Advertisement

Characterization of a New Thermophilic Spore Photoproduct Lyase from Geobacillus stearothermophilus (SplG) with Defined Lesion Containing DNA Substrates*

  • J. Carsten Pieck
    Affiliations
    Department of Chemistry and Biochemistry, Ludwig Maximilians University Munich, D-81377 Munich
    Search for articles by this author
  • Ulrich Hennecke
    Affiliations
    Department of Chemistry and Biochemistry, Ludwig Maximilians University Munich, D-81377 Munich
    Search for articles by this author
  • Antonio J. Pierik
    Affiliations
    Department of Microbiology, Philipps University Marburg, D-35032 Marburg, Germany
    Search for articles by this author
  • Marcus G. Friedel
    Affiliations
    Department of Chemistry and Biochemistry, Ludwig Maximilians University Munich, D-81377 Munich
    Search for articles by this author
  • Thomas Carell
    Correspondence
    To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, Ludwig Maximilians University Munich, Butenandtstr. 5-13, D-81377 Munich, Germany. Tel.: 49-89-2180-77750; Fax: 49-89-2180-77756;
    Affiliations
    Department of Chemistry and Biochemistry, Ludwig Maximilians University Munich, D-81377 Munich
    Search for articles by this author
  • Author Footnotes
    * This work was supported by Deutsche Forschungsgemeinschaft, Volkswagen Foundation, and the European Union Marie Curie Network (CLUSTOX DNA). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:September 11, 2006DOI:https://doi.org/10.1074/jbc.M607053200
      The Geobacillus stearothermophilus splG gene encodes a thermophilic spore photoproduct lyase (SplG) that belongs to the family of radical S-adenosylmethionine (AdoMet) enzymes. The aerobically purified apo-SplG forms a homodimer, which contains one [4Fe-4S] cluster per monomer unit after reconstitution to the holoform. Formation of the [4Fe-4S] cluster was proven by quantification of the amount of iron and sulfur per homodimer and by UV and EPR spectroscopy. The UV spectrum features a characteristic absorbance at 420 nm typical for [4Fe-4S] clusters, and the EPR data were found to be identical to those of other proteins containing an [4Fe-4S]+ center. Probing of the activity of the holo-SplG with oligonucleotides containing one spore photoproduct lesion at a defined site proved that the enzyme is able to turn over substrate. In addition to repair, we observed cleavage of AdoMet to generate 5′-deoxyadenosine. In the presence of aza-AdoMet the SplG is completely inhibited, which provides direct support for the repair mechanism.
      Spores of various Bacillus and Clostridium species are extremely resistant to harsh physical, chemical, and biological conditions allowing them to survive even under extreme conditions (
      • Setlow P.
      ,
      • Nicholson W.L.
      • Munakata N.
      • Horneck G.
      • Melosh H.J.
      • Setlow P.
      ). The oldest known viable spore was discovered from a Bacillus species, designated 2-9-3, in a 250 million-year-old salt crystal from the Permian Salado Formation (
      • Vreeland R.H.
      • Rosenzweig W.D.
      • Powers D.W.
      ). The resistance of spores from Geobacillus stearothermophilus toward heat is even so high that the survival of the organism during heat sterilization is used as a bioindicator for insufficient heat treatment (
      • Penna T.
      • Ishii M.
      • Machoshvili I.
      • Marques M.
      ).
      Particularly noteworthy is the unusually high stability of spores in the presence of UV light. For example under typical UV sterilization conditions, only about 70% of thermophilic G. stearothermophilus spores are inactivated. Under the same conditions, typical pathogens such as herpes simplex or polio viruses are fully destroyed (
      • Gerhardt P.
      • Marquis R.E.
      ,
      • Devine D.A.
      • Keech A.P.
      • Wood D.J.
      • Killington R.A.
      • Boyes H.
      • Doubleday B.
      • Marsh P.D.
      ). In addition, UV irradiation of spores gives rise to different DNA lesions (
      • Setlow P.
      ). Although in normal cells mostly cyclobutane pyrimidine dimers and (
      • Devine D.A.
      • Keech A.P.
      • Wood D.J.
      • Killington R.A.
      • Boyes H.
      • Doubleday B.
      • Marsh P.D.
      -
      • Penna T.
      • Ishii M.
      • Machoshvili I.
      • Marques M.
      ) lesions (
      • Cadet J.
      • Anselmino C.
      • Douki T.
      • Voituriez L.
      ) are formed, in spores the unusual photoproduct 5-thyminyl-5,6-dihydrothymine (SP),
      The abbreviations used are: SP, spore photoproduct (5-thyminyl-5,6-dihydrothymine); IPTG, isopropyl β-d-thiogalactopyranoside; AdoMet, S-adenosylmethionine; SplB, spore photoproduct lyase B. subtilis; SplG, spore photoproduct lyase G. stearothermophilus; TCEP, Tris(2-carboxyethyl)phosphine hydrochloride; ssDNA, single-stranded DNA; rp-HPLC, reverse phase-high pressure liquid chromatography; DPA, dipicolinic acid; aRNR, anaerobic ribonucleotide reductase; DTT, dithiothreitol; MS, mass spectrometry; MS/MS, tandem mass spectrometry; MALDI, matrix-assisted laser desorption ionization; ICP-AES, inductively coupled plasma atomic emission spectroscopy; dAdoH, 5′-deoxyadenosine; FTICR, Fourier transformation-ion-cyclotron resonance.
      2The abbreviations used are: SP, spore photoproduct (5-thyminyl-5,6-dihydrothymine); IPTG, isopropyl β-d-thiogalactopyranoside; AdoMet, S-adenosylmethionine; SplB, spore photoproduct lyase B. subtilis; SplG, spore photoproduct lyase G. stearothermophilus; TCEP, Tris(2-carboxyethyl)phosphine hydrochloride; ssDNA, single-stranded DNA; rp-HPLC, reverse phase-high pressure liquid chromatography; DPA, dipicolinic acid; aRNR, anaerobic ribonucleotide reductase; DTT, dithiothreitol; MS, mass spectrometry; MS/MS, tandem mass spectrometry; MALDI, matrix-assisted laser desorption ionization; ICP-AES, inductively coupled plasma atomic emission spectroscopy; dAdoH, 5′-deoxyadenosine; FTICR, Fourier transformation-ion-cyclotron resonance.
      depicted in Scheme 1 (
      • Setlow P.
      ,
      • Donnellan J.E.
      • Stafford R.S.
      ), is exclusively generated (
      • Douki T.
      • Setlow B.
      • Setlow P.
      ). These differences in the photoreactivity may be because of an unusual packing of the DNA in spores (
      • Setlow P.
      ,
      • Nicholson W.L.
      • Setlow B.
      • Setlow P.
      ,
      • Nicholson W.L.
      • Setlow B.
      • Setlow P.
      ) and the high amounts of dipicolinic acid (DPA) present in spores (
      • Douki T.
      • Setlow B.
      • Setlow P.
      ).
      Figure thumbnail gr1
      SCHEME 1Proposed repair mechanism of the spore photoproduct. The repair reaction is proposed to proceed by hydrogen abstraction from C-6 of the spore photoproduct followed by β-scission of the bond linking the two pyrimidines and back transfer of the hydrogen atom.
