Lily Y. Li

Endonuclease G (endoG) is released from mitochondria during apoptosis and is in part responsible for internucleosomal DNA cleavage. Here we report the action of the purified human recombinant form of this endonuclease on naked DNA and chromatin substrates. The addition of the protein to isolated nuclei from non-apoptotic cells first induces higher order chromatin cleavage into DNA fragments ≥ 50 kb in length, followed by inter- and intranucleosomal DNA cleavages with products possessing significant internal single-stranded nicks spaced at nucleosomal (∼190 bases) and subnucleosomal (∼10 bases) periodicities. We demonstrate that both exonucleases and DNase I stimulate the ability of endoG to generate double-stranded DNA cleavage products at physiological ionic strengths, suggesting that these activities work in concert with endoG in apoptotic cells to ensure efficient DNA breakdown. Endonuclease G (endoG) is released from mitochondria during apoptosis and is in part responsible for internucleosomal DNA cleavage. Here we report the action of the purified human recombinant form of this endonuclease on naked DNA and chromatin substrates. The addition of the protein to isolated nuclei from non-apoptotic cells first induces higher order chromatin cleavage into DNA fragments ≥ 50 kb in length, followed by inter- and intranucleosomal DNA cleavages with products possessing significant internal single-stranded nicks spaced at nucleosomal (∼190 bases) and subnucleosomal (∼10 bases) periodicities. We demonstrate that both exonucleases and DNase I stimulate the ability of endoG to generate double-stranded DNA cleavage products at physiological ionic strengths, suggesting that these activities work in concert with endoG in apoptotic cells to ensure efficient DNA breakdown. second mitochondria-derived activator of caspase/(direct IAP binding protein with low pI) apoptosis-inducing factor caspase-activated deoxyribonuclease (also termed DFF40) DNA fragmentation factor exonuclease III micrococcal nuclease Nonidet P-40 topoisomerase II long terminal repeat human immunodeficiency virus 1 Endonuclease G inhibitor of CAD. Apoptosis, or programmed cell death, plays an important role in both development and maintenance of tissue homeostasis (reviewed in Refs. 1Jacobson M.D. Weil M. Raff M.C. Cell. 1997; 88: 347-354Abstract Full Text Full Text PDF PubMed Scopus (2428) Google Scholar and 2Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4578) Google Scholar). Two apoptotic pathways have been identified: the death-receptor pathway and the mitochondrial pathway (3Green D.R. Cell. 2000; 102: 1-4Abstract Full Text Full Text PDF PubMed Scopus (900) Google Scholar). Mitochondria have been shown to harbor multiple apoptogenic factors including cytochrome c, procaspases, SMAC/DIABLO,1 AIF, and endoG (4Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4452) Google Scholar, 5Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1815) Google Scholar, 6Du C., C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2974) Google Scholar, 7Verhagen A. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1998) Google Scholar, 8Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Nature. 1999; 397: 441-446Crossref PubMed Scopus (3494) Google Scholar, 9Li L.Y. Xu L. Wang X. Nature. 2001; 412: 95-99Crossref PubMed Scopus (1422) Google Scholar, 10Parrish J. Li L. Klotz K. Ledwich D. Wang X. Xue D. Nature. 2001; 412: 90-94Crossref PubMed Scopus (355) Google Scholar). Both cytochrome c and SMAC/DIABLO are involved in caspase activation, whereas AIF and endoG have been associated with one of the hallmarks of the terminal stages of apoptosis, DNA breakdown (11Wyllie A.H. Nature. 1980; 284: 555-556Crossref PubMed Scopus (4235) Google Scholar, 12Wyllie A.H. Morris R.G. Smith A.L. Dunlop D. J. Pathol. 1984; 142: 66-77Crossref Scopus (1483) Google Scholar). Apoptotic cell genomic DNA cleavage occurs in at least two stages: initial cleavage at intervals of ≥50 kb, consistent with the size of chromatin loop domains, followed by a second stage of internucleosomal DNA cleavage (also called DNA laddering) (13Oberhammer F. Wilson J.W. Dive C. Morris I.D. Hickman J.A. Waleling A.E. Walker P.R. Sirorska M. EMBO J. 1993; 12: 3679-3684Crossref PubMed Scopus (1169) Google Scholar). AIF (8Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Nature. 