Research Article Gamma Radiation-Induced Damage in the Zinc …downloads.hindawi.com/journals/bca/2016/1642064.pdf · oxidation state of zinc (II). In animal cells, zinc (II) ions

Research ArticleGamma Radiation-Induced Damage in the Zinc Finger ofthe Transcription Factor IIIA

XiaoHong Zhang123 YuJi Miao1 XiaoDan Hu1 Rui Min4

PeiDang Liu2 and HaiQian Zhang123

1Department of Nuclear Science and Engineering Nanjing University of Aeronautics and Astronautics Nanjing 210016 China2Jiangsu Key Laboratory for Biomaterials and Devices Southeast University Nanjing 210009 China3Collaborative Innovation Center of Radiation Medicine Jiangsu Higher Education Institutions Suzhou UniversitySuzhou 215123 China4Division of Radiation Medicine Department of Naval Medicine Second Military Medical University Shanghai 200433 China

Correspondence should be addressed to XiaoHong Zhang zhangxiaohongnuaaeducn

Received 31 July 2016 Accepted 18 September 2016

Academic Editor Massimiliano F Peana

Copyright copy 2016 XiaoHong Zhang et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

A zinc fingermotif is an element of proteins that can specifically recognize and bind toDNA Because they containmultiple cysteineresidues zinc finger motifs possess redox properties Ionizing radiation generates a variety of free radicals in organisms Zinc fingermotifs therefore may be a target of ionizing radiation The effect of gamma radiation on the zinc finger motifs in transcriptionfactor IIIA (TFIIIA) a zinc finger protein was investigated TFIIIA was exposed to different gamma doses from 60Co sources Thedose rates were 020Gymin and 800Gyh respectivelyThe binding capacity of zinc finger motifs in TFIIIA was determined usingan electrophoretic mobility shift assay We found that 1000Gy of gamma radiation impaired the function of the zinc finger motifsin TFIIIAThe sites of radiation-induced damage in the zinc finger were the thiol groups of cysteine residues and zinc (II) ionsThethiol groups were oxidized to form disulfide bonds and the zinc (II) ions were indicated to be reduced to zinc atoms These resultsindicate that the zinc finger motif is a target domain for gamma radiation which may decrease 5S rRNA expression via impairmentof the zinc finger motifs in TFIIIA

1 Introduction

Zinc finger proteins are one of the largest protein familiesin eukaryotes and approximately 2-3 of all human genesencode zinc finger proteins [1]The term ldquozinc fingerrdquo denotesa variety of compact protein domains with a tetrahedralgeometry stabilized by structural zinc II ions that interactwith cysteine thiol and histidine imidazole groups [2] Thezinc finger motif promotes specific protein-nucleic acidrecognition and binding [3] and most zinc finger proteinsfunction as transcription factorsThus it can be inferred thatagents that damage the zinc finger motif may also impairgene expression [4 5] Hence investigations of factors thatdamage zinc finger motifs have received much attention inresearch on zinc finger protein-related regulation of geneexpression Zinc finger motifs can be divided into Cys

8 Cys4

Cys3His and Cys

2His2types and others The presence of

multiple cysteine residues indicates that zinc finger motifspossess redox properties because most redox reactions inproteins occur at the thiol group of cysteine residues [6]Based on their redox properties agents that might impairthe activity of zinc finger motifs have been investigatedSeveral studies have demonstrated that zinc finger motifs aresusceptible to in vitro oxidation induced by agents such as3-nitrosobenzamide hydrogen peroxide and disulfiram [7ndash10] These observations were also confirmed in vivo whenrepression of a gene specific to Sp-1 a zinc finger proteinwas observed in cultured HeLa cells subjected to oxidativestress [11] The biological effects of ionizing radiation includeoxidation or reduction of biological materials As an indirecteffect many water radiolysis products are produced Thuszinc finger motifs are likely targets of ionizing radiation

Hindawi Publishing CorporationBioinorganic Chemistry and ApplicationsVolume 2016 Article ID 1642064 7 pageshttpdxdoiorg10115520161642064

2 Bioinorganic Chemistry and Applications

A zinc atom is a relatively redox-inert element with anoxidation state of zinc (II) In animal cells zinc (II) ions arestored in cytosolic cysteine-rich proteins ormetallothioneinsin the form of zinc-sulfur clustersThe level of ldquofreerdquo intracel-lular zinc (II) ions is very low that is in the nanopicