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• W2029122696 abstract "DNA exonucleases are critical for DNA replication, repair, and recombination. In the bacteriumEscherichia coli there are 14 DNA exonucleases including exonucleases I-IX (including the two DNA polymerase I exonucleases), RecJ exonuclease, SbcCD exonuclease, RNase T, and the exonuclease domains of DNA polymerase II and III. Here we report the discovery and characterization of a new E. coli exonuclease, exonuclease X. Exonuclease X is a member of a superfamily of proteins that have homology to the 3′-5′ exonuclease proofreading subunit (DnaQ) ofE. coli DNA polymerase III. We have engineered and purified a (His)6-exonuclease X fusion protein and characterized its activity. Exonuclease X is a potent distributive exonuclease, capable of degrading both single-stranded and duplex DNA with 3′-5′ polarity. Its high affinity for single-strand DNA and its rapid catalytic rate are similar to the processive exonucleases RecJ and exonuclease I. Deletion of the exoX gene exacerbated the UV sensitivity of a strain lacking RecJ, exonuclease I, and exonuclease VII. When overexpressed, exonuclease X is capable of substituting for exonuclease I in UV repair. As we have proposed for the other single-strand DNA exonucleases, exonuclease X may facilitate recombinational repair by pre-synaptic and/or post-synaptic DNA degradation. DNA exonucleases are critical for DNA replication, repair, and recombination. In the bacteriumEscherichia coli there are 14 DNA exonucleases including exonucleases I-IX (including the two DNA polymerase I exonucleases), RecJ exonuclease, SbcCD exonuclease, RNase T, and the exonuclease domains of DNA polymerase II and III. Here we report the discovery and characterization of a new E. coli exonuclease, exonuclease X. Exonuclease X is a member of a superfamily of proteins that have homology to the 3′-5′ exonuclease proofreading subunit (DnaQ) ofE. coli DNA polymerase III. We have engineered and purified a (His)6-exonuclease X fusion protein and characterized its activity. Exonuclease X is a potent distributive exonuclease, capable of degrading both single-stranded and duplex DNA with 3′-5′ polarity. Its high affinity for single-strand DNA and its rapid catalytic rate are similar to the processive exonucleases RecJ and exonuclease I. Deletion of the exoX gene exacerbated the UV sensitivity of a strain lacking RecJ, exonuclease I, and exonuclease VII. When overexpressed, exonuclease X is capable of substituting for exonuclease I in UV repair. As we have proposed for the other single-strand DNA exonucleases, exonuclease X may facilitate recombinational repair by pre-synaptic and/or post-synaptic DNA degradation. exonuclease I exonuclease VII exonuclease X single-strand double strand dithiothreitol bovine serum albumin polymerase chain reaction kilobase(s) Examination of multiple sequence alignments by both BLAST (1Koonin E.V. Curr. Biol. 1997; 7: 604-606Abstract Full Text Full Text PDF PubMed Google Scholar) and hidden Markov model (2Moser M.J. Holley W.R. Chatterjee A. Mian I.S. Nucleic Acids Res. 1997; 25: 5110-5118Crossref PubMed Scopus (203) Google Scholar) have helped define a large family of proteins that share sequence homology with the 3′-5′ exodeoxyribonuclease domain of DNA polymerases. The ε proofreading subunit of Escherichia coli DNA polymerase III, encoded by dnaQ, is the archetypal member of this family. Other family members include the bacterial proteins RNase T, RNase D, exonuclease I (ExoI),1 oligoribonuclease (3Zhang X. Zhu L. Deutscher M.P. J. Bacteriol. 