12Lee-Kirsch M.A. Chowdhury D. Harvey S. Gong M. Senenko L. Engel K. Pfeiffer C. Hollis T. Gahr M. Perrino F.W. Lieberman J. Hubner N. J. Mol. Med. 2007; 85: 531-537Crossref PubMed Scopus (169) Google Scholar, 14Rice G. Newman W.G. Dean J. Patrick T. Parmar R. Flintoff K. Robins P. Harvey S. Hollis T. O'Hara A. Herrick A.L. Bowden A.P. Perrino F.W. Lindahl T. Barnes D.E. Crow Y.J. Am. J. Hum. Genet. 2007; 80: 811-815Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 19Ramantani G. Kohlhase J. Hertzberg C. Innes A.M. Engel K. Hunger S. Borozdin W. Mah J.K. Ungerath K. Walkenhorst H. Richardt H.H. Buckard J. Bevot A. Siegel C. von Stülpnagel C. Ikonomidou C. Thomas K. Proud V. Niemann F. Wieczorek D. Häusler M. Niggemann P. Baltaci V. Conrad K. Lebon P. Lee-Kirsch M.A. Arthritis Rheum. 2010; 62: 1469-1477Crossref PubMed Scopus (150) Google Scholar). The dominant phenotypes caused by these TREX1 alleles and the metal binding functions of these residues suggest a common mechanism of dysfunction. TREX1 is a homodimer, so TREX1 dimers in cells of these heterozygous individuals could be TREX1MUT/MUT and TREX1WT/WT homodimers and TREX1WT/MUT heterodimers. Therefore, the TREX1D200H/D200H and TREX1WT/WT homodimers and TREX1WT/D200H heterodimers were prepared, and the ssDNA degradation activities of these enzymes were compared with the activities of the TREX1 D18N and D200N mutants that we had previously determined (Fig. 2 and Table 1). The ssDNA exonuclease activities of the dominant TREX1 D18N, D200N, and D200H homodimers were reduced by more than 104-fold when compared with TREX1WT. The activities of the TREX1 D18N, D200N, and D200H heterodimers are reduced by only 1.5-, 2.6-, and 1.5-fold when compared with TREX1WT (Fig. 2 and Table 1). The ∼2-fold loss in activity of the TREX1 heterodimers indicates that the TREX1WT protomer within the dimer retains fully functional ssDNA degradation activity. This ∼50% reduction in ssDNA degradation activity has been demonstrated in patient cells carrying the D200N allele (14Rice G. Newman W.G. Dean J. Patrick T. Parmar R. Flintoff K. Robins P. Harvey S. Hollis T. O'Hara A. Herrick A.L. Bowden A.P. Perrino F.W. Lindahl T. Barnes D.E. Crow Y.J. Am. J. Hum. Genet. 2007; 80: 811-815Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). The ssDNA degradation activities of the TREX1 dominant mutants suggest that TREX1 protomers within the dimer can act independently during the degradation of small ssDNA substrates.TABLE 1Relative ssDNA exonuclease activities of TREX1WT and dominant variantsTREX1Relative activityaActivities were derived from reactions in Fig. 2 or as previously reported. Relative activities are calculated as: relative activity = 100 × ((fmol of dNMP released/s/fmol of mutant enzyme)/(fmol of dNMP released/s/fmol WT enzyme)).StudyWTbWT = wild type.1This study and Refs. 4Mazur D.J. Perrino F.W. J. Biol. Chem. 2001; 276: 17022-17029Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 21de Silva U. Choudhury S. Bailey S.L. Harvey S. Perrino F.W. Hollis T. J. Biol. Chem. 2007; 282: 10537-10543Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, and 23Lehtinen D.A. Harvey S. Mulcahy M.J. Hollis T. Perrino F.W. J. Biol. Chem. 2008; 283: 31649-31656Abstract Full Text Full Text PDF PubMed Scopus (90) Google ScholarWT/D200H1/1.5This studyD200H/D200HNone detectedThis studyWT/D18N1/2.6Ref. 12Lee-Kirsch M.A. Chowdhury D. Harvey S. Gong M. Senenko L. Engel K. Pfeiffer C. Hollis T. Gahr M. Perrino F.W. Lieberman J. Hubner N. J. Mol. Med. 2007; 85: 531-537Crossref PubMed Scopus (169) Google ScholarD18N/D18N1/160,000Ref. 