      During germination, the SP lesion is repaired either by the general nucleotide excision repair pathway (
      • Setlow P.
      ,
      • Munakata N.
      • Rupert C.S.
      ) or by a single enzyme, called spore photoproduct lyase, which is able to split the SP lesions directly back into two thymidines. Recent studies by Nicholsen et al. (
      • Nicholson W.L.
      • Fajardocavazos P.
      • Pedrazareyes M.
      • Sun Y.B.
      • Zazuetasandoval R.
      ) and Broderick and co-workers (
      • Cheek J.
      • Broderick J.B.
      ) performed with the SP-lyase from Bacillus subtilis showed that the enzyme requires S-adenosylmethionine (AdoMet) as a cofactor for repair. A detailed sequence comparison, spectroscopic studies (
      • Rebeil R.
      • Nicholson W.L.
      ,
      • Rebeil R.
      • Sun Y.B.
      • Chooback L.
      • Pedraza-Reyes M.
      • Kinsland C.
      • Begley T.P.
      • Nicholson W.L.
      ), and a recent labeling experiment (
      • Cheek J.
      • Broderick J.B.
      ) all provide evidence that the SplG is a member of the radical AdoMet enzyme superfamily, which also includes anaerobic ribonucleotide reductase (aRNR), pyruvate formate-lyase, lysine-2,3-aminomutase, benzylsuccinate synthase-activating enzyme, and biotin synthase (BioB) (
      • Cheek J.
      • Broderick J.B.
      ). The DNA repair mechanism postulated by Begley and Mehl (
      • Mehl R.A.
      • Begley T.P.
      ) is depicted in Scheme 1. It is assumed that the 5′-dAdo radical (
      • Cheek J.
      • Broderick J.B.
      ,
      • Rebeil R.
      • Nicholson W.L.
      ), formed after electron transfer from the [4Fe-4S] cluster to the AdoMet, initiates the repair by abstracting the C-6 hydrogen of the SP lesion. The C-C bond linking the two pyrimidines undergoes β-fragmentation to give an allyl-type radical. The thermodynamically problematic last step is the transfer of the hydrogen atom back from the 5′-dAdoH to the thymine monomer radical, which completes the repair process (
      • Mehl R.A.
      • Begley T.P.
      ). HPLC and electrospray ionization-MS analyses showed that the SplG cleaves AdoMet under anaerobic and reductive conditions to generate ∼2 molecules of 5′-deoxyadenosine per homodimer SplG in the absence of the SP lesion substrate (
      • Rebeil R.
      • Nicholson W.L.
      ). In the presence of the SP lesion substrate the 5′-deoxyadenosine production was found to be strongly increased (
      • Rebeil R.
      • Nicholson W.L.
      ). The organization of the [4Fe-4S] cluster and of the AdoMet-binding site requires three specific cysteine residues (Cys-91, Cys-95, and Cys-98), which form the typical motif, CXXXCXXC-, of proteins belonging to this radical enzyme superfamily (
      • Rebeil R.
      • Nicholson W.L.
      ). The SplB from B. subtilis contains a fourth cysteine (Cys-141), which was found to be essential for in vivo activity of the protein similar to that observed for biotin synthase.
      Here we describe the isolation and characterization of the first thermophilic spore photoproduct lyase (G. stearothermophilus). The stability of the enzyme allowed us to purify the apoprotein under aerobic conditions. Full reconstitution of the protein was possible under strict anaerobic conditions. Using a novel assay with a synthetic dinucleotide SP lesion substrate and with small DNA single strands, which contain one SP lesion at a defined site, we show that the fully reconstituted protein is active in the presence of AdoMet. We observed a specific activity of 2.6 μmol of SP repaired per min/mg of SplG. In the presence of aza-AdoMet, the SplG is completely inhibited, which provides direct support for the mentioned mechanism. An interesting feature of the enzyme is that it forms an extremely stable, catalytically active homodimer.

      EXPERIMENTAL PROCEDURES

      ChemicalsS-Adenosylmethionine was purchased from Sigma and further purified by HPLC to remove additional S-adenosylhomocysteine. All bacteria media were purchased from Roth, and all other chemicals were obtained from commercial suppliers and used without further purification.
      Bacterial Strains and Growth ConditionsG. stearothermophilus strain 10 (DSM number 13240) was grown in nutrient broth medium (meat peptone 5.00 g/liter and meat extract 3.00 g/liter, pH 7.0) under aerobic conditions in a shaker (300 rpm) at 60 °C. Escherichia coli strain Tuner™ pLysS (DE3) (Novagen) was used for overexpression of SplG.
      Construction of Plasmid—Genomic DNA of G. stearothermophilus was isolated using the DNeasy® tissue kit (Qiagen). The splG gene was obtained by PCR amplification from the genomic G. stearothermophilus DNA using AccuPrime™ Pfx DNA polymerase (Invitrogen) with the two primers, 5′-CACCATGCATCACCATCACCATCACATGAAACCGTTTGTGCCAAAACT-3′ and 5′-TTACGTAAAATACTGCACTTGGG-3′. The resulting 1.0-kb splG gene PCR product was cloned into the vector pET101-D (Invitrogen) by the Topo reaction.
      Overproduction and Purification of Recombinant ProteinE. coli strain Tuner™ pLysS (DE3), which is a lacYZ deletion mutant of BL21, enables the adjustment of the levels of protein expression throughout all cells in a culture, because the lac permease (lacY) mutation allows uniform entry of IPTG into all cells in the population. Recombinant SplG was expressed with an N-terminal His6 tag. Cells were cultivated with shaking at 150 rpm in 1 liter of LB medium (
      • Sambrock J.
      • Russel D.
      ) containing 100 μm Fe(III)-citrate at 37 °C to an A600 = 0.6. IPTG was added to 1 mm, and incubation was continued at 22 °C for 12 h. Cells were harvested (10000 × g, 8 min, 4 °C) and stored at -20 °C. Subsequently the cell pellet was resuspended in 30 ml of 60 mm Na2HPO4 (pH 8.0), 300 mm NaCl, 10 mm imidazole, 5 mm l-cysteine, 20 mm ammonium ferrous sulfate, 10 mm ATP, and 1 tablet of protease inhibitor mixture (Roche Applied Science). The crude cell extract was solubilized in a French press at 1000 psi. The cell debris was removed by centrifugation (38,000 × g, 35 min, 4 °C), and the supernatant was applied to a nickel-nitrilotriacetic acid-agarose column (Qiagen) equilibrated with 50 mm Tris-HCl (pH 8.0), 300 mm NaCl, and 10 mm imidazole. SplG was eluted from the column using the same buffer containing 500 mm imidazole. In a second purification step, apo-SplG dimer was separated by a gel permeation chromatography with 50 mm Tris-HCl (pH 8.0), 300 mm NaCl. The purified SplG, up to 2 mg/liter expression, was concentrated and transferred into a glovebox (MBraun, Unilab) containing a 5% H2, 95% N2 atmosphere, and less than 2 ppm O2. Inside the anaerobic chamber the aerobic buffer containing SplG apoprotein was exchanged by gel filtration to the reconstitution buffer.