1998; 180: 2779-2781Crossref PubMed Google Scholar), the Saccharomyces cerevisiae PAN2 protein, and the human Werner syndrome protein (WRN) (1Koonin E.V. Curr. Biol. 1997; 7: 604-606Abstract Full Text Full Text PDF PubMed Google Scholar, 2Moser M.J. Holley W.R. Chatterjee A. Mian I.S. Nucleic Acids Res. 1997; 25: 5110-5118Crossref PubMed Scopus (203) Google Scholar, 4Mian I.S. Nucleic Acids Res. 1997; 25: 3187-3195Crossref PubMed Scopus (180) Google Scholar). These proteins share a conserved tripartite set of “Exo” motifs containing negatively charged aspartate and glutamate residues (5Bernad A. Blanco L. Lazaro J.M. Martin G. Salas M. Cell. 1989; 59: 219-228Abstract Full Text PDF PubMed Scopus (337) Google Scholar). These hallmark residues can be visualized in the crystal structure of the Klenow (proofreading) subunit of E. coli DNA polymerase I to coordinate two divalent cations that catalyze DNA phosphodiester bond cleavage (6Freemont P.S. Friedman J.M. Beese L.S. Sanderson M.R. Steitz T.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8924-8928Crossref PubMed Scopus (328) Google Scholar, 7Derbyshire V. Freemont P.S. Sanderson M.R. Beese L. Friedman J.M. Joyce C.M. Steitz T.A. Science. 1988; 240: 199-201Crossref PubMed Scopus (301) Google Scholar, 8Beese L.S. Steitz T.A. EMBO J. 1991; 10: 25-33Crossref PubMed Scopus (919) Google Scholar, 9Beese L.S. Derbyshire V. Steitz T.A. Science. 1993; 260: 352-355Crossref PubMed Scopus (450) Google Scholar). Comparison of the crystal structures of the Klenow fragment and bacteriophage T4 DNA polymerase suggests that the Exo motifs are diagnostic of functional conservation, since both proteins share the same active site structure despite the lack of sequence identity outside of the Exo motifs (2Moser M.J. Holley W.R. Chatterjee A. Mian I.S. Nucleic Acids Res. 1997; 25: 5110-5118Crossref PubMed Scopus (203) Google Scholar, 10Wang J., Yu, P. Lin T.C. Konigsberg W.H. Steitz T.A. Biochemistry. 1996; 35: 8110-8119Crossref PubMed Scopus (105) Google Scholar). Presumably, other proteins that share these motifs adopt a catalytic site structure and mechanism of action similar to the polymerase exonuclease domain. It has recently been demonstrated that the Werner syndrome protein (WRN) has a 3′-5′ DNA exonuclease activity (11Huang S. Li B. Gray M.D. Oshima J. Mian I.S. Campisi J. Nat. Genet. 1998; 20: 114-116Crossref PubMed Scopus (377) Google Scholar, 12Kamath-Loeb A.S. Shen J.C. Loeb L.A. Fry M. J. Biol. Chem. 1998; 273: 34145-34150Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 13Shen J.C. Gray M.D. Oshima J. Kamath-Loeb A.S. Fry M. Loeb L.A. J. Biol. Chem. 1998; 273: 34139-34144Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Originally identified as a 3′-5′ RecQ-like DNA helicase (14Yu C.E. Oshima J. Fu Y.H. Wijsman E.M. Hisama F. Alisch R. Matthews S. Nakura J. Miki T. Ouais S. Martin G.M. Mulligan J. Schellenberg G.D. Science. 1996; 272: 258-262Crossref PubMed Scopus (1487) Google Scholar, 15Suzuki N. Shimamoto A. Imamura O. Kuromitsu J. Kitao S. Goto M. Furuichi Y. Nucleic Acids Res. 1997; 25: 2973-2978Crossref PubMed Scopus (195) Google Scholar, 16Gray M.D. Shen J.C. Kamath-Loeb A.S. Blank A. Sopher B.L. Martin G.M. Oshima J. Loeb L.A. Nat. Genet. 1997; 17: 100-103Crossref PubMed Scopus (519) Google Scholar), the Werner syndrome protein also has an N-terminal DnaQ-like nuclease domain (17Mushegian A.R. Bassett Jr., D.E. Boguski M.S. Bork P. Koonin E.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5831-5836Crossref PubMed Scopus (216) Google Scholar,18Morozov V. Mushegian A.R. Koonin E.V. Bork P. Trends Biochem. Sci. 1997; 22: 417-418Abstract Full Text PDF PubMed Scopus (138) Google Scholar). WRN possesses a weak exonuclease activity with specificity for the 3′-ending recessed strand of a partial DNA duplex but is unable to degrade single-strand DNA alone (12Kamath-Loeb A.S. Shen J.C. Loeb L.A. Fry M. J. Biol. Chem. 1998; 273: 34145-34150Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). We recently reported that RNase T of E. coli, previously described as a 3′-5′ ribonuclease (19Li Z. Deutscher M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6883-6886Crossref PubMed Scopus (99) Google Scholar, 20Deutscher M.P. Marlor C.W. Zaniewski R. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6427-6430Crossref PubMed Scopus (51) Google Scholar, 21Deutscher M.P. Marlor C.W. J. Biol. Chem. 1985; 260: 7067-7071Abstract Full Text PDF PubMed Google Scholar), also possessed a potent 3′ to 5′ distributive single-strand (ss) DNA-specific exonuclease activity (22Viswanathan M. Dower K.W. Lovett S.T. J. Biol. Chem. 1998; 273: 35126-35131Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). When overexpressed, RNase T was capable of complementing DNA repair defects caused by a deficiency in E. coli Exo I. Unlike exonucleases associated with DNA polymerase, which can degrade from the 3′ end of double-strand (ds) DNA molecules, RNase T had no activity on dsDNA substrates (22Viswanathan M. Dower K.W. Lovett S.T. J. Biol. Chem. 1998; 273: 35126-35131Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Clearly, the architecture of proteins within the DnaQ superfamily allows for different modalities of function, since the same active site configuration can be used to accommodate various substrates (ssDNA, dsDNA, RNA) while retaining the 3′-5′ polarity of degradation. In addition to the bacterial members of the DnaQ superfamily listed above is an open reading frame of unknown function designatedyobC (also known as o220 and b1844) at 41.5 min on theE. coli chromosome. We have cloned, overexpressed, and characterized the protein product of this gene. Overexpression of this gene concomitantly induces high levels of a DNase activity on both ssDNA and dsDNA. We have renamed this open reading frameexoX and the native 25-kDa protein product exonuclease X (ExoX). We have purified a (His)6-ExoX fusion protein to homogeneity and characterized its nuclease activity on various DNA substrates. ExoX is an extremely potent 3′ to 5′ distributive nuclease capable of degrading 40-kilobase bacteriophage T7 ssDNA and dsDNA to completion. Its affinity for ssDNA ends is greater than for dsDNA, and it appears to have no affinity for RNA. When overexpressed, ExoX, like RNase T (23Viswanathan M. Lanjuin A. Lovett S.T. Genetics. 1999; 151: 929-934Crossref PubMed Google Scholar), is capable of substituting for ExoI in vivo, as measured by its ability to increase the UV survival of an ExoI-deficient strain. A mutation in exoX did not by itself cause sensitivity to UV but strongly augmented the UV sensitivity of a strain deficient in ssDNA exonucleases RecJ, ExoI, and ExoVII. The exoX gene was amplified by PCR usingPfu polymerase (Stratagene Inc.) from wild type E. coli strain MG1655 genomic DNA using primers 5′-CGGAATTCTAAGGAGGGATCCATGTTGCGCATTATC-3′ and 5′-GCTCTAGACTAAGTATTTTCCAG-3′ and buffer conditions recommended by the manufacturer. Primers were annealed to the genomic DNA at 50 °C for 30 s and extended for 2 min at 72 °C; 25 cycles of PCR were performed. The PCR product was subsequently digested with restriction endonucleases EcoRI and XbaI and ligated into the compatible sites of pBSSK− (Stratagene Inc.), producing pExoX. Sequence analysis verified the construct was error-free. The EcoRI-XbaI fragment from pExoX was cloned into compatible sites within pBSKS− (Stratagene Inc.) creating pExoXKS−. Plasmid pExoXfs, a derivative of pExoXKS− with a frameshift mutation 171 base pairs downstream from the initiation codon of exoX, was constructed by cleavage of pExoXKS− DNA with restriction endonuclease NcoI, “fill-in” synthesis with Klenow fragment (DNA polymerase I), and blunt end DNA ligation. AnexoX (His)6-tagged gene fusion was constructed by cloning the 693-base pair BamHI-SacI fragment of pExoX into the same sites within pET28a(+) (Novogen), producing the plasmid pExoX-His. A 2,919-base pair region of the E. coli chromosome containing the exoX gene was amplified by PCR using primers beginning 1,088 base pairs upstream (5′-GGGAATTCGTACCCGTATGCGTGATG-3′) and 1,167 base pairs downstream (5′-GGTCTAGACGAGGATCATCAATTCCGG-3′) ofexoX. The PCR was performed using Turbo Pfupolymerase (Stratagene, Inc.) in buffer conditions recommended by the manufacturer. Primers