12Lee-Kirsch M.A. Chowdhury D. Harvey S. Gong M. Senenko L. Engel K. Pfeiffer C. Hollis T. Gahr M. Perrino F.W. Lieberman J. Hubner N. J. Mol. Med. 2007; 85: 531-537Crossref PubMed Scopus (169) Google ScholarWT/D200N1/1.5Ref. 23Lehtinen D.A. Harvey S. Mulcahy M.J. Hollis T. Perrino F.W. J. Biol. Chem. 2008; 283: 31649-31656Abstract Full Text Full Text PDF PubMed Scopus (90) Google ScholarD200N/D200N1/20,000Ref. 23Lehtinen D.A. Harvey S. Mulcahy M.J. Hollis T. Perrino F.W. J. Biol. Chem. 2008; 283: 31649-31656Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholara Activities were derived from reactions in Fig. 2 or as previously reported. Relative activities are calculated as: relative activity = 100 × ((fmol of dNMP released/s/fmol of mutant enzyme)/(fmol of dNMP released/s/fmol WT enzyme)).b WT = wild type. Open table in a new tab The TREX1 dsDNA Exonuclease Activities of Dominant MutantsThe dominant negative effects of the TREX1 D18N, D200N, and D200H alleles are apparent upon examination of the dsDNA degradation activities. The TREX1 D18N, D200N, and D200H protomers within the heterodimers exhibit a dominant inhibitory effect on the dsDNA degradation activities of the TREX1WT protomer. Incubation of nicked plasmid dsDNA with TREX1WT homodimer results in the degradation of the nicked polynucleotide strand and the accumulation of the un-nicked ssDNA strand (Fig. 3A, lane 2). In contrast, the TREX1WT/D18N, TREX1WT/D200H, and TREX1WT/D200N heterodimers do not degrade the nicked dsDNA plasmid (Fig. 3A, lanes 3–5). Additions of up to 10-fold higher concentrations of the TREX1 mutant heterodimers resulted in no detectable dsDNA degradation (Ref. 23Lehtinen D.A. Harvey S. Mulcahy M.J. Hollis T. Perrino F.W. J. Biol. Chem. 2008; 283: 31649-31656Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar and data not shown). These data indicate that the dominant TREX1 D18N, D200N, and D200H heterodimers exhibit at least a 200-fold decreased level of dsDNA degradation activity relative to TREX1WT in contrast to the modest ∼2-fold level of reduced ssDNA degradation activity by these mutant heterodimers (Table 1).The TREX1 Dominant Mutants Inhibit the TREX1WT dsDNA Degradation ActivityThe TREX1 D200H protomers in the TREX1D200H/D200H homodimers and TREX1WT/D200H heterodimers exhibit a dominant inhibitory effect on the dsDNA degradation activity of TREX1WT. The TREX1WT enzyme was mixed with increased amounts of the TREX1D200H/D200H and TREX1WT/D200H enzymes and incubated with the nicked dsDNA plasmid (Fig. 3B). In these reactions, the TREX1WT competes with the mutant TREX1 enzyme to degrade the nicked dsDNA plasmid. The amount of TREX1WT (76 nm) added in these reactions is 10-fold higher than the amount required to degrade the nicked polynucleotide of the dsDNA (23Lehtinen D.A. Harvey S. Mulcahy M.J. Hollis T. Perrino F.W. J. Biol. Chem. 2008; 283: 31649-31656Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The presence of increased amounts of the TREX1D200H/D200H (Fig. 3B, lanes 8–12) and TREX1WT/D200H (Fig. 3B, lanes 13–17) results in decreased dsDNA degradation activity by the TREX1WT enzyme as evidenced by the increased amount of remaining nicked dsDNA. The inhibition of TREX1WT dsDNA degradation activity by the TREX1D200H/D200H and TREX1WT/D200H enzymes is similar to that previously demonstrated with the TREX1 D18N and D200N mutations (23Lehtinen D.A. Harvey S. Mulcahy M.J. Hollis T. Perrino F.W. J. Biol. Chem. 2008; 283: 31649-31656Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The potent inhibition of TREX1WT dsDNA