1999; 397: 441-446Crossref PubMed Scopus (3494) Google Scholar), topo II (14Li T.-K. Chen A.Y., Yu, C. Mao Y. Wang H. Liu L.F. Genes Dev. 1999; 13: 1553-1560Crossref PubMed Scopus (150) Google Scholar), and caspase-treated DFF/CAD-ICAD (15Sakahira H. Enari M. Ossawa Y. Uchiyama Y. Nagata S. Curr. Biol. 1999; 9: 543-546Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 16Zhang J. Lee H. Lou D.W. Bovin G.P. Xu M. Biochem. Biophys. Res. Commun. 2000; 274: 225-229Crossref PubMed Scopus (25) Google Scholar, 17Widlak P. Cell. Mol. Biol. Lett. 2000; 5: 373-379Google Scholar) have each been implicated in the higher order DNA cleavage reaction. Nucleosomal DNA laddering, on the other hand, has been associated with several endonucleases, including caspase-activated DFF/CAD-ICAD (18Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1655) Google Scholar, 19Liu X. Li P. Widlak P. Zou H. Luo X. Garrard W.T. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8461-8466Crossref PubMed Scopus (505) Google Scholar, 20Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2834) Google Scholar, 21Sakahira H. Enari M. Nagata S. Nature. 1998; 391: 96-99Crossref PubMed Scopus (1442) Google Scholar, 22Halenbeck R. MacDonald H. Roulston A. Chen T.T. Conroy L. Williams L.T. Curr. Biol. 1998; 8: 537-540Abstract Full Text Full Text PDF PubMed Google Scholar, 23Liu X. Zou H. Widlak P. Garrard W. Wang X. J. Biol. Chem. 1999; 274: 13836-13840Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 24Widlak P. Li P. Wang X. Garrard W.T. J. Biol. Chem. 2000; 275: 8226-8232Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 25Widlak P. Garrard W.T. Mol. Cell Biochem. 2001; 218: 125-130Crossref PubMed Scopus (46) Google Scholar), endoG (9Li L.Y. Xu L. Wang X. Nature. 2001; 412: 95-99Crossref PubMed Scopus (1422) Google Scholar, 10Parrish J. Li L. Klotz K. Ledwich D. Wang X. Xue D. Nature. 2001; 412: 90-94Crossref PubMed Scopus (355) Google Scholar), and DNase I (26Oliveri M. Daga A. Cantoni C. Lunardi C. Millo R. Puccetti A. Eur. J. Immunol. 2001; 31: 743-751Crossref PubMed Scopus (84) Google Scholar). Although some of the catalytic properties of endoG have been reported previously, nucleic acid, not chromatin substrates, had been employed. In addition, most of these studies used various partially purified forms of the protein from different tissue sources, and the possible contributions of impurities remain uncertain (27Ruiz-Carrillo A. Renaud J. EMBO J. 1987; 6: 401-407Crossref PubMed Scopus (95) Google Scholar, 28Côté J. Renaud J. Ruiz-Carrillo A. J. Biol. Chem. 1989; 264: 3301-3310Abstract Full Text PDF PubMed Google Scholar, 29Côté J. Ruiz-Carrillo A. Science. 1993; 261: 765-769Crossref PubMed Scopus (202) Google Scholar, 30Gerschenson M. Houmiel K.L. Low R.L. Nucleic Acids Res. 1995; 23: 88-97Crossref PubMed Scopus (48) Google Scholar, 31Ikeda S. Ozaki K. Biochem. Biophys. Res. Commun. 1997; 235: 291-294Crossref PubMed Scopus (46) Google Scholar). Furthermore, this nuclease was originally thought to play a role in mitochrondrial DNA replication (29Côté J. Ruiz-Carrillo A. Science. 1993; 261: 765-769Crossref PubMed Scopus (202) Google Scholar), which seems unlikely because a yeast knockout exhibits no phenotype (32Zassenhaus H.P. Hofmann T.J. Uthayshanker R. Vincet R.D. Zona M. Nucleic Acids Res. 1988; 16: 3283-3296Crossref PubMed Scopus (62) Google Scholar), and the enzyme co-localizes with cytochromec in the intermembrane space as opposed to the matrix where DNA replication occurs (9Li L.Y. Xu L. Wang X. Nature. 2001; 412: 95-99Crossref PubMed Scopus (1422) Google Scholar). One newly recognized function for endoG is as a caspase-independent pathway for DNA breakdown during apoptosis (9Li L.Y. Xu L. Wang X. Nature. 2001; 412: 95-99Crossref PubMed Scopus (1422) Google Scholar,10Parrish J. Li L. Klotz K. Ledwich D. Wang X. Xue D. Nature. 