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• W2029320528 abstract "DNA fragmentation/degradation is an important step for apoptosis. However, in unicellular organisms such as yeast, this process has rarely been investigated. In the current study, we revealed eight apoptotic nuclease candidates in Saccharyomyces cerevisiae, analogous to the Caenorhabditis elegans apoptotic nucleases. One of them is Tat-D. Sequence comparison indicates that Tat-D is conserved across kingdoms, implicating that it is evolutionarily and functionally indispensable. In order to better understand the biochemical and biological functions of Tat-D, we have overexpressed, purified, and characterized the S. cerevisiae Tat-D (scTat-D). Our biochemical assays revealed that scTat-D is an endo-/exonuclease. It incises the double-stranded DNA without obvious specificity via its endonuclease activity and excises the DNA from the 3′- to 5′-end by its exonuclease activity. The enzyme activities are metal-dependent with Mg2+ as an optimal metal ion and an optimal pH around 5. We have also identified three amino acid residues, His185, Asp325, and Glu327, important for its catalysis. In addition, our study demonstrated that knock-out of TAT-D in S. cerevisiae increases the TUNEL-positive cells and cell survival in response to hydrogen hyperoxide treatment, whereas overexpression of Tat-D facilitates cell death. These results suggest a role of Tat-D in yeast apoptosis. DNA fragmentation/degradation is an important step for apoptosis. However, in unicellular organisms such as yeast, this process has rarely been investigated. In the current study, we revealed eight apoptotic nuclease candidates in Saccharyomyces cerevisiae, analogous to the Caenorhabditis elegans apoptotic nucleases. One of them is Tat-D. Sequence comparison indicates that Tat-D is conserved across kingdoms, implicating that it is evolutionarily and functionally indispensable. In order to better understand the biochemical and biological functions of Tat-D, we have overexpressed, purified, and characterized the S. cerevisiae Tat-D (scTat-D). Our biochemical assays revealed that scTat-D is an endo-/exonuclease. It incises the double-stranded DNA without obvious specificity via its endonuclease activity and excises the DNA from the 3′- to 5′-end by its exonuclease activity. The enzyme activities are metal-dependent with Mg2+ as an optimal metal ion and an optimal pH around 5. We have also identified three amino acid residues, His185, Asp325, and Glu327, important for its catalysis. In addition, our study demonstrated that knock-out of TAT-D in S. cerevisiae increases the TUNEL-positive cells and cell survival in response to hydrogen hyperoxide treatment, whereas overexpression of Tat-D facilitates cell death. These results suggest a role of Tat-D in yeast apoptosis. Apoptosis is an important biological process required for maintaining tissue homeostasis, removal of redundant or damaged cells, normal developmental progression, and response to various toxic stimuli (1Wyllie A.H. Kerr J.F.R. Currie A.R. Int. Rev. Cytol. 1980; 68: 251-306Crossref PubMed Scopus (6725) Google Scholar, 2Rodriguez-Tarduchy G. Collins M. Lopez-Rivas A. EMBO J. 1990; 9: 2997-3002Crossref PubMed Scopus (207) Google Scholar, 3McConkey D.J. Hartzell P. Chow S.C. Orrenius S. Jonkal M. J. Biol. Chem. 1990; 265: 3009-3011Abstract Full Text PDF PubMed Google Scholar, 4Kawabe Y. Ochi A. Nature. 1991; 349: 245-248Crossref PubMed Scopus (662) Google Scholar, 5Hengartner M.O. Riddle D.L. Blumenthal T. Meyer B.J. Press J.R. C. elegans II. Cold Spring Harbor Laboratory Press, Plainview, NY1997: 383-415Google Scholar, 6Metzstein M.M. Stanfield G.M. Horvitz H.R. Trends Genet. 