degradation activity exhibited by TREX1 dimers containing D18N, D200N, and D200H protomers could explain the dominant phenotypes exhibited by these TREX1 mutant alleles described in FCL and AGS (12Lee-Kirsch M.A. Chowdhury D. Harvey S. Gong M. Senenko L. Engel K. Pfeiffer C. Hollis T. Gahr M. Perrino F.W. Lieberman J. Hubner N. J. Mol. Med. 2007; 85: 531-537Crossref PubMed Scopus (169) Google Scholar, 14Rice G. Newman W.G. Dean J. Patrick T. Parmar R. Flintoff K. Robins P. Harvey S. Hollis T. O'Hara A. Herrick A.L. Bowden A.P. Perrino F.W. Lindahl T. Barnes D.E. Crow Y.J. Am. J. Hum. Genet. 2007; 80: 811-815Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). These data suggest that FCL and AGS TREX1 D18N, D200N, and D200H heterozygote patients likely have varying mixtures of TREX1 WT and mutant homo- and heterodimers. The TREX1 D18N-, D200N-, and D200H-containing dimers would likely inhibit the TREX1WT dsDNA degradation activity in these cells.Identification of TREX1 Residues Contributing to DNA BindingThe TREX1 Arg-174, Lys-175, and Arg-128 residues are positioned within the enzyme to participate in DNA binding. To measure the contribution of these residues in the TREX1-catalyzed reaction, a series of variant enzymes was generated in which each of these residues was changed to alanine individually or in combinations. The TREX1 and variant proteins were tested to confirm the presence of nuclease activity using a 30-mer oligonucleotide and to establish the relative ssDNA excision activities (Fig. 4 and Table 2). The TREX1R174A and TREX1K175A exhibit no loss of activity, and the TREX1R174A,K175A shows a modest ∼3-fold reduced excision activity relative to the TREX1WT. These data indicate some contribution to ssDNA binding by the Arg-174 and Lys-175 that is satisfied by the presence of one of these residues on the flexible loop. The TREX1R128A exhibits an ∼2-fold reduced excision activity relative to the TREX1WT, also indicating a modest contribution to ssDNA binding by the Arg-128 located in the catalytic core. The TREX1R128A,R174A and TREX1R128,K175A double mutants exhibit ∼3-fold reduced excision activities, consistent with the requirement for one of the positively charged flexible loop residues (Arg-174 or Lys-175) and Arg-128 in the core for full ssDNA exonuclease activity. The TREX1R128A,R174A,K175A triple mutant exhibits an ∼30-fold reduced excision activity, further demonstrating the requirement for a single positively charged residue positioned on the flexible loop and the Arg-128 within the catalytic core for full ssDNA degradation activity. Also, a steady-state kinetic analysis indicated an ∼35-fold higher Km value for the TREX1R128A,R174A,K175A when compared with the TREX1WT protein and similar kcat values, confirming the structural integrity of the mutant enzyme and further supporting the diminished DNA binding potential (data not shown). However, the ∼3-fold magnitude in loss of TREX1 catalytic function upon mutation of the flexible loop Arg-174 and Lys-175 residues contrasts sharply with the ∼200-fold loss of TREX2 catalytic function upon mutation of the comparable Arg-163, Arg-165, and Arg-167 flexible loop residues (Refs. 28Perrino F.W. de Silva U. Harvey S. Pryor Jr., E.E. Cole D.W. Hollis T. J. Biol. Chem. 2008; 283: 21441-21452Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar and 29Perrino F.W. Harvey S. McMillin S. Hollis T. J. Biol. Chem. 2005; 280: 15212-15218Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar and data not shown). Furthermore, the TREX1

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