      [4Fe-4S] Cluster Reconstitution—The apo-SplG was dissolved in 800 μl of reconstitution buffer, 50 mm Tris-HCl (pH 8.0), with 5 mm DTT for 0.5 h, followed by the addition of FeCl2 and Na2S to a final concentration of 100 μm. The SplG was then incubated anaerobically for 16 h at 4 °C. The holo-protein solution was finally concentrated in an Ultrafree®-0.5 centrifugal filter device (Millipore) up to a volume of 200 μl, and the buffer was exchanged.
      Synthesis of 5S- and 5R-configured Spore Lesion—Both diastereomers of the spore photoproduct were synthesized as described previously (
      • Friedel M.G.
      • Berteau O.
      • Pieck J.C.
      • Atta M.
      • Olliagnier-de-Choudens S.
      • Fontecave M.
      • Carell T.
      ).
      Synthesis of the aza-AdoMet—The synthesis and purification was similar to previously described procedures (
      • Jackson R.F.W.
      • Fraser J.L.
      • Wishart N.
      • Porter B.
      • Wythes M.J.
      ,
      • Thompson M.J.
      • Mekhalfia A.
      • Hornby D.P.
      • Blackburn G.M.
      ,
      • Yang M.
      • Ye W.
      • Schneller S.W.
      ).
      Synthesis of ssDNA Containing One SP Lesion—500 nmol of a 6-mer ssDNA (5′-GGTTGG-3′) was resuspended in 4 ml of buffer containing 50 mm NaCl, 5 mm pyridine-2,6-dicarboxylic acid, and 5 mm CaCl2 (pH 7.0). The solution was lyophilized in a 6-well cell culture plate. The obtained dry film was subsequently irradiated at 254 nm under anaerobic conditions. The SP containing DNA was separated by rp-HPLC from residual undamaged DNA. Oligonucleotides were concentrated in vacuo using a Savant SpeedVac and were desalted with Sep-Pak® cartridges (Waters) before use.
      Analyses of the SP in ssDNA—To prove that the SP was formed in the oligonucleotide, the oligonucleotide with the sequence 5′-GGTspTGG-3′ was enzymatically digested and analyzed by rp-HPLC-MS/MS. The results were compared with an analysis of previous studies (
      • Douki T.
      • Cadet J.
      ). To 100 μl of a 20 μm solution of the oligonucleotide to be digested, 10 μl of a buffer containing 300 mm ammonium acetate, 100 mm CaCl2, and 1 mm ZnSO4 (pH 5.7) was added, followed by addition of 22 units of nuclease P1 (Penicillium citrinum) and 0.05 units of calf spleen phosphodiesterase. The solution was incubated at 37 °C for 3 h. To the resulting solution 12 μl of a buffer containing 500 mm Tris-HCl, 1 mm EDTA (pH 8.0), 10 units of alkaline phosphatase (calf intestinal phosphatase), and 9.1 units of snake venom phosphodiesterase were added sequentially followed by incubation at 37 °C for another 3 h. The solution thus obtained was added to 6 μl of 0.1 m HCl heat-denatured for 5 min. The solution was then centrifuged at 3000 rpm for 5 min. 30 μl of the solution was transferred into an HPLC vial, and the sample was injected into an HPLC-MS/MS system.
      Assay for ssDNA Repair, Cleavage of AdoMet, Inhibition with aza-AdoMet and the 5S- and 5R-configured Spore Lesion—To prove enzyme activity, the amount of 5′-deoxyadenosine and repaired SP was determined and analyzed by rp-HPLC. Each reaction mixture contains 100 mm Tris-HCl (pH 7.0), 200 mm KCl, 3 mm sodium dithionite, 5 mm DTT, and 0.45 mm AdoMet. All solutions were degassed, and the chemicals were transferred into the glovebox as solids. One sample contained only the components described above. To the second sample 50 μm holo-SplG was added. The third sample contained 50 μm SplG and the 5S-configured spore lesion (1 mm). The fourth sample was identical to the third sample but instead of the 5S-configured spore lesion it contained the 5R-configured spore lesion (1 mm). The reaction conditions for the ssDNA assay are identical to those described above. The reaction vial contained additionally 15 μm of the purified SP DNA strand and a 10-fold decreased SplG concentration. A sample was taken every 20 min for the kinetic measurement. All other reaction mixtures were incubated for 12 h at 4 °C. For the inhibition assay aza-AdoMet was used instead of AdoMet at the same concentration. After incubation, the reaction mixture was frozen in liquid nitrogen and stored at -80 °C. All samples were thawed and centrifuged to remove precipitated protein before HPLC purification.
      rp-HPLC Analyses—For rp-HPLC analysis, the samples were injected directly after centrifugation into the rp-HPLC system to decrease the amount of AdoMet cleaved to S-adenosylhomocysteine. Up to 50 μl of the enzyme reaction mixture were injected onto the rp-HPLC column (Nucleosil C18, 250 mm, 5 μm, 300 Å, Macherey & Nagel) equilibrated with 0.1% trifluoroacetic acid in H2O. The products were separated using a 20-ml linear gradient 30% solvent B (50% CH3CN with 0.1% trifluoroacetic acid) (flow rate 0.7 ml/min at 22 °C). The HPLC gradient was chosen to allow rapid separation and hence easy detection of thymidine in the assay solution, which is the only expected product of the repair reaction. The peaks were assigned by co-injection of thymidine and 5′-deoxyadenosine, and also by further analysis of the newly formed peak by rp-HPLC coupled to a FTICR spectrometer to get a high resolution mass of the newly formed product.
      LC-FTICR Spectrometer Analysis—Fractions of the rp-HPLC analyses were lyophilized and resuspended with double distilled water. All measurements were run on a Thermo Finnigan LTQ FT (Thermo Finnigan, Bremen, Germany). The resolution was attuned to 100,000 at m/z = 400. The mass range was adjusted up to 1000. The measurements were conducted using an IonMax ion source with an electrospray ionization head (Thermo Finnigan, Bremen, Germany). The spray capillary voltage was 3 kV, the heating capillary temperature 300 °C, and the flow rate 200 μl/min.
      UV-visible Spectroscopy—UV-visible spectroscopy was performed in 150 mm Tris-HCl (pH 8.0). The holo-SplG (0.8 mg/ml) was transferred from the glovebox with a UV cuvette (precision cell made of quartz Suprasil®; Helma) airtight with a plug. The cluster reduction was performed previously in the glovebox with 3.6 mm sodium dithionite. All measurements were performed with a UV-visible spectrometer (Varian 100 Bio, Cary).
      EPR Spectroscopy—Purified holo-SplG was concentrated, placed anaerobically in EPR tubes, and frozen in liquid nitrogen. EPR spectra at X-band (9.45 GHz) were obtained with a Bruker ESP spectrometer. Cooling was performed with an Oxford Instruments ESR-900 helium flow cryostat with an ITC-4 temperature controller.