1998; 14: 410-416Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). It is a programmed cell death characterized by cell shrinkage, membrane blebbing, chromatin condensation around the periphery of the nucleus, and DNA fragmentation and degradation (1Wyllie A.H. Kerr J.F.R. Currie A.R. Int. Rev. Cytol. 1980; 68: 251-306Crossref PubMed Scopus (6725) Google Scholar). Failure of this process could lead to developmental defects, immunological and neuro-degenerative disorders, and formation of cancers in mammals and other higher organisms (7Kawane K. Fukuyama H. Kondoh G. Takeda J. Ohsawa Y. Uchiyama Y. Nagata S. Science. 2001; 292: 1546-1549Crossref PubMed Scopus (299) Google Scholar, 8Kawane K. Fukuyama H. Yoshida H. Nagase H. Ohsawa Y. Uchiyama Y. Okada K. Iida T. Nagata S. Nat. Immunol. 2003; 4: 138-144Crossref PubMed Scopus (194) Google Scholar, 9Walport M.J. Nat. Genet. 2000; 25: 135-136Crossref PubMed Scopus (126) Google Scholar, 10Yasutomo K. Horiuchi T. Kagami S. Tsukamoto H. Hashimura C. Urushihara M. Kuroda Y. Nat. Genet. 2001; 28: 313-314Crossref PubMed Scopus (473) Google Scholar). A critical and late step in apoptosis is DNA degradation, which requires participation of endo- and exonucleases. So far, at least three nucleases have been well characterized to play a role in DNA degradation for apoptosis, including DFF40/CAD, Cps-6/endonuclease G, and Nuc 1/DNase II (11Liu 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 (502) Google Scholar, 12Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2807) Google Scholar, 13Li L.Y. Luo X. Wang X. Nature. 2001; 412: 95-99Crossref PubMed Scopus (1399) Google Scholar, 14Parrish J. Li L. Klotz K. Ledwich D. Wang X. Xue D. Nature. 2001; 412: 90-94Crossref PubMed Scopus (353) Google Scholar, 15Barry M.A. Eastman A. Arch. Biochem. Biophys. 1993; 300: 440-450Crossref PubMed Scopus (433) Google Scholar, 16Lyon C.J. Evans C.J. Bill B.R. Otsuka A.J. Aguilera R.J. Gene (Amst.). 2000; 252: 147-154Crossref PubMed Scopus (35) Google Scholar, 17Evans C.J. Merriam J.R. Aguilera R.J. Gene (Amst.). 2002; 295: 61-70Crossref PubMed Scopus (10) Google Scholar, 18Scovassi A.I. Torrigila A. Eur. J. Histochem. 2003; 47: 185-194Crossref PubMed Scopus (34) Google Scholar). Of the three nucleases, DFF40/CAD is caspase-dependent, whereas the other two are caspase-independent. More recently, seven additional nucleases have been identified for apoptosis in Caenorhabditis elegans, including Crn-1/FEN-1 and Crn-2/Tat-D (19Parrish J.Z. Xue D. Mol. Cell. 2003; 11: 987-996Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Further analysis indicated that Crn-1/FEN-1, a flap endo- and 5′ to 3′ exonuclease critical for DNA replication, repair, and recombination (20Liu Y. Kao H.I. Bambara R.A. Annu. Rev. Biochem. 2004; 73: 589-615Crossref PubMed Scopus (303) Google Scholar), degrades DNA through coordination with Cps-6/endonuclease G in C. elegans cells (19Parrish J.Z. Xue D. Mol. Cell. 2003; 11: 987-996Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Evidence is accumulating that apoptosis also exists in unicellular organisms, such as in budding yeast Saccharomyces cerevisiae (21Burhans W.C. Weinberger M. Marchetti M.A. Ramachandran L. D'Urso G. Huberman J.A. Mutat Res. 2003; 532: 227-243Crossref PubMed Scopus (94) Google Scholar, 22Madeo F. Herker E. Wissing S. Jungwirth H. Eisenberg T. Frohlich K.U. Curr. Opin. Microbiol. 2004; 7: 655-660Crossref PubMed Scopus (261) Google Scholar, 23Frohlich K.U. Madeo F. Exp. Gerontol. 2001; 37: 27-31Crossref PubMed Scopus (41) Google Scholar). The apoptotic response of S. cerevisiae has been observed in aging cells (24Laun P. Pichova A. Madeo F. Fuchs J. Ellinger A. Kohlwein S. Dawes I. Frohlich K.U. Breitenbach M. Mol. Microbiol. 