2001; 412: 90-94Crossref PubMed Scopus (355) Google Scholar). Here we study the action of homogenous human recombinant endoG on DNA and chromatin substrates. We have found that the enzyme possesses novel properties including cooperation with exonuclease and DNase I for more efficient DNA breakdown under physiological ionic strengths. Full-length human endoG cDNA with an additional six histidine residues appended to its C terminus and cloned into pFastBacI (Life Technologies, Inc.), was transformed into DH10Bac cells (Life Technologies, Inc.), and the recombinant viral DNA was purified according to the Bac-to-Bac baculovirus expression procedure. The purified bacmids were used to transfect Sf21 insect cells using CellFECTIN reagent (Life Technologies, Inc.). Transfected cells were grown in IPL41 medium with 10% fetal calf serum, 2.6 g/liter tryptose phosphate, 4 g/liter yeastolate, and 0.1% Pluronic F-68 plus penicillin (100 units/ml), streptomycin (100 μg/ml), and Fungizone (0.25 g/ml). Forty milliliters of the amplified viral stock was used to infect 1 liter of cells at 2 × 106 cells/ml. The infected cells were harvested 2 days later, and resuspended and homogenized in 5 volumes of buffer T (20 mm Tris-HCl (pH 8.0), 50 mm NaCl, 1 mm β-mercaptoethanol, and 0.1 mm phenylmethylsulfonyl fluoride) with 0.5% Nonidet P-40. These and all subsequent operations were conducted at 4 °C. The cell homogenate was centrifuged at 10,000 × g for 30 min, and the supernatant was loaded onto a 3-ml nickel affinity column. The column was washed with 30 ml of buffer T with 0.5% Nonidet P-40, then 30 ml of buffer T, and followed by 200 ml of buffer T plus 1m NaCl. The column was washed once more with buffer T, and proteins were eluted with buffer T plus 250 mm imidazole. The eluted proteins were loaded onto a Superdex 200 column (Amersham Biosciences, Inc.) and eluted with buffer A (20 mmHepes-KOH, pH 7.0, 10 mm KCl, 1.5 mmMgCl2, 1 mm NaEDTA, 1 mm NaEGTA, 1 mm dithiothreitol, and 0.1 mmphenylmethylsulfonyl fluoride). The peak fractions were loaded onto a Mono S column (Amersham Biosciences, Inc.) and eluted with a 20-ml linear gradient from 0 to 300 mm NaCl in buffer A. The peak of endoG nuclease activity, eluting at ∼80 mm NaCl, was stored at −20 °C in 50% glycerol. Protein purity was assessed by SDS, 15% polyacrylamide gel electrophoresis. Plasmid pWLTR11 DNA (33Widlak P. Gaynor R.B. Garrard W.T. J. Biol. Chem. 1997; 272: 17654-17661Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), ϕX174 virion DNA (New England BioLabs), or high molecular weight RNA from wheat germ (Calbiochem) were employed as non-chromatin substrates. Nuclei were purified from HeLa S3 cells. Cells were lysed in a buffer consisting of 10 mm KCl, 0.25 m sucrose, 4 mm MgCl2, 1 mm dithiothreitol, 20 mm Hepes, pH 7.5, 0.5% Nonidet P-40, and CompleteTM (Roche Molecular Biochemicals) protease inhibitors set and then washed two times in the same buffer without Nonidet P-40. One microgram of naked DNA was incubated for 30 min at 37 °C with endoG (final concentration: 0.5 α unit/ml) in buffer consisting of 10 mm KCl, 3 mm MgCl2, 0.5 mm dithiothreitol, 20 mm Hepes, pH 7.5 (final volume: 15 μl), if not stated otherwise. Two micrograms of DNA (as nuclei) were incubated for varying times (5–45 min) at 37 °C with either recombinant purified endoG (final concentration 10 units/ml), recombinant purified activated DFF (50 units/ml, Ref. 24Widlak P. Li P. Wang X. Garrard W.T. J. Biol. Chem. 2000; 275: 8226-8232Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), MNase (Worthington, 40 units/ml), or DNase I (Worthington, 10 units/ml) in a buffer consisting of 100 mmKCl, 3 mm MgCl2, 1 mmCaCl2, 0.5 mm dithiothreitol, 20 mmHepes, pH 7.5 (final volume 20 μl). Nuclease reactions were terminated by mixing with one-half volume of stop solution (0.6% SDS, 50 mm EDTA, and 6-mg/ml non-chromatin reactions gel buffer was and were then on using as the buffer and with reactions were incubated for 1 at °C with and then DNA was purified by and DNA was in buffer and incubated with a of and DNA was then on X. Zou H. Widlak P. Garrard W. Wang X. J. Biol. Chem. 1999; 274: 13836-13840Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), polyacrylamide