      Iron and Sulfur Content Determination—Iron and sulfur content was analyzed either by inductively coupled plasma atomic emission spectroscopy (ICP-AES), using a Varian Vista RL CCD simultaneous ICP-AES spectrometer, or by a colorimetric assay. ICP-AES protein samples were dialyzed against two changes of buffer (50 mm Tris-HCl (pH 8.0), and 300 mm NaCl) containing 300 nmol/ml holo-SplG. The emission of iron and sulfur was monitored. In the colorimetric iron assay, 600 μl of holo-SplG was incubated for 10 min with 100 μl of HCl (8 m) and for further 10 min with 100 μl of trichloroacetic acid (80% w/v). Subsequently, 200 μl of ammonium acetate was added (75% w/v) to adjust the pH to 4.5. 80 μl of hydroxylamine hydrochloride (10% w/v) and 2,4,6-tripyridyl-S-triazine (4 mm) were added. The absorbance was measured at 593 nm with a UV-visible spectrometer (Varian 100 Bio, Cary) (
      • Fischer D.S.
      • Price D.C.
      ). In the colorimetric S2- assay, 700 μl of holo-SplG was incubated and vortexed with 100 μl of NaOH (6% w/v) and 500 μl of Zn(CH3COO)2. Then 250 μl of N,N-dimethyl-p-phenyldiamine (0.1% w/v in 5 m HCl) and 100 μl of FeCl3 (11.5 mm in 0.6 m HCl) were added and incubated for 0.5 h. The absorbance was measured at 670 nm with a UV-visible spectrometer (Varian 100 Bio, Cary) (
      • King T.E.
      • Morris R.O.
      ). The average value of the iron and sulfur concentration, determined from a standard curve, was divided by the protein concentration in the assay to determine the moles of iron and sulfur bound per mol of protein monomer.
      Dimer and Monomer Structure Analysis—The aerobically purified SplG monomer and dimer were separated by gel permeation chromatography as described above. To probe the contribution of an intrasubunit and intersubunits disulfide bond formation between Cys-141 and Cys-326, the apo-SplG monomer and dimer were incubated with increasing concentrations of reductant tris(2-carboxyethyl)phosphine (TCEP) (
      • Doucette P.A.
      • Whitson L.J.
      • Cao X.
      • Schirf V.
      • Demeler B.
      • Valentine J.S.
      • Hansen J.C.
      • Hart P.J.
      ) from 1 to 250 mm. The structure modifications were analyzed by SDS-PAGE or gel permeation chromatography.

      RESULTS

      Based on the sequence from G. stearothermophilus, oligonucleotides were designed to amplify the splG gene by PCR with chromosomal DNA from G. stearothermophilus. The obtained 1054-bp fragment was cloned into the pET101-D vector, and the expression of the gene was successfully performed in E. coli.
      We first wanted to clarify whether the enzyme exists in a monomeric or dimeric form (
      • Rebeil R.
      • Nicholson W.L.
      ). To this end, the SP lyase from G. stearothermophilus was isolated and purified under aerobic and anaerobic conditions. In both cases the protein had a dark brown color indicative of the presence of an intact Fe/S cluster. This result provided the first evidence that the cluster survives aerobic handling of the protein. Fig. 1 shows the SDS-PAGE results. In Fig. 1, lane 2, a new band (marked with an arrow) was detected, which corresponded to the SplG monomer after induction with IPTG and also stained in Western blot experiments. Subsequent affinity chromatography to purify apo-SplG was performed. However, only a protein mixture as shown in Fig. 1, lane 3, was obtained. The molecular mass marker indicated for the major band a mass of ∼64 kDa, already indicating that the SplG protein dimerizes under aerobic and anaerobic conditions. In order to clarify the molecular sizes in more detail, MALDI-MS analysis (data not shown) of this band was performed. This MS analysis revealed molecular mass values of 74 and 81 kDa for the isolated proteins. The 81-kDa protein possesses the correct molecular mass for the dimerized form of apo-SplG, whereas the molecular mass at 74 kDa is a proteolytically cleaved apo-SplG dimer.
      Figure thumbnail gr2
      FIGURE 1Analysis of purified and reconstituted SplG in different purification steps by SDS-PAGE. The SplG solutions were heated up to 95 °C for 10 min in SDS buffer containing 5% (v/v) β-mercaptoethanol and electrophoresed through 10% SDS-PAGE. The lane marked M contains molecular weight markers. Lanes 1 and 2 contain the crude cell extract before (lane 1) and after induction (lane 2) with IPTG. The arrow indicates the SplG monomer. Lane 3 shows the nickel-nitrilotriacetic acid-purified apo-SplG before reconstitution; lane 4 shows the holo-SplG after reconstitution. Lane 5 shows the holo-SplG dimer after size exclusion purification.
      The second set of bands (Fig. 1, lane 3) between 36 and 50 kDa on the gel was also further analyzed by MALDI-MS mass spectrometry (data not shown). The two analyzed protein fractions featured molecular masses of 37 and 40.5 kDa, which are the correct molecular masses for the apo-SplG monomer (40.5 kDa) and again of a proteolytically shortened version of the protein (37 kDa). The last band on the SDS-polyacrylamide gel (Fig. 1, lane 3) corresponded to a small, N-terminally His6-tagged 27-kDa fragment of the SplG. All the proteolytically shortened fragments with molecular weights smaller than those calculated from the DNA sequence are clearly C-terminal deletion fragments because all fragments are positively recognized by the anti-His6 tag monoclonal antibody in Western blotting experiments.
      In order to convert the functionally inactive apo-SplG into the catalytically competent form, the [4Fe4S] cluster needed to be reconstituted. To this end, the apo-SplG was treated with FeCl2, Na2S, and DTT in the glovebox. The fully reconstituted form was highly oxygen-sensitive as expected. Handling of the reconstituted enzyme required strict anaerobic conditions. Fig. 1, lane 5, shows the fully reconstituted holo-SplG dimer after purification by affinity chromatography of the apoprotein. Interestingly, holo-SplG showed the same behavior as in the gel electrophoresis experiments (Fig. 1, lane 4). The protein exists as a full-length and a C-terminally truncated dimer and monomer. A more detailed SDS-PAGE analysis (Fig. 2) of the protein monomers and dimers showed that the bands are in all cases rather broad. In order to investigate whether this effect is caused by potential residual secondary structure elements, perhaps induced by the Fe/S cluster, we added increasing amounts of TCEP to the protein solution (up to 250 mm). Indeed, this procedure significantly sharpened the bands indicating that residual secondary structural elements were resolved. Most surprisingly, however, is the observation that the ratio between the protein in the monomeric and dimeric state did not change. This shows that the SplG protein dimer is extremely stable.
      Figure thumbnail gr3
      FIGURE 2Analysis of secondary SplG structure. The SplG monomer and dimer were treated as described under “Experimental Procedures” and electrophoresed through 12% SDS-PAGE. A, SplG dimer treated with increased TCEP concentrations. B, SplG monomer treated with increase TCEP concentrations. The first lanes in A and B contain molecular weight markers.
      We next started to investigate the presence of the [4Fe-4S]+/2+ cluster in more detail using UV-visible spectroscopy. The absorption spectra of the reconstituted SplG dimer exhibited a broad absorption peak at around 420 nm (Fig. 3A), characteristic for a [4Fe-4S]2+ cluster. Based on the UV-visible spectrum we can fully rule out that the protein contains an [2Fe-2S]2+ cluster, as such a cluster would possess absorption peaks/shoulders around 330, 420, 460, and 560 nm (
      • Orme-Johnson W.H.
      • Orme-Johnson N.R.
      ,
      • Imai T.
      • Taguchi K.
      • Ogawara Y.
      • Ohmori D.
      • Yamakura F.