2001; 39: 1166-1173Crossref PubMed Google Scholar) and mutants with mutations in ATPase CDC48 (25Madeo F. Frohlich E. Frohlich K.U. J. Cell Biol. 1997; 139: 729-734Crossref PubMed Scopus (680) Google Scholar) or anti-silencing protein ASF1 (26Yamaki M. Umehara T. Chimura T. Horikoshi M. Genes Cells. 2001; 6: 1043-1054Crossref PubMed Scopus (88) Google Scholar). In addition, weak acidic (27Ludovico P. Sousa M.J. Silva M.T. Leao C. Corte-Real M. Microbiology. 2001; 147: 2409-2415Crossref PubMed Scopus (412) Google Scholar, 28Ludovico P. Rodrigues F. Almeida A. Silva M.T. Barrientos A. Corte-Real M. Mol. Biol. Cell. 2002; 13: 2598-2606Crossref PubMed Scopus (319) Google Scholar), oxidative stress (29Madeo F. Frohlich E. Ligr M. Grey M. Sigrist S.J. Wolf D.H. Frohlich K.U. J. Cell Biol. 1999; 145: 757-767Crossref PubMed Scopus (868) Google Scholar), salt stress (30Huh G.H. Damsz B. Matsumoto T.K. Reddy M.P. Rus A.M. Ibeas J.I. Narasimhan M.L. Bressan R.A. Hasegawa P.M. Plant J. 2002; 29: 649-659Crossref PubMed Scopus (237) Google Scholar, 31Wadskog I. Maldener C. Proksch A. Madeo F. Adler L. Mol. Biol. Cell. 2004; 15: 1436-1444Crossref PubMed Scopus (80) Google Scholar), UV irradiation (32Del Carratore R. Della Croce C. Simili M. Taccini E. Scavuzzo M. Sbrana S. Mutat. Res. 2002; 513: 183-191Crossref PubMed Scopus (89) Google Scholar), and mating pheromone treatment (33Severin F.F. Hyman A.A. Curr. Biol. 2002; 12: 233-235Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) can also induce apoptosis in yeast cells. Dying yeast cells under these conditions display several markers that are characteristic of apoptosis. These include the rapid exposure of phosphatidylserine on the outer cell membrane, the margination of chromatin in nuclei, nuclear fragmentation, and the degradation of DNA. Exposure of the cells to the protein translation inhibitor, cycloheximide, prevents these death-associated changes, indicating that the death response requires active protein synthesis (29Madeo F. Frohlich E. Ligr M. Grey M. Sigrist S.J. Wolf D.H. Frohlich K.U. J. Cell Biol. 1999; 145: 757-767Crossref PubMed Scopus (868) Google Scholar). Although yeast has been a good model system for apoptotic studies (34Manon S. Antioxid. Redox Signal. 2004; 6: 259-267Crossref PubMed Scopus (20) Google Scholar), the process and mechanism of apoptotic DNA fragmentation/degradation in yeast has rarely been investigated. It is unclear which nucleases are involved in yeast apoptosis. To gain knowledge on apoptotic nucleases in yeast cells, we started with a comprehensive homology search based on presently available information on apoptotic nucleases of multicellular organisms, including caspase-dependent DFF40/CAD and caspase-independent nucleases, such as Cps-6/endo-nuclease G, Nuc-1/DNase IIa, Crn-1/FEN-1, Crn-2/Tat-D, Crn-3/PM/ScI-100, Crn-4/RNase T, Crn-5/Rrp46, Crn-6/DNase IIb, Cyp-13/Cyp E, DNase I, and DNase γ. The search resulted in eight candidate nucleases for yeast apoptotic DNA fragmentation/degradation, which correspond to the above listed except for DFF40/CAD, Nuc-1/DNase IIa, DNase I, and DNase γ. Among the eight potential apoptotic nucleases in yeast, Tat-D appears most conserved. It exists in organisms across all kingdoms. However, the biochemical and biological functions of Tat-D are still poorly understood. Tat-D was first implicated in Schizosaccharomyces pombe to have a role in spindle elongation and chromosome decondensation during progression of late anaphase (35Samejima I. Yanagida M. J. Cell Biol. 1994; 127: 1655-1670Crossref PubMed Scopus (90) Google Scholar). It was later, however, considered to be an integral membrane component of the