or of and and with of at cleavage was as P. Li P. Wang X. Garrard W.T. J. Biol. Chem. 2000; 275: 8226-8232Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). a of was from with and then incubated with either purified recombinant activated DFF or cleavage on both DNA was with I or I products were on polyacrylamide with the Nuclei were incubated with purified recombinant endoG or activated DFF for and min at 37 and the nuclease reactions were terminated by (final concentration: 0.1 nuclei were incubated for 30 min in the of 0.1 mm topo II inhibitor were in low and were then incubated for 3 at 37 °C in solution SDS, 20 mm EDTA, and with the DNA was in a gel and then with Nuclei were incubated with either purified recombinant purified recombinant activated or MNase and then DNA was DNA was isolated on a low DNA with terminal and on a polyacrylamide DNA was with (Roche Molecular with (Roche Molecular and on an We a baculovirus expression to a endoG which was purified to and from insect cell by on nickel affinity Superdex and Mono as by gel with The molecular of the protein was whereas the size for the recombinant protein to the and of the (29Côté J. Ruiz-Carrillo A. Science. 1993; 261: 765-769Crossref PubMed Scopus (202) Google Scholar) is the we found that the had been from the purified recombinant protein not that the in is has pH for double-stranded DNA at pH and pH The higher pH is for by DNA and the that endoG has on single-stranded nucleic In with (27Ruiz-Carrillo A. Renaud J. EMBO J. 1987; 6: 401-407Crossref PubMed Scopus (95) Google Scholar, 30Gerschenson M. Houmiel K.L. Low R.L. Nucleic Acids Res. 1995; 23: 88-97Crossref PubMed Scopus (48) Google Scholar), the enzyme either or and not as its and is at physiological ionic and The of or in with enzyme In with (29Côté J. Ruiz-Carrillo A. Science. 1993; 261: 765-769Crossref PubMed Scopus (202) Google Scholar, 30Gerschenson M. Houmiel K.L. Low R.L. Nucleic Acids Res. 1995; 23: 88-97Crossref PubMed Scopus (48) Google Scholar, 31Ikeda S. Ozaki K. Biochem. Biophys. Res. Commun. 1997; 235: 291-294Crossref PubMed Scopus (46) Google Scholar), both single-stranded DNA and RNA are double-stranded DNA 2 that are first by single-stranded by endoG We the endoG cleavage for naked DNA cleavage in with DFF as a The cleavage products DNA of the and were on We cleavage at the and found that which DNA cleavages endoG single-stranded of G residues 3 in with a report J. Renaud J. Ruiz-Carrillo A. J. Biol. Chem. 1989; 264: 3301-3310Abstract Full Text PDF PubMed Google Scholar). We cleavages of C and A and of G residues In addition, the DNA by activated DFF by because by terminal 3 in with studies J. Renaud J. Ruiz-Carrillo A. J. Biol. Chem. 1989; 264: 3301-3310Abstract Full Text PDF PubMed Google Scholar). In studies on naked nucleic that the catalytic properties of the recombinant protein are in with studies (27Ruiz-Carrillo A. Renaud J. EMBO J. 1987; 6: 401-407Crossref PubMed Scopus (95) Google Scholar, 28Côté J. Renaud J. Ruiz-Carrillo A. J. Biol. Chem. 1989; 264: 3301-3310Abstract Full Text PDF PubMed Google Scholar, 29Côté J. Ruiz-Carrillo A. Science. 1993; 261: 765-769Crossref PubMed Scopus (202) Google Scholar, 30Gerschenson M. Houmiel K.L. Low R.L. Nucleic Acids Res. 1995; 23: 88-97Crossref PubMed Scopus (48) Google Scholar, 31Ikeda S. Ozaki K. Biochem. Biophys. Res. Commun. 1997; 235: 291-294Crossref PubMed Scopus (46) Google Scholar). We on the action of the protein on chromatin substrates. apoptosis, initial DNA cleavage occurs at intervals the size of chromatin loop domains, ≥50 kb (13Oberhammer F. Wilson J.W. Dive C. Morris I.D. Hickman J.A. Waleling A.E. Walker P.R. Sirorska M. EMBO J. 1993; 12: 3679-3684Crossref PubMed Scopus (1169) Google Scholar). endoG higher order cleavage we the protein to isolated HeLa cell nuclei and the cleavage products by gel electrophoresis. nuclei were with DFF or with the topo II inhibitor because each have been shown to generate higher order DNA cleavage (13Oberhammer F. Wilson J.W. Dive C. Morris I.D. Hickman J.A. Waleling A.E. Walker P.R. Sirorska M. EMBO J. 1993; 12: 3679-3684Crossref PubMed Scopus (1169) Google Scholar, 17Widlak P. Cell. Mol. Biol. Lett. 2000; 5: 373-379Google Scholar). 4 that endoG higher order DNA cleavage in nuclei from non-apoptotic cells. the ability of endoG to generate DNA we its action with that of MNase and shown in of isolated HeLa cell nuclei with MNase or DFF in DNA by as by and the Furthermore, cleavage was for endoG which subnucleosomal DNA fragments the and of single-stranded we gel electrophoresis. the products on a gel in the first a second of was under shown in 5 this that in to caspase-activated which double-stranded DNA fragments internal endoG DNA fragments internal nicks spaced at a significant of fragments as in in the first nicks in under single-stranded and DNA fragments 5 the action of endoG at the subnucleosomal we HeLa cell nuclei DNA products on a high shown in endoG chromatin with the same as DNase at L. Nucleic Acids Res. 6: PubMed Scopus Google Scholar, Scholar). that this cleavage occurs the was by first DNA fragments by and then the for on a shown in that was with endoG exhibits internal nicks spaced at nicks are from MNase or DFF A of that other proteins the ability of endoG to DNA during the of the enzyme is low at physiological ionic 1 C and Refs. A. Renaud J. EMBO J. 1987; 6: 401-407Crossref PubMed Scopus (95) Google Scholar and 30Gerschenson M. Houmiel K.L. Low R.L. Nucleic Acids Res. 1995; 23: 88-97Crossref PubMed Scopus (48) Google Scholar). the enzyme is on single-stranded nucleic and Refs. 30Gerschenson M. Houmiel K.L. Low R.L. Nucleic Acids Res. 1995; 23: 88-97Crossref PubMed Scopus (48) Google Scholar and 31Ikeda S. Ozaki K. Biochem. Biophys. Res. Commun. 1997; 235: 291-294Crossref PubMed Scopus (46) Google Scholar). is a high internucleosomal DNA endoG of isolated not of the DNA during apoptosis in DFF knockout cells (9Li L.Y. Xu L. Wang X. Nature. 2001; 412: 95-99Crossref PubMed Scopus (1422) Google Scholar). DNase I knockout cells to chromatin under apoptotic that DFF (26Oliveri M. Daga A. Cantoni C. Lunardi C. Millo R. Puccetti A. Eur. J. Immunol. 2001; 31: 743-751Crossref PubMed Scopus (84) Google Scholar). the by DNase I of chromatin exhibits a high internucleosomal DNA which is not as as the reported DNase apoptotic (26Oliveri M. Daga A. Cantoni C. Lunardi C. Millo R. Puccetti A. Eur. J. Immunol. 2001; 31: 743-751Crossref PubMed Scopus (84) Google Scholar). these to nicks by DNase I for endoG action because of single-stranded and exonuclease of nicks by endoG or DNase I stimulate DNA under physiological ionic strengths. A that the DNA products are more of naked DNA with DNase I and Furthermore, endoG by of on a naked DNA on chromatin to more DNA In the of nucleosomal fragments is either DNase or both were with endoG not stimulate DNase I because DNase I not single-stranded DNA In these into that are to in DNA during is released from the intermembrane space of mitochondria during apoptosis in a caspase-independent and a novel pathway for DNA breakdown (9Li L.Y. Xu L. Wang X. Nature. 2001; 412: 95-99Crossref PubMed Scopus (1422) Google Scholar, 10Parrish J. Li L. Klotz K. Ledwich D. Wang X. Xue D. Nature. 2001; 412: 90-94Crossref PubMed Scopus (355) Google Scholar). that the in properties of endoG cleavage the phenotype of the DNA products by first higher order DNA cleavage into fragments kb followed by nucleosomal DNA laddering, with fragments These have been used in for cells We have found that the action of human recombinant endoG on naked nucleic is in with (27Ruiz-Carrillo A. Renaud J. 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Contact dermatitis is a common reason for patient visits to primary-care clinics and represents up to 7% of all dermatologic consultations in the US. Substantial progress has been made in elucidating the pathophysiology of contact dermatitis, particularly the allergic form. A better understanding of pathologic mechanisms has led to improved management of cases and will continue to advance treatment modalities. The present paper reviews the pathogenesis and current treatment of allergic contact dermatitis and speculates on the prospects for improved future therapy.