      • Ikezawa H.
      • Urushiyama A.
      ). In addition, the molar extinction coefficient (ϵ410 ≈ 15,000 m -1 cm-1) for the [4Fe-4S]2+ cluster fits nicely to the calculated value of ϵ410 ≈ 13,500 m-1 cm-1 per enzyme monomer. Based on the extinction coefficient, we concluded that the holo-SplG dimer contains one [4Fe-4S]2+ cluster per monomer. A value of 3.8 ± 0.3 mol of iron per mol of holo-SplG monomer was found by the method of Fischer and Price (
      • Fischer D.S.
      • Price D.C.
      ). ICP-AES revealed a value of 4.5 ± 0.4 mol of iron per mol of holo-SplG. In addition, we determined 3.7 ± 0.3 mol of acid-labile sulfur per mol of enzyme using the method of King and Morris (
      • King T.E.
      • Morris R.O.
      ). The iron sulfur cluster is likely bound to the protein via the well established motif 91CXXXCXXC98.
      Figure thumbnail gr4
      FIGURE 3UV-visible absorption spectra of reconstituted SplG in 150 mmTris-HCl (pH 8.0). A, spectrum of SplG (0.8 mg/ml) recorded after reconstitution of the SplG dimer. B, spectrum of SplG recorded (i) prior to dithionite addition and (ii) 5 and 30 min after dithionite addition.
      In order to reduce the [4Fe-4S]2+ to the catalytically active [4Fe-4S]+ form, 3.6 mm sodium dithionite was added to the protein solution in the UV cuvette. This resulted immediately in a strong reduction of the absorbance in the entire visible region (Fig. 4B), demonstrating that the reconstituted iron sulfur cluster is indeed redox-active. Addition of the dithionite solution caused an almost full loss of all absorption bands λ > 350 nm within 30 min (Fig. 4B) indicating full conversion of the cluster into the active [4Fe-4S]+ redox state.
      Figure thumbnail gr5
      FIGURE 4EPR spectra of SplG. EPR spectra were measured under the following conditions: microwave frequency 9.459 GHz; modulation frequency 100 kHz; modulation amplitude 1.25 millitesla; microwave power 20 milliwatt; temperature 10 K. A shows the SplG protein reduced with dithionite (10 mm) and the simulation of this spectrum (above) performed with an EPR simulation program (
      • Albracht S.P.J.
      • Beinert H.
      ). B, the spectrum of SplG after AdoMet addition is shown. C, spectra for SplG after addition of AdoMet and 5S-configured substrate. D shows the reconstituted SplG after exposure to air (amplitude 2.5-fold reduced).
      We next performed EPR spectroscopy to obtain a deeper insight into the [4Fe-4S] cluster. The obtained data are in full agreement with the presence of two [4Fe-4S]+ clusters per SplG homodimer. The axial EPR spectrum with g values of 2.04, 1.93, and 1.89 (Fig. 4A) are typical of [4Fe-4S]+ clusters and are very similar to spectra obtained for aRNR and SplB (
      • Ollagnier S.
      • Meier C.
      • Mulliez E.
      • Gaillard J.
      • Schuenemann V.
      • Trautwein A.
      • Mattioli T.
      • Lutz M.
      • Fontecave M.
      ,
      • Ugulava N.B.
      • Gibney B.R.
      • Jarrett J.T.
      ). Addition of the coenzyme AdoMet to the reduced SplG showed a strong decrease of the signals (Fig. 4B) indicating an interaction of AdoMet with the cluster. Further reduction of the signal intensity was obtained by addition of the synthetic 5S-configured SP (Fig. 4C). SplG treated with oxygen after reconstitution shows an extreme change of the spectrum (Fig. 4D) typical for decomposed oxidized Fe/S proteins. All these features show that SplG is very similar to SplB. SplG is, however, more stable and has a strongly increased tendency to form a stable dimer.
      The main objective was the characterization of the catalytic potential of the spore photoproduct lyase using for the first time well defined substrates. To this end we analyzed formation of the reduced 5′-deoxyadenosine (5′-dAdoH) because this compound is readily detectable by rp-HPLC at 260 nm, and formation of 5′-dAdoH is a typical indicator used to monitor spore photoproduct lyase activity (
      • Ollagnier S.
      • Mulliez E.
      • Schmidt P.P.
      • Eliasson R.
      • Gaillard J.
      • Deronzier C.
      • Bergman T.
      • Gräslund A.
      • Reichard P.
      • Fontecave M.
      ). When we added AdoMet to the reconstituted SplG and analyzed the solution by rp-HPLC, we indeed detected cleavage of AdoMet to 5′-dAdoH (Scheme 2A). AdoMet was cleaved even in the absence of any SP substrate in a time-dependent manner (data not shown). However, the cleavage ceases with time indicating that the formed methionine is able to bind competitively to the Fe/S cluster (
      • Jarrett J.T.
      ). Because 5′-dAdoH formation proceeded in our hands even in the absence of substrate, we believe that formation of this molecule is not a good indicator of enzyme activity. We therefore studied the ability of holo-SplG to repair the spore photoproduct. We investigated the activity with monomeric substrate analogs that were recently synthesized in our group (
      • Friedel M.G.
      • Berteau O.
      • Pieck J.C.
      • Atta M.
      • Olliagnier-de-Choudens S.
      • Fontecave M.
      • Carell T.
      ). Addition of the 5S-configured lesion substrate to a solution containing the enzyme SplG resulted in a clear increase in 5′-dAdoH formation (Scheme 2D). In addition, the appearance of a new peak in the rp-HPLC at 12.5 min was observed. Co-injection of thymidine, which co-eluted with the new peak, showed formation of the expected product of the enzymatic repair reaction. In order to get further information about the newly formed compound, the fractions were pooled, lyophilized, and resuspended in double distilled water. The fractions were then analyzed by rp-HPLC coupled to MS/MS using a Finnigan LC-FTICR system (Fig. 5). For the peak eluting at 13.5 min, the molecular ion [5′-dAdoH + trifluoroacetic acid]+ at 364.09 was clearly detected. The peak that eluted at 12.5 min gave a mass of m/z = 355.08, corresponding to [dT + trifluoroacetic acid]+. These MS results prove the formation of 5′-dAdoH and production of deoxythymidine in the presence of the 5S-configured spore lesion analog. In accord with earlier work using the enzyme SplB (
      • Friedel M.G.
      • Berteau O.
      • Pieck J.C.
      • Atta M.
      • Olliagnier-de-Choudens S.
      • Fontecave M.
      • Carell T.
      ), no increase in 5′-AdoH formation and no deoxythymidine production were observed in the presence of the 5R-configured substrate showing that SplG, as well as SplB, is a highly stereospecific enzyme, which recognizes and repairs only one of the two potential diastereomers of the lesion, namely the 5S-configured lesion.
      Figure thumbnail gr6
      SCHEME 2Repair of the 5 S- and 5R-configured spore photoproducts by SplG. A repair of 5S-configured spore photoproducts to thymidine was observed. No repair of the 5R-configured spore photoproducts was detected. HPLC analysis of AdoMet conversion to 5′-deoxyadenosine (AdoH) and repair of SP to thymidine by SplG. The enzyme reaction and HPLC analysis were performed as described under “Experimental Procedures.” A, reaction contained 50 μm SplG and AdoMet. B, reaction containing 50 μm SplG, AdoMet, and the 5R-configured SP. C, negative control with only AdoMet and the 5S-configured SP. D, reaction containing 5 nmol of SplG, AdoMet and the 5S-configured SP. In all four traces, the y axes showed the absorbance at 260 nm.