Sec-independent protein export complex that is the so-called twin arginine translocation (Tat) system in Escherichia coli (36Sargent F. Bogsch E.G. Stanley N.R. Wexler M. Robinson C. Berks B.C. Palmer T. EMBO J. 1998; 17: 3640-3650Crossref PubMed Scopus (443) Google Scholar). Further analysis indicated that Tat-D is actually a DNase rather than a membrane protein (37Wexler M. Sargent F. Jack R.L. Stanley N.R. Bogsch E.G. Robinson C. Berks B.C. Palmer T. J. Biol. Chem. 2000; 275: 16717-16722Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). This result is consistent with the recent finding that Tat-D has a role in apoptotic DNA degradation in C. elegans (19Parrish J.Z. Xue D. Mol. Cell. 2003; 11: 987-996Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). In the current study, we characterized the biochemical functions of Tat-D using purified yeast recombinant Tat-D protein and determined its role in apoptotic DNA degradation in yeast cells. Tat-D was revealed to be a metal-dependent endo-/exonuclease with optimal activities under acidic conditions. The TAT-D gene knock-out in yeast has an insignificant effect on cell growth and DNA repair but shows alteration in apoptosis induced by hydrogen superoxide. Furthermore, overexpression of Tat-D in yeast cells facilitates cell death. These results indicate that Tat-D is involved in yeast apoptosis. Protein Blast Search and Sequence Alignment—For the protein Blast search, we obtained nine C. elegans (Cps-6 Nuc 1, Crn-1, Crn-2, Crn-3, Crn-4, Crn-5, Crn-6, and Cyp-13) and three human (DFF40/CAD, DNase I, and DNase γ) nuclease proteins as query elements. We searched their counterparts in the S. cerevisiae data base (available on the World Wide Web at www.yeastgenome.org). In addition, we searched for all of the available Tat-D homologues in the NCBI data base using Crn-2 protein as the query sequence. The relevant proteins were aligned, and their sequence similarities were calculated using the ClustalW 1.8 multiple sequence alignment algorithm at the BCM Search Launcher, Baylor College of Medicine HGSC (available on the World Wide Web at searchlauncher.bcm.tmc.edu). Protein Overexpression, Purification, and Site-directed Mutagenesis—The ScTAT-D DNA fragment was generated by PCR using S. cerevisiae genomic DNA and the primers listed in Table I. The DNA fragment was then inserted into the pET-28b overexpression vector. The three mutant proteins created for this study were prepared using the QuikChange™ site-directed mutagenesis kit from Stratagene (La Jolla, CA) and with primers listed in Table I. The pET-28b plasmid with the insertion of yTAT-D DNA was used as a template. Site-directed mutagenesis, overexpression, and purification of wild type and mutant FEN-1 enzymes were carried out based on our previously published procedures (38Qiu J. Qian Y. Frank P. Wintersberger U. Shen B. Mol. Cell. Biol. 1999; 19: 8361-8371Crossref PubMed Scopus (136) Google Scholar, 39Qiu J. Bimston D.N. Partikian A. Shen B. J. Biol. Chem. 2002; 277: 24659-24666Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 40Qiu J. Liu R. Chapados B.R. Sherman M. Tainer J.A. Shen B. J. Biol. Chem. 2004; 279: 24394-24402Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar).Table IDNA oligonucleotides used for making DNA substrates and yTat-D mutantsOligonucleotide nameOligonucleotide sequenceOligonucleotide useD15′-GGACTCTGCCTCAAGACGGTAGTCAACGTG-3′SubstrateD25′-CACGTTGACTACCGTC-3′SubstrateD35′-TTGAGGCAGAGTCC-3′SubstrateD45′-GGACTCTGCCTCAA-3′SubstrateD55′-AGGACTCTGCCTCAA-3′SubstrateD65′-ACTCTGCCTCAA-3′SubstrateE185A-F5′-AATGTCAAGCAGTCCTAACTTTGAT-3′E185A mutationE185A-R5′-GGACTCTGCCTCAAGACGGTAGTCA-3′E185A mutationD325A-F5′-GGGCATGTTCTACGCGACCATTCGC-3′D325A