were annealed to MG1655 E. coligenomic DNA at 60 °C for 30 s and extended for 3 min at 72 °C; 25 cycles of PCR were performed. The PCR product was digested with XbaI and EcoRI and ligated into compatible sites within the Litmus 29 vector (New England Biolabs Inc.), creating the plasmid pExoXFlank. A precise deletion of the exoX open reading frame from the plasmid pExoXFlank was performed by PCR. The primers utilized for the PCR flanked exoX and were oriented to replicate the entire plasmid except for the exoX open reading frame. Both primers; 5′-GGGGGCGGCCGCGGCATGCTCCAGGCCG-3′ and 5′-GGGGGCGGCCGCTCCGCAGGCGTAGCGGG-3′ contained NotI sites at the primer 5′ terminus. The PCR was performed as above except that primer annealing was at 45 °C, and extensions were performed for 6 min. The resulting 5-kb PCR product was treated withDpnI to remove any original methylated template DNA and then digested with NotI and ligated to a 2.1-kb NotI fragment from plasmid pCK155 (24Kristensen C.S. Eberl L. Sanchez-Romero J.M. Givskov M. Molin S. De Lorenzo V. J. Bacteriol. 1995; 177: 52-68Crossref PubMed Google Scholar) containing a Tn5 npt gene, which confers kanamycin resistance. Flanking npt on both sides are 140-base pair resolution sites from the broad host range plasmid RP4 multimer resolution system, which allows for the precise excision of npt (24Kristensen C.S. Eberl L. Sanchez-Romero J.M. Givskov M. Molin S. De Lorenzo V. J. Bacteriol. 1995; 177: 52-68Crossref PubMed Google Scholar). The ligation was transformed by electroporation into XL1-Blue (Stratagene Inc.) cells selecting kanamycin-resistance. The resulting plasmid pExoXΔ had a complete deletion of exoX and a 2.1-kilobase insertion as verified by restriction analysis. STL2350 (xonA2 recJ284::Tn10 Δ(xseA-guaB) zff-3139::Tn10kan) was used for ExoX protein expression. Plasmid pTJH30 (25Haggerty T.J. Lovett S.T. J. Bacteriol. 1997; 179: 6705-6713Crossref PubMed Google Scholar), carrying a heat shock-inducible T7 RNA polymerase, was introduced into STL2350 by transformation (26Chung C.T. Niemela S.L. Miller R.H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2172-2175Crossref PubMed Scopus (1160) Google Scholar) and selection for chloramphenicol resistance. For protein-labeling experiments pExoX or pBSSK− were transformed into STL2350/pTJH30 cells selecting ampicillin and chloramphenicol resistance. Strain STL4239 is the pTJH30/pExoX transformant of STL2350. STL2329 (λDE3 sbcB15 recJ284::Tn10 endA Δ(xth-pncA)gal thi) (27Studier F.W. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4820) Google Scholar) and BL21 (E. coli B λDE3 F− dcm ompT hsdS(rB−mB−) gal), both carrying a chromosomally integrated isopropyl-1-thio-β-d-galactopyranoside-inducible T7 RNA polymerase gene (27Studier F.W. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4820) Google Scholar), were used to express and purify a NH2-(His)6-tagged ExoX fusion protein expressed from plasmid pExoX-His. For protein-labeling experiments, pExoX-His or pET28a(+) were transformed into STL2329, selecting kanamycin resistance. pExoXΔ was transformed into the E. coli strain JC7623 (28Horii Z. Clark A.J. J. Mol. Biol. 1973; 80: 327-344Crossref PubMed Scopus (310) Google Scholar) (recB recC sbcB sbcC) by electroporation and grown nonselectively overnight. Kanamycin-resistant and ampicillin-sensitive isolates were analyzed by Southern blot (29Southern E.M. J. Mol. Biol. 1975; 98: 503-517Crossref PubMed Scopus (21462) Google Scholar) and were found to have the appropriate deletion/insertion at the exoX locus. One such isolate, STL4525 (ΔexoX1::npt recB recC sbcB sbcC), was used as a donor for further strain constructions by P1virA-mediated transductions (Ref. 30Miller J.H. A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory, Plainview, NY1992: 268-274Google Scholar and see below). With the exception of STL2329 and STL4525, all strains designated STL were derived from BT199 and carry additional genetic