      Figure thumbnail gr7
      FIGURE 5LC-FTICR analysis of the peaks (retention time = 13.5 and 12.5 min) from . Displayed are the molecular weights of thymidine associated with trifluoroacetic acid [M + trifluoroacetic acid (TFA)]+ = 356.08 (A) and 5′-deoxyadenosine associated with trifluoroacetic acid [M + trifluoroacetic acid]+ = 364.09 (B). a.u., absorption units; amu, atomic mass units.
      In order to analyze the activity of SplG in more detail and to determine whether the enzyme is capable of turnover, we next investigated how the protein repairs small DNA strands containing a defined SP lesion at a defined site. In order to generate the substrate, we irradiated DNA with UV light. Based on the observation that irradiation of calf thymus DNA in the presence of DPA generates SP lesions (
      • Douki T.
      • Setlow B.
      • Setlow P.
      ), we prepared a solution of single strand 5′-GGTTGG-3′ and DPA, which was subsequently concentrated to dryness. The obtained DNA film was irradiated under strictly anaerobic conditions with UV light (λ = 254 nm). Analysis of the DNA after defined intervals by rp-HPLC showed that the UV light converted the DNA into several unknown products. After about 300 min of irradiation, product formation ceased. The DNA was dissolved in water, and the DNA product strands were isolated by semi-preparative rp-HPLC. The largest fraction was enzymatically digested, and the digest was further analyzed by rp-HPLC-MS (Fig. 6). We indeed obtained the signal with the correct molecular mass for the SP lesion. In addition, the fragmentation pattern of the spore photoproduct lesion was exactly as described previously (
      • Douki T.
      • Cadet J.
      ). Our irradiation procedure therefore produced a small single-stranded DNA strand containing one SP lesion in the middle (5′-GGTspTGG-3′). This pure substrate was finally used in kinetic measurements. The reconstituted holo-SplG together with AdoMet was added under strictly anaerobic conditions to the solution containing 5′-GGTspTGG-3′. The obtained mixture was analyzed by rp-HPLC every 20 min. Indeed, addition of the enzyme and AdoMet slowly converted the SP lesion containing strand (Fig. 7, blue) into a new DNA strand (red). Coinjection proved that the new signal (red) was caused by the ssDNA strand 5′-GGTTGG-3′, proving efficient repair (Fig. 7). Based on these data, we calculated a specific enzyme activity of 2.6 μmol of repaired SP lesion containing ssDNA per min and per mg of SplG. The calculated turnover number is ∼100, which shows that the SplG is catalytically competent. Indeed, using aza-AdoMet instead of AdoMet in the ssDNA repair assay (Fig. 8) fully blocked the repair reaction thus providing strong support for the repair mechanism proposed by Cheek and Broderick (
      • Cheek J.
      • Broderick J.B.
      ), Rebeil and Nicholsen (
      • Rebeil R.
      • Nicholson W.L.
      ), and Mehl and Begley (
      • Mehl R.A.
      • Begley T.P.
      ). Most interesting is the fact that the repair is an absolutely clean process. No side reactions such as DNA cleavage were observed, showing that the enzyme has the ability to tightly control the reactive radical so close to the DNA.
      Figure thumbnail gr8
      FIGURE 6LC-FTICR analysis of the spore photoproduct after enzymatic total digestion. Displayed is the molecular weight of spore photoproduct [M + H]- = 545,12 (A) as the parent ion which is further fragmented to give the daughter ions shown in B. Here the ions were detected in a molecular weight range between 160 and 555 units (dR, 2-deoxyribose; P, 2-deoxyribose and the spore photoproduct without 2-deoxyribose and 2-deoxyribose are shown as structure). amu, atomic mass units.
      Figure thumbnail gr9
      FIGURE 7Repair of the ssDNA containing spore photoproducts by SplG. A repair of the 6-mer ssDNA containing spore photoproducts to undamaged DNA was observed. The enzyme reaction and HPLC analysis were performed as described under “Experimental Procedures.” The red peak series shows the decreasing amount of DNA containing the spore photoproduct. The blue peak series shows the growing amount of repaired DNA. The peaks marked with × are from the buffer. a.u., absorption units.
      Figure thumbnail gr10
      FIGURE 8The enzyme assay with aza-AdoMet instead of AdoMet shows no spore photoproduct repair. The peaks marked with × are from the buffer. a.u., absorption units.

      DISCUSSION

      The repair of the spore DNA lesion (SP) is performed in nature by an unusual repair enzyme called DNA spore photoproduct lyase. So far the repair mechanism has been studied with the enzyme isolated from B. subtilis. It was found that the enzyme belongs to the class of radical AdoMet enzymes, which use the 5′-adenosyl radical to initiate a radical reaction. Labeling studies by Broderick (
      • Cheek J.
      • Broderick J.B.
      ) provided support for the repair mechanism proposed by Mehl and Begley (
      • Mehl R.A.
      • Begley T.P.
      ). More detailed studies of the repair mechanism and particularly of the lesion recognition step are needed in order to answer the question of how the enzyme controls radical processes so close to a DNA duplex. Here, radical-induced DNA damage is always a possible and likely side reaction. In order to address these questions, we isolated a spore photoproduct lyase enzyme from the thermophilic G. stearothermophilus organism. This enzyme turned out to be stable enough for detailed analysis. We also developed a new procedure that allows preparation of small oligonucleotides containing one SP lesion at a defined site for detailed repair and binding studies. Using this substrate we could provide direct evidence that the enzyme is sufficiently stable to turn over the lesion substrate.
      SplG dimerizes during the aerobic purification process. The protein is inherently stable as a dimer under anaerobic conditions after cluster reconstitution (Figs. 2 and 3). The dimeric state was found to be so stable that even the addition of 250 mm TCEP does not induce monomerization. SplG shares these properties with several other well characterized members of the radical AdoMet protein family. Proteins such as BioB, aRNR-AE, and pyruvate formate-lyase (
      • Voegtli W.C.
      • Ge J.
      • Perlstein D.L.
      • Stubbe J.
      • Rosenzweig A.C.
      ,
      • Berkovitch F.
      • Nicolet Y.
      • Wan J.T.
      • Jarrett J.T.
      • Drennan C.L.
      ,
      • Becker A.
      • Fritz-Wolf K.
      • Kabsch W.
      • Knappe J.
      • Schultz S.
      • Volker Wagner A.F.
      ) also form protein dimers. The preferred dimer state and unusual stability of secondary intra- and intermolecular structures of the SplG in comparison to SplB is probably a consequence of the thermophilic origin of G. stearothermophilus strain whose temperature optimum is around 60 °C. The dimerization of the full-length SplG monomer is presumably facilitated by the fifth cysteine at position 326, which is lacking in the SplB protein. This would indicate that the dimerization involves formation of at least two intermolecular disulfide bridges. This dual linkage of the monomers together, perhaps with other intramolecular forces, seems to be responsible for the unusual stability of the homodimer.