mutationD325A-R5′-GCGAATGGTCGCGTAGAACATGCCC-3′D325A mutationD327A-F5′-CCGCACCATTGCGATGATGGAGAACGGC-3′D327A mutationD327A-R5′-GCCGTTCTCCATCATCGCAATGGTGCGG-3′D327A mutation1257NF5′-TGGACACTTTCGCTGCCAAGGCATTTTTTTGAGGCG-3′ScTAT-D CCAGTACTGTTATAGTTCGTACGCTGCAGGTCGAC-3′ disruption1257NR5′-TTACAGGTCGTCTTCCACGTGGTATCTATCAAAGTA GCCAGATCCACGTCCTATCGATGAATTCGAGCTCG-3′ScTAT-D CCAGTACTGTTATAGTTCGTACGCTGCAGGTCGAC-3′ disruption1257F5′-TCCTGGTAGCAATGGACAACTA-3′ScTAT-D CCAGTACTGTTATAGTTCGTACGCTGCAGGTCGAC-3′ disruption1257R5′-CTTTGCTCTTTGCACCAATAGA-3′ScTAT-D CCAGTACTGTTATAGTTCGTACGCTGCAGGTCGAC-3′ disruption Open table in a new tab DNA Substrate Preparation and scTat-D Nuclease Activity Assays— Protocols for DNA substrate preparation and nuclease activity assays were performed as previously published (40Qiu J. Liu R. Chapados B.R. Sherman M. Tainer J.A. Shen B. J. Biol. Chem. 2004; 279: 24394-24402Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Oligonucleotides used to construct the substrates are listed in Table I. Briefly, oligonucleotides shown in the figures were individually phosphorylated at the 5′-end. This was done by incubating 40 pmol of the oligonucleotide with 10 μCi of [γ-32P]ATP and 1 μl (10 units/μl) of polynucleotide kinase at 37 °C for 60 min. Polynucleotide kinase was then inactivated by heating at 72 °C for 10 min. 80 pmol of each remaining oligonucleotide comprising the substrates were added to the labeled oligonucleotides. The samples were incubated at 70 °C for 5 min followed by slow cooling to 25 °C, thus allowing the oligonucleotides to anneal and form the flap and nick-duplex substrates. Substrates were precipitated at -20 °C overnight after adding 20 μl of 3 m NaOAc and 1 ml of 100% ethanol. Substrates were collected by centrifugation and washed once with 70% ethanol and resuspended in sterile water. Reactions were carried out with the indicated amount of ScTat-D and 0.1 or 0.2 μm of DNA substrates in a reaction buffer containing 50 mm Tris (pH 8.0) and 10 or 5 mm MgCl2. Each reaction was then brought to a total volume of 10 μl with water. All reactions were incubated at 30 °C for 15 min and terminated by adding an equal volume of stop solution (95% formamide, 20 mm EDTA, 0.05% bromphenol blue, 0.05% xylene cyanol). An aliquot of each reaction was then run on a 15% denaturing polyacrylamide gel at 1900 V for 1 h. The gel was dried at 70 °C for 50 min, and the bands were visualized by autoradiography. Yeast Strains and Gene Disruption—Two yeast strains, RKY2672 and BY4741, were used for the disruption of ScTAT-D. The gene was knocked out using the PCR-mediated gene disruption method (41Baudin A. Ozier-Kalogeropopulous O. Deanouel A LaCroute F Cullin C. Nucleic Acids Res. 1993; 21: 3329-3330Crossref PubMed Scopus (1120) Google Scholar, 42Lorenz M.C. Muir R.S. Lim E. McElver J. Weber S.C. Heitman J. Gene (Amst.). 1995; 158: 113-117Crossref PubMed Scopus (257) Google Scholar). We used the plasmid pFA6a-KanMX4 (kindly provided by Dr. K. Kuchler) for amplifying the marker gene, Kanr. A DNA fragment of the marker gene flanked on the upstream side by a 53-bp upstream sequence of ScTAT-D and on the downstream side by a 53-bp downstream sequence of ScTAT-D was produced by PCR and used to transform yeast cells as indicated. The transformed cells were plated on selection medium, YPD with 200 μg/ml G418. ScTAT-D disruption was confirmed by PCR for the G418-resistant colonies. The RKY2672-based strains were used to examine the spontaneous mutation status due to the disruption of ScTAT-D. The By4741-based strains were used for assays on cell growth and survival rate with H2O2, 4′,6-diamidino-2-phenylindole, and annexin V staining and TdT-mediated dUTP nick end labeling (TUNEL) 1The abbreviations used are: TUNEL, TdT-mediated dUTP nick end labeling; PBS, phosphate-buffered saline. assays. Yeast Cell Survival Tests—Yeast cells were grown in YPD medium overnight and adjusted to identical density at A600. The cells were diluted by 100 times and grown in 10 ml of YPD medium until they reached exponential phase (A600 = 0.6). H2O2 was added to the desired concentrations. After a 2-h treatment with H2O2, cells were washed twice with distilled water, and the sample volume was adjusted to 5 ml. Cells treated with 0, 0.5, 1, 2, 3, and 6 mm H2O2 were diluted by 4000, 2000, 1000, and 500 times, respectively. 100 μl of diluted sample was plated onto YPD plates. The number of surviving colonies was determined after a 2-day incubation at 30 °C. The survival rate was calculated using the number of colonies with H2O2 treatment, multiplying by the dilutions during cell plating, and dividing by the number of control cells (without H2O2 treatment). Assays were repeated three times. TUNEL Assays on Yeast Cells—The procedure for the TUNEL assay follows Madeo et al. (29Madeo F. Frohlich E. Ligr M. Grey M. Sigrist S.J. Wolf D.H. Frohlich K.U. J. Cell Biol. 1999; 145: 757-767Crossref PubMed Scopus (868) Google Scholar). Cells were grown to early log phase (A600 = 0.6), and hydrogen superoxide was then added with the indicated amounts in Fig. 7. After 2 h, cells were fixed by adding 3.7% formaldehyde into the cell cultures. Cells were fixed for at least 1 h and then collected, washed, and digested with lyticase. The digested cells were applied to a polylysine-coated slide as described for immunofluorescence (43Adams A.E. Pringle J.R. J. Cell Biol. 1984; 98: 934-945Crossref PubMed Scopus (598) Google Scholar). The slides were rinsed with PBS and incubated with 0.3% H2O2 in methanol for 30 min at room temperature to block endogenous peroxidases. The slides were further rinsed with PBS, incubated in permeabilization solution (0.1% Triton X-100 and 0.1% sodium citrate) for 2 min on ice, rinsed twice with PBS, incubated with 10 μl of TUNEL reaction mixture (terminal deoxynucleotidyl transferase 200 units/ml, fluorescein isothiocyanate-labeled dUTP 10 mm, 25 mm Tris-HCl, 200 mm sodium cacodylate, 5 mm cobalt chloride; Roche Applied Science) for 60 min at 37 °C, and then rinsed 3 times with PBS. For the detection of peroxidase, cells were incubated with 10 μl of Converter-POD (anti-fluorescein isothiocyanate antibody, Fab fragment from sheep, conjugated with horseradish peroxidase) for 30 min at 37 °C, rinsed three times with PBS, and then stained with DAB-substrate solution (Roche Applied Science) for 10 min at room temperature. A coverslip was mounted with a drop of Kaiser's glycerol gelatin (Merck). Since staining intensity varies, only samples from the same slide were compared. 4′,6-Diamidino-2-phenylindole staining was performed as described previously (25Madeo F. Frohlich E. Frohlich K.U. J. Cell Biol. 1997; 139: 729-734Crossref PubMed Scopus (680) Google Scholar). To determine frequencies of TUNEL-positive cells, at least 300 cells of three independent experiments were evaluated. Presence of Potential Apoptotic Nucleases in Yeast—In order to identify apoptotic nucleases in yeast cells, we performed a Blast search using the yeast protein data base based on 12 known apoptotic nucleases so far identified from multicellular organisms as query sequences. The 12 known nucleases are listed in Table II, including DFF40/CAD, Cps-6/endonuclease G, Nuc-1/DNase IIa, Crn-1/FEN-1, Crn-2/Tat-D, Crn-3/PM/ScI-100, Crn-4/RNase T, Crn-5/Rrp46, Crn-6/DNase IIb, Cyp-13/Cyp E, DNase I, and DNase γ. Most of them are recently screened from C. elegans. After an extensive