markers (F− λ− thi-1 Δ(gpt-proA)62thr-1 leuB6 kdgK51 rfbD1 ara-14 lacY1 galK2 xyl-5 mtl-1 tsx-33 supE44 rpsL31 rac −). pExoXKS, pExoXfs, and pBSKS− were transformed (26Chung C.T. Niemela S.L. Miller R.H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2172-2175Crossref PubMed Scopus (1160) Google Scholar) into either STL2701 (ΔxonA300::cat Δ(xseA-guaB) zff-3139::Tn10kan recJ2052::Tn10kan) or STL2348 (Δ(xseA-guaB) zff-3139::Tn10kan recJ284::Tn10, selecting ampicillin. In addition, various isogenic exonuclease-deficient mutants in the BT199 genetic background (listed in Fig. 7) were assayed for UV survival. The mutant alleles used in the construction of the various exonuclease-deficient strains were: ΔexoX1::npt, for ExoX; ΔxonA300::cat, for ExoI;recJ284::Tn10 for RecJ; andxseA18::amp for ExoVII. Details of the construction of these strains will be published elsewhere and are available by request from the authors. Single colonies were grown in LB or LB + ampicillin (for strains containing plasmids) liquid medium to exponential stage (A 600 = 0.4–0.5), serially diluted in 56/2 buffer (60 mm Na2HPO4, 40 mmKH2PO4, 0.02% MgSO4·7H2O, 0.2% (NH4)2SO4, 0.01% Ca(NO3)2·4H2O, 0.5 μmFeSO4), and plated on LB or LB + ampicillin plates. Plates were immediately irradiated with varying doses of UV (254 nm) irradiation and incubated at 37 °C in the dark overnight. Total viable cells were determined from serial-diluted unirradiated cells. ExoX protein expression was induced from the T7 φ10 promoter of pExoX by 42 °C heat shock induction of the T7 RNA polymerase gene on plasmid pTJH30. T7 promoter-mediated expression of the (His)6-ExoX fusion protein from plasmid pExoX-His was induced by the addition of isopropyl-1-thio-β-d-galactopyranoside (1 mm) to STL4337 (pExoX-His transformant of STL2329). Culture growth, [35S]methionine protein labeling in the presence of rifampicin and crude extract preparations for both proteins were performed as described previously (25Haggerty T.J. Lovett S.T. J. Bacteriol. 1997; 179: 6705-6713Crossref PubMed Google Scholar). Protein concentrations were measured by the method of Bradford (31Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215589) Google Scholar) with standard reagent (Bio-Rad) and bovine serum albumin (BSA) as standard. All proteins were resolved by 15% SDS-polyacrylamide gel electrophoresis (32Ausubel F.A. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Short Protocols in Molecular Biology. Greene Publishing and Wiley-Interscience, New York1989Google Scholar) and visualized by Coomassie stain and/or autoradiography. All steps were performed at 4 °C using ultrapure reagents. Buffer A contained 50 mm Tris-HCl (pH 7.5), 200 mm NaCl, and 1 mm dithiothreitol. Buffer B contained 10% glycerol, 20 mm Tris-HCl (pH 8.0), 500 mm NaCl, 25 mm imidazole, and 1 mmβ-mercaptoethanol. Buffer C contained 10% glycerol, 20 mm Tris-HCl (pH 7.5), 1 mm EDTA, and 1 mm dithiothreitol (DTT). STL4533 (pExoX-His transformant of BL21) was cultured at 37 °C in 34 liters of LB + Km to anA 600 of 0.6, then induced with 1 mmisopropyl-1-thio-β-d-galactopyranoside for 1 h. Cells were harvested and frozen as described previously (33Lovett S.T. Kolodner R.D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2627-2631Crossref PubMed Scopus (221) Google Scholar) in a volume of 700 ml. A crude extract was prepared by lysing cells in 10% sucrose, 50 mm Tris-HCl (pH 8.0), 50 mm NaCl, 5 mm MgCl2, 1 mm DTT, 1 mm EDTA, and 1 mg/ml lysozyme for 1 h on ice. Three cycles of freeze/thawing (10 min 37 °C, 10 min 0 °C) were performed before a supernatant was obtained by high speed centrifugation at 130,000 × g for 20 min. The crude extract (700 ml, 1.8 g of protein) was adjusted to 200 mm NaCl and 5 mm imidazole before adding 15 ml of Ni2+-nitrilotriacetic acid-agarose resin (Qiagen) equilibrated in Buffer A. The resin and extract slurry was allowed to mix for 8 h with stirring at 4 °C. The resin was washed by repeated low