      Comparison of the deduced amino acid sequence of SplB and SplG reveals four conserved cysteine residues (Cys-91, Cys-95, Cys-98, and Cys-141). Three cysteines are needed to coordinate the [4Fe-4S] cluster. The function of the fourth conserved cysteine is unknown. Recently published results, however, indicate that this conserved cysteine is vital for the function of the protein. Site-directed mutagenesis showed that a mutation at this position in the splB gene (C141A) is associated with a loss of enzyme activity in vivo (
      • Fajardo-Cavazos P.
      • Rebeil R.
      • Nicholson W.L.
      ). Potentially, the conserved fourth cysteine is required for dimer formation.
      The [4Fe-4S] cluster formation of SplB and SplG is coordinated by the first three cysteines, which demonstrates a high degree of similarity to other proteins belonging to the radical AdoMet super family (
      • Hanzelmann P.
      • Schindelin H.
      ,
      • Petrovich R.M.
      • Ruzicka F.J.
      • Reed G.H.
      • Frey P.A.
      ). We could confirm the existence of a [4Fe-4S] cluster after quantification of the sulfur and iron content, which was measured by using two independent methods. We measured the iron content to be ∼4 mol of iron per mol for the holo-SplG monomer. In addition a value of 4 mol of acid-labile sulfide per mol of enzyme was measured and is characteristic for a [4Fe-4S] cluster (
      • Tamarit J.
      • Mulliez E.
      • Meier C.
      • Trautwein A.
      • Fontecave M.
      ). The presence of such a cluster is supported by UV and EPR spectroscopic data. We could observe an intense absorption at 420 nm in the UV spectra, typical for [4Fe-4S] clusters (
      • Jakimowicz P.
      • Cheesman M.R.
      • Bishai W.R.
      • Chater K.F.
      • Thomson A.J.
      • Buttner M.J.
      ). Moreover, we obtained no spectroscopic evidence for the presence of a [2Fe-2S] cluster after reduction with dithionite (
      • Duin E.C.
      • Lafferty M.E.
      • Crouse B.R.
      • Allen R.M.
      • Sanyal I.
      • Flint D.H.
      • Johnson M.K.
      ). Dithionite reduction proceeds very slowly over a period of 30 min.
      The EPR spectra concur with the results of UV spectroscopy and the quantification of the iron and sulfur content in the SplG. Here we obtained clear evidence for the presence of the [4Fe-4S] cluster in the reconstituted SplG homodimer. The dramatic decrease of the EPR signals after adding the cofactor AdoMet and the 5S-configured substrate is evidence for the binding of AdoMet and the 5S-configured SP to the [4Fe-4S]+ cluster.
      Regarding the functional analysis of SplG, we could show that the purified homodimer is able to cleave AdoMet to 5′-deoxyadenosine and methionine using rp-HPLC-MS. This feature was frequently used to characterize the activity of the enzyme (
      • Cheek J.
      • Broderick J.B.
      ,
      • Rebeil R.
      • Nicholson W.L.
      ,
      • Rebeil R.
      • Sun Y.B.
      • Chooback L.
      • Pedraza-Reyes M.
      • Kinsland C.
      • Begley T.P.
      • Nicholson W.L.
      ,
      • Friedel M.G.
      • Berteau O.
      • Pieck J.C.
      • Atta M.
      • Olliagnier-de-Choudens S.
      • Fontecave M.
      • Carell T.
      ). However, 5′-deoxyadenosine formation is not a reliable indicator for the repair mechanism. For the B. subtilis Spl, formation of about two molecules of 5′-deoxyadenosine per molecule of (His10) SplB dimer was observed. Comparably, Ollagnier et al. (
      • Ollagnier S.
      • Mulliez E.
      • Schmidt P.P.
      • Eliasson R.
      • Gaillard J.
      • Deronzier C.
      • Bergman T.
      • Gräslund A.
      • Reichard P.
      • Fontecave M.
      ) observed that ∼3 molecules of AdoMet were cleaved per dimer by the aRNR-AE of E. coli. The AdoMet cleavage of the holo-SplG is half compared with SplB. Based on these data we are currently not sure if AdoMet cleavage is indeed a proper indicator for SplG enzyme activity. Cheek and Broderick (
      • Cheek J.
      • Broderick J.B.
      ) used an SP specifically 3H-labeled at C-6. They observed that the C-6 tritium label from SP is finally found in AdoMet and not in 5′-deoxyadenosine. This suggests that the AdoMet is indeed needed as a catalytic cofactor to reversibly generate the putative 5′-deoxyadenosine radical intermediate and not as a co-substrate. It could well be that the background 5′-deoxyadenosine formation proceeds via a shunt pathway.
      We therefore decided to use synthetic SP substrates to confirm the activity of SplG. We first studied enzyme activity using synthetic SP lesions. With these substrates, formation of thymidine, and hence repair, was clearly observed. However, only with the 5S-configured substrate was product formation detected. No repair could be seen if the 5R-substrate was used, similar to a recent study that we performed with SplB. Most interesting are the studies with a defined DNA single strand containing an SP lesion at a defined site. This ssDNA substrate was prepared in a novel way by irradiation of a dry film of ssDNA in the presence of DPA under strict anaerobic conditions at 254 nm. The DNA containing the SP lesion was isolated by rp-HPLC, and the presence of the lesion was confirmed by rp-HPLC-MS and rp-HPLC-MS/MS after a total digest of the ssDNA. This novel substrate was added to a buffered solution of SplG in the presence of AdoMet. The ssDNA substrate was readily accepted by the repair enzyme, which converted the lesion-containing DNA with a turnover number of about 100 into the repaired ssDNA. The usage of aza-AdoMet instead of AdoMet blocked the repair reaction as expected.
      In conclusion, we show that the purified holo-SplG dimer of G. stearothermophilus repairs the 5S-configured SP DNA lesions in the dimeric state. We were able to generate a pure 6-mer ssDNA strand containing an SP lesion at a defined site, which can be used to determine the enzymatic activity. The calculated specific enzyme activity is 2.6 μmol of SP repaired per min/mg of SplG. This enzyme activity is completely inhibited by the cofactor mimic aza-AdoMet. The thermophilic enzyme apo-SplG was purified under aerobic conditions and fully reconstituted under conditions favoring the cluster formation to the holo-SplG. This holo-SplG contains the correct amount of iron and sulfur for two [4Fe-4S] clusters per homodimer of holo-SplG. The presence of this cluster was confirmed by EPR and UV spectroscopy. Transcriptional regulation of SplG is not performed by a homolog of SplA in G. stearothermophilus. The presented results provide a more stable SplG enzyme and a new method that allows synthesis of defined SP lesion containing substrates. Both achievements should now allow a detailed biochemical and structural analysis of the repair reaction similar to what was recently achieved in the case of the cyclobutane pyrimidine dimer DNA photolyase.

      Acknowledgments

      We thank Dr. Werner Spahl and Helmut Hartel for LP-FTICR and ICP-AES measurements; Dr. Thorsten Selmer, Dr. David Hammond, Ralf Strasser, and Dr. Glenn Burley for discussions and critical comments on the manuscript.

      References

        • Setlow P.
        Annu. Rev. Microbiol. 1995; 49: 29-54
        • Nicholson W.L.