search, no homologue of DFF40/CAD in yeast cells was found, although this is considered the critical caspase-dependent nuclease in multicellular organisms, such as mammals and Drosophila. Among the other nucleases, eight nucleases have their homologues in yeast cells, including Cps-6/endonuclease G, Crn-1/FEN-1, Crn-2/Tat-D, Crn-3/PM/SCI-100, Crn-4/RNase T, Crn-5/Rrp46, Crn-6/DNase IIb, and Cyp-13/Cyp E (Table II). Except for Crn-2/Tat-D, Crn-3/PM/SCI-100, and Crn-6/DNase IIb, the remaining five nucleases were shown to form a degradesome in C. elegans (19Parrish J.Z. Xue D. Mol. Cell. 2003; 11: 987-996Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Since Cps-6/endonuclease G is translocated from the mitochondrion to the nucleus during apoptosis (14Parrish J. Li L. Klotz K. Ledwich D. Wang X. Xue D. Nature. 2001; 412: 90-94Crossref PubMed Scopus (353) Google Scholar, 44Parrish J.Z. Yang C. Shen B. Xue D. EMBO J. 2003; 22: 3451-3460Crossref PubMed Scopus (104) Google Scholar), its DNA fragmentation/degradation is mitochondrion-dependent. This pathway may play a critical role in yeast apoptosis due to the absence of DFF40/CAD endonuclease in yeast cells. The absence of DFF40/CAD is in accordance with the previous finding that yeast cells lack some classical apoptotic elements, including BCL-2 family members and caspases.Table IIPutative yeast (S. cerevisae) apoptotic nucleases corresponding to those revealed in multicellular organismsPreviously proposed apoptotic nucleasesIdentified or possible activitiesCorresponding proteinsSimilarityaProtein sequence similarity between C. elegans and yeast proteinsHumanC. elegansYeast%Cps-6/endonuclease GbSummarized from Ref. 19Endonuclease+c+, presence++57Nuc1/DnaseIIabSummarized from Ref. 19Endonuclease++-d-, absenceCrn-1/FEN-1bSummarized from Ref. 19Flap endonuclease and (possibly) exonuclease+++73Crn-2/Tat-DbSummarized from Ref. 19Nuclease+++55Crn-3/PM/ScI-100bSummarized from Ref. 19Ribonuclease+++53Crn-4/RNaseTbSummarized from Ref. 19Ribonuclease+++39Crn-5/Rrp46bSummarized from Ref. 19Ribonuclease+++48Crn-6/DNase IIbbSummarized from Ref. 19Endonuclease++-33Cyp13/CypEbSummarized from Ref. 19Nuclease+++63DFF40/CADEndonuclease+--DNase IEndonuclease+--DNase γEndonuclease+--a Protein sequence similarity between C. elegans and yeast proteinsb Summarized from Ref. 19Parrish J.Z. Xue D. Mol. Cell. 2003; 11: 987-996Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholarc +, presenced -, absence Open table in a new tab Sequence Similarity and Evolutionary Relationship of Tat-D Proteins—Among the eight putative apoptotic nuclease candidates in yeast cells, Tat-D is the most interesting to us, since the protein is the most conserved but is functionally poorly characterized. We searched all of the available Tat-D protein sequences from the National Institutes of Health protein data base and revealed that more than 31 organisms have their Tat-D sequence information. These organisms cover all the kingdoms from Archaeabacteria, Eubacteria to eukaryotes, indicating that Tat-D is evolutionarily selected and could have important biological functions. Fig. 1A shows the protein sequence alignment of Tat-D homologues from 12 representative organisms. The alignment identified 21 amino acid residues identical among different Tat-D proteins (indicated by the asterisks in Fig. 1A). Of these residues, five are negatively charged, which could be important for Tat-D catalysis, and five others are positively charged, possibly important for substrate binding. Because the conserved amino acid residues are scattered along the protein sequence, no clear motifs could be defined for Tat-D catalysis or substrate binding without further information on its