speed centrifugation and resuspension in 175 ml of Buffer A, 85 ml of Buffer B, and finally with 50 ml of Buffer C + 200 mm NaCl. Proteins were eluted from the resin with 35 ml of Buffer C + 500 mm NaCl + 400 mm imidazole. The resulting fraction (6.0 mg of protein) was concentrated to 10 ml using a Centriprep 10 (Amicon) cartridge and then dialyzed against 2 1-liter changes of Buffer C. This fraction was then applied to a 0.5-ml dsDNA cellulose column equilibrated in Buffer C + 25 mm NaCl and washed with 10 ml of Buffer C. Bound proteins were eluted in a single step to Buffer C + 500 mm NaCl. Fractions containing nuclease activity were pooled and dialyzed overnight against 0.5 liters of 60% glycerol, 500 mm NaCl, and 1 mm DTT, and then again against 60% glycerol, 1 mm DTT, 1 mm EDTA. The purified protein (0.75 mg at 0.41 mg/ml) was stored at −20 °C. Uniformly labeled bacteriophage T7 [3H]DNA with a specific activity of 2.5 × 104 cpm/nmol of nucleotide was prepared as described previously (33Lovett S.T. Kolodner R.D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2627-2631Crossref PubMed Scopus (221) Google Scholar) using [3H]thymidine (NEN Life Science Products). 3′ end-labeled substrate was generated by Klenow fragment fill-in synthesis ofHindIII-digested pBSSK− DNA with [32P]dATP. 5′ end-labeled substrate was generated fromHindIII-digested pBSSK− DNA, treated with shrimp alkaline phosphatase, and phosphorylated with T4 polynucleotide kinase and [γ-32P]ATP. Both 3′ and 5′ end-labeled substrates were purified by G-50 Sephadex quick spin column (Roche Molecular Biochemicals). Unless otherwise stated, enzyme assays employed 0.5 μg of T7 [3H]DNA (1.5 nmol) or 0.5 μg (0.5 pmol of ends) of 3′ or 5′ end-labeled substrate. For ssDNA assays, substrates were incubated for 5 min at 100 °C then quenched on ice. Standard reactions contained 10 mm Tris-HCl (pH 8.0), 10 mm MgCl2, 1 mm NaCl, and 2 mg/ml BSA in 50 μl (for T7 substrates) or 30 μl (for end-labeled substrates). Competition assays were performed with 50 ng (50 fmol) of 3′ end-labeled substrate, varying the amount of either single-strand or double-strand cold competitor DNA. Competition experiments using RNA competitors utilized E. coli tRNA XXI (Sigma) and Torula yeast RNA (molecular mass range 3 × 103–4 × 104 g/mol, Sigma). Both RNAs are used by Sigma as ribonuclease assay substrates. Cold RNA stocks were prepared before each competition experiment in cold sterile water. TheA 260 of the RNA stocks was determined immediately before use to verify the optical density provided by the manufacturer and to ensure that noticeable degradation had not occurred. When required, protein samples were diluted in a buffer containing 60% glycerol, 10 mm Tris-HCl (pH 8.0), 1 mm DTT, and 1 mm EDTA. Sample incubation, trichloroacetic acid precipitation, and soluble count determination were performed as described previously (34Viswanathan M. Lovett S.T. Genetics. 1998; 149: 7-16Crossref PubMed Google Scholar). One unit of DNase activity with T7 DNA substrate corresponds to the release of 1 nmol of acid-soluble product in a 20-min reaction at 37 °C. For agarose gel electrophoresis assays, 0.5 μg (0.5 pmol) of either 3′ or 5′ 32P end-labeled ssDNA substrate was incubated with various amounts of (His)6-ExoX protein. Reactions were quenched at various time points on ice by the addition of EDTA to 5.0 mm. Standard assay conditions were employed, except that BSA was omitted to allow DNA electrophoresis without further manipulation of the samples after the reactions were quenched. ssDNA substrate samples were boiled for 5 min and cooled on ice before loading onto a 1.0% agarose-NA gel (Amersham Pharmacia Biotech); double-strand substrates were loaded directly onto the gels. Gels were run at 80 V for 45 min in Tris acetate + EDTA buffer (32Ausubel F.A. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Short Protocols in Molecular Biology. Greene Publishing and Wiley-Interscience, New York1989Google Scholar), dried at 80 °C for 30 min, and exposed to film. Shrimp alkaline phosphatase was obtained from Amersham Pharmacia Biotech. Lysozyme was obtained from U. S. Biochemical Corp. All other enzymes were obtained from New England Biolabs, Inc. The antibiotics ampicillin, kanamycin, and chloramphenicol were used at 100, 60, and 15 μg/ml, respectively. The E. coli open reading frame designated yobC, hereafter known as exoX, was amplified by PCR and cloned directionally into pBSSK−, placing its expression under T7 promoter control. Induction ofexoX expression from the T7 promoter of pExoX led to the production of a single 25-kDa protein, consistent with the expected molecular mass of the protein (Fig.1 A, lane 4). The 25-kDa protein was absent in uninduced extracts (Fig. 1 A,lane 3) and from cells carrying vector only (Fig.1 A, lane 2). Crude extracts prepared from these induced cells were tested for DNase activity using uniformly3H-labeled T7 DNA substrates. Strains induced for expression of exoX exhibited a 150-fold increase in ssDNase activity and a 280-fold increase in dsDNase activity compared with cells carrying vector alone (Table I). Both ssDNase and dsDNase activities were Mg2+-dependent, as no increase in activity was noted in the absence of the divalent cation (data not shown).Table IMg2+-dependent DNase activity associated with ExoX or (His)6-ExoX overexpressionPlasmidSpecific activityssDNAdsDNAUnits/mgpBSSK−5010pExoX76002800pET28a(+)11020pExoX-His52003400Nuclease assays were performed using crude extracts prepared from cells carrying the indicated plasmids. Cells were induced for protein expression 20 min before assay. Assays were performed in the presence of Mg2+ using both single-strand and double-strand T7 [3H]DNA substrates. One unit of DNase corresponds to the release of 1 nmol of acid-soluble product in a 20-min reaction at 37 °C. Results are the average of two determinations. Open table in a new tab Nuclease assays were performed using crude extracts prepared from cells carrying the indicated plasmids. Cells were induced for protein expression 20 min before assay. Assays were performed in the presence of Mg2+ using both single-strand and double-strand T7 [3H]DNA substrates. One unit of DNase corresponds to the release of 1 nmol of acid-soluble product in a 20-min reaction at 37 °C. Results are the average of two determinations. An N-terminal (His)6-tagged ExoX protein fusion was constructed using the pET28a(+) vector (Novogen). (His)6-ExoX protein was expressed from the T7 promoter on plasmid pExoX-His (Fig. 1 A). Crude extracts prepared from induced cells carrying pExoX-His were tested for DNase activity. Strains induced for expression of (His)6-ExoX exhibited a 47-fold increase in ssDNase activity and a 170-fold increase in dsDNase activity compared with cells carrying vector alone (Table I). Comparison of uninduced levels of expression between the pET28a(+) vector and pExoX-His revealed a basal level of expression of the (His)6 fusion protein without induction of the T7 promoter. Upon induction, increased expression of two [35S]methionine-labeled proteins was noted. The identity of the second band is unknown; however, it did not appear (Fig.1 A, lane 4) when the native protein was expressed in a different strain background. The (His)6-ExoX protein was overexpressed in E. coli strain BL21 and was purified using nickel-agarose chromatography (Fig. 1 B). Three high molecular weight contaminants and a lower molecular weight protein were removed by dsDNA cellulose affinity chromatography. The identity of the abundant lower molecular weight band is not known; however, it may be a proteolytic product of the (His)6-ExoX protein, since it bound to the Ni2+-nitrilotriacetic acid resin yet failed to bind dsDNA cellulose affinity column. Furthermore, the protein that bound to the dsDNA cellulose column had significant nuclease activity, whereas the flow-through fraction from the