        • Munakata N.
        • Horneck G.
        • Melosh H.J.
        • Setlow P.
        Microbiol. Mol. Biol. Rev. 2000; 64: 548-572
        • Vreeland R.H.
        • Rosenzweig W.D.
        • Powers D.W.
        Nature. 2000; 407: 897-900
        • Penna T.
        • Ishii M.
        • Machoshvili I.
        • Marques M.
        Appl. Biochem. Biotechnol. 2002; 98: 525-538
        • Gerhardt P.
        • Marquis R.E.
        Smith I. Slepecky R.A. Setlow P. Regulation of Prokaryotic Development. American Society for Microbiology, Washington, D. C.1989: 43-63
        • Devine D.A.
        • Keech A.P.
        • Wood D.J.
        • Killington R.A.
        • Boyes H.
        • Doubleday B.
        • Marsh P.D.
        J. Appl. Microbiol. 2001; 91: 786-794
        • Setlow P.
        Environ. Mol. Mutagen. 2001; 38: 97-104
        • Cadet J.
        • Anselmino C.
        • Douki T.
        • Voituriez L.
        J. Photochem. Photobiol. 1992; 15: 277-298
        • Donnellan J.E.
        • Stafford R.S.
        Biophys. J. 1968; 8: 17-28
        • Douki T.
        • Setlow B.
        • Setlow P.
        Photochem. Photobiol. 2005; 81: 163-169
        • Nicholson W.L.
        • Setlow B.
        • Setlow P.
        Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8288-8292
        • Nicholson W.L.
        • Setlow B.
        • Setlow P.
        J. Bacteriol. 1990; 172: 6900-6906
        • Douki T.
        • Setlow B.
        • Setlow P.
        Photochem. Photobiol. 2005; 4: 591-597
        • Setlow P.
        Comments Mol. Cell Biophys. 1988; 5: 253-264
        • Munakata N.
        • Rupert C.S.
        Mol. Gen. Genet. 1974; 130: 239-250
        • Nicholson W.L.
        • Fajardocavazos P.
        • Pedrazareyes M.
        • Sun Y.B.
        • Zazuetasandoval R.
        J. Cell Biochem. 1995; 19: 275
        • Cheek J.
        • Broderick J.B.
        J. Am. Chem. Soc. 2002; 124: 2860-2861
        • Rebeil R.
        • Nicholson W.L.
        Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9038-9043
        • Rebeil R.
        • Sun Y.B.
        • Chooback L.
        • Pedraza-Reyes M.
        • Kinsland C.
        • Begley T.P.
        • Nicholson W.L.
        J. Bacteriol. 1998; 180: 4879-4885
        • Cheek J.
        • Broderick J.B.
        J. Biol. Inorg. Chem. 2001; 6: 209-226
        • Mehl R.A.
        • Begley T.P.
        Org. Lett. 1999; 1: 1065-1066
        • Sambrock J.
        • Russel D.
        Molecular Cloning: A Laboratory Manual,3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001: 1-125
        • Friedel M.G.
        • Berteau O.
        • Pieck J.C.
        • Atta M.
        • Olliagnier-de-Choudens S.
        • Fontecave M.
        • Carell T.
        Chem. Commun. 2006; 4: 445-447
        • Jackson R.F.W.
        • Fraser J.L.
        • Wishart N.
        • Porter B.
        • Wythes M.J.
        J. Chem. Soc. Perkin Trans. I. 1998; 12: 1903-1912
        • Thompson M.J.
        • Mekhalfia A.
        • Hornby D.P.
        • Blackburn G.M.
        J. Org. Chem. 1999; 64: 7467-7473
        • Yang M.
        • Ye W.
        • Schneller S.W.
        J. Org. Chem. 2004; 69: 3993
        • Douki T.
        • Cadet J.
        Photochem. Photobiol. Sci. 2003; 2: 433-436
        • Fischer D.S.
        • Price D.C.
        Clin. Chem. 1964; 10: 21-31
        • King T.E.
        • Morris R.O.
        Methods Enzymol. 1964; 10: 634-641
        • Doucette P.A.
        • Whitson L.J.
        • Cao X.
        • Schirf V.
        • Demeler B.
        • Valentine J.S.
        • Hansen J.C.
        • Hart P.J.
        J. Biol. Chem. 2004; 279: 54558-54566
        • Orme-Johnson W.H.
        • Orme-Johnson N.R.
        Spiro T.G. Iron Sulfur Proteins. Wiley Interscience, New York1982: 67-96
        • Imai T.
        • Taguchi K.
        • Ogawara Y.
        • Ohmori D.
        • Yamakura F.
        • Ikezawa H.
        • Urushiyama A.
        J. Biochem. (Tokyo). 2001; 130: 649-655
        • Ollagnier S.
        • Meier C.
        • Mulliez E.
        • Gaillard J.
        • Schuenemann V.
        • Trautwein A.
        • Mattioli T.
        • Lutz M.
        • Fontecave M.
        J. Am. Chem. Soc. 1999; 121: 6344-6350
        • Ugulava N.B.
        • Gibney B.R.
        • Jarrett J.T.
        Biochemistry. 2000; 39: 5206-5214
        • Ollagnier S.
        • Mulliez E.
        • Schmidt P.P.
        • Eliasson R.
        • Gaillard J.
        • Deronzier C.
        • Bergman T.
        • Gräslund A.
        • Reichard P.
        • Fontecave M.
        J. Biol. Chem. 1997; 272: 24216-24223
        • Jarrett J.T.
        Arch. Biochem. Biophys. 2005; 433: 312-321
        • Voegtli W.C.
        • Ge J.
        • Perlstein D.L.
        • Stubbe J.
        • Rosenzweig A.C.
        Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10073-10078
        • Berkovitch F.
        • Nicolet Y.
        • Wan J.T.
        • Jarrett J.T.
        • Drennan C.L.
        Science. 2004; 303: 76-79
        • Becker A.
        • Fritz-Wolf K.
        • Kabsch W.
        • Knappe J.
        • Schultz S.
        • Volker Wagner A.F.
        Nat. Struct. Biol. 1999; 6: 969-975
        • Fajardo-Cavazos P.
        • Rebeil R.
        • Nicholson W.L.
        Curr. Microbiol. 2005; 51: 331-335
        • Hanzelmann P.
        • Schindelin H.
        Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12870-12875
        • Petrovich R.M.
        • Ruzicka F.J.
        • Reed G.H.
        • Frey P.A.
        Biochemistry. 1992; 31: 10774-10781
        • Tamarit J.
        • Mulliez E.
        • Meier C.
        • Trautwein A.
        • Fontecave M.
        J. Biol. Chem. 1999; 274: 31291-31296
        • Jakimowicz P.
        • Cheesman M.R.
        • Bishai W.R.
        • Chater K.F.
        • Thomson A.J.
        • Buttner M.J.
        J. Biol. Chem. 2005; 280: 8309-8315
        • Duin E.C.
        • Lafferty M.E.
        • Crouse B.R.
        • Allen R.M.
        • Sanyal I.
        • Flint D.H.
        • Johnson M.K.
        Biochemistry. 1997; 36: 11811
        • Albracht S.P.J.
        • Beinert H.
        Biochim. Biophys. Acta. 1982; 683: 245-277