three-dimensional structure. Nevertheless, a motif close to the C terminus, marked by a box in Fig. 1A, might be evolved for subcellular localization, since the motif sequence appears only in the eukaryotic cells. It contains multiple positively charged amino acid residues, which is similar to the nuclear localization signal of nuclear proteins such as FEN-1 (45Qiu J. Li X.W. Frank G. Shen B. J. Biol. Chem. 2001; 276: 30909-30914Google Scholar). Fig. 1B shows the reconstruction of evolutionary relationships among the 12 Tat-D proteins based on the above aligned sequences. Overall, the tree exhibits an evolutionary trend from lower to higher organisms. The bacterial Tat-Ds are more ancestral except for E. coli Tat-D that groups with C. elegans. In eukaryotes, the two yeast Tat-Ds are more ancestral to those from mammalian, plant, and fruit fly. This result indicates that Tat-D is evolutionarily conserved, possibly due to its important biological functions. Purified Yeast Tat-D Has a Nuclease Activity—In order to better understand the biochemical and biological roles of Tat-D, we cloned the S. cerevisiae TAT-D gene, overexpressed it, and purified its protein (ScTat-D) to homogeneity (Fig. 2A). Although the overall overexpression level was low in the BL21 (DE3) expression system, the His-tagged protein is soluble and can be purified through a nickel column. As shown in Fig. 2A, the purified protein is about 45 kDa in size. We then tested whether this protein could cleave circular plasmid DNA, which would allow us to easily determine whether it has endonuclease activity. Our result shows that ScTat-D can efficiently degrade plasmid DNA, indicating that ScTat-D is a DNase (Fig. 2B). This result implies that Tat-D could be a candidate for apoptotic DNA degradation. Tat-D Has Both Endonuclease Activity Making Random Cleavages and Exonuclease Activity Excising from 3′ to 5′—To reveal the cleavage pattern of ScTat-D endonuclease activity and to determine if the enzyme also has an exonuclease activity, we used single-stranded or blunt end double-stranded DNA substrates labeled at the 5′- or 3′-end. Fig. 3A shows the DNA cleavage pattern of ScTat-D using substrates labeled at the 5′-end. Clearly, ScTat-D has enzyme activity on both the double- and single-stranded substrates, whereas the activity on the single-stranded DNA is at least 20 times weaker that on the double-stranded substrate. In addition, the cleavage pattern could rule out the 5′ to 3′ enzyme activity, since no single nucleotides were cut from the 5′-end. Once the endonuclease activity cleaves, the products could be substrates suitable for exonuclease activity as well. Fig. 3B shows the DNA cleavage pattern of ScTat-D using substrates labeled at the 3′-end. It is clear that ScTat-D makes a strong excision at the 3′-end. The activity on the double-stranded substrate is about 5 times stronger than that on the single-stranded substrate. This result suggests that ScTat-D has 3′ to 5′ exonuclease activity. To confirm that ScTat-D has both endo- and exonuclease activities, we performed nuclease activity assays based on a substrate with a biotin at the 3′-end. Since the biotin is able to interact with streptavidin, we used it to block the 3′ to 5′ exonuclease activity of ScTat-D in the presence of bound streptavidin. Our result (Fig. 4A) shows that with increasing amounts of streptavidin, the 3′ to 5′ exonuclease activity of ScTat-D was completely abolished, whereas its endonuclease activity was not affected. This result indicates that Sc" @default.
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• W2029320528 title "Search for Apoptotic Nucleases in Yeast" @default.
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