dsDNA cellulose column containing the lower molecular weight protein had nearly none. The purified protein was analyzed by mass spectrometry and was determined to have a molecular mass of 28,552 mass units. The molecular mass determined by mass spectrometry is commensurate with the expected mass of the fusion protein by amino acid sequence. Purified (His)6-ExoX was used to determine optimal conditions for ssDNase activity. ExoX showed optimal activity at pH 8.0 in the presence of Mg2+ (Fig.2 A). Similar to other nucleases, the ssDNase activity of ExoX was dependent upon the presence of the divalent cation Mg2+; no detectable degradation was seen in its absence or in the presence of Mn2+ (Fig.2 B). BSA enhanced the ssDNase activity of ExoX (Fig.2 C). The ssDNase activity of ExoX was enhanced with low salt concentrations, but above 5 mm salt, the activity decreased with increasing concentration (Fig. 2 D). The addition of the sulfhydryl reducing agent, DTT (1–5 mm), did not alter levels of ssDNase activity (data not shown). In reactions with denatured bacteriophage T7 DNA (40 kb in length), 1 ng (34 fmol) of purified (His)6-ExoX linearly degraded 35% of the total DNA (0.5 μg, 1.5 nmol in nucleotides) in 20 min. The addition of 10 ng of protein resulted in 100% of the ssDNA substrate being degraded (Fig. 3 A). Similarly, in reactions with T7 dsDNA, 5 ng (0.17 pmol) of purified (His)6-ExoX linearly degraded 30% of the total DNA in 20 min, and the addition of 40 ng (1.4 pmol) of (His)6-ExoX resulted in 100% of the dsDNA substrate being degraded (Fig.3 B). Using data points in the linear range of T7 DNA degradation from Fig. 3, the calculated rate of nucleotide release/protein monomer is 800 nucleotide/min for ssDNA, and 150 nucleotides/min for dsDNA. From its ability to degrade T7 DNA completely, we conclude that ExoX has little or no specificity for DNA sequence or structure. No endonuclease activity was observed in reactions with φX174 circular ssDNA, supercoiled dsDNA, or relaxed dsDNA, suggesting that" @default.
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• W2029122696 title "Exonuclease X of Escherichia coli" @default.
• W2029122696 cites W1519899019 @default.
• W2029122696 cites W1538603370 @default.
• W2029122696 cites W1542472052 @default.
• W2029122696 cites W1547438680 @default.
• W2029122696 cites W1547905266 @default.
• W2029122696 cites W1577713059 @default.
• W2029122696 cites W1582490677 @default.
• W2029122696 cites W1585911315 @default.
• W2029122696 cites W1606628568 @default.
• W2029122696 cites W1619666977 @default.
• W2029122696 cites W1842766745 @default.
• W2029122696 cites W1913308298 @default.
• W2029122696 cites W1966347623 @default.
• W2029122696 cites W1971681144 @default.
• W2029122696 cites W1972974900 @default.
• W2029122696 cites W1975920595 @default.
• W2029122696 cites W1975993749 @default.
• W2029122696 cites W1976194927 @default.
• W2029122696 cites W1980624564 @default.
• W2029122696 cites W2001189955 @default.
• W2029122696 cites W2012105278 @default.
• W2029122696 cites W2035834598 @default.
• W2029122696 cites W2040035676 @default.
• W2029122696 cites W2042355200 @default.
• W2029122696 cites W2052337263 @default.
• W2029122696 cites W2058317622 @default.
• W2029122696 cites W2063450941 @default.
• W2029122696 cites W2064310741 @default.
• W2029122696 cites W2071444861 @default.
• W2029122696 cites W2073534733 @default.
• W2029122696 cites W2084712319 @default.
• W2029122696 cites W2102428326 @default.
• W2029122696 cites W2120980434 @default.
• W2029122696 cites W2124912613 @default.
• W2029122696 cites W2138524348 @default.
• W2029122696 cites W2144164911 @default.
• W2029122696 cites W2145304961 @default.
• W2029122696 cites W2147019339 @default.
• W2029122696 cites W2153043528 @default.
• W2029122696 cites W2153389989 @default.
• W2029122696 cites W2165639777 @default.
• W2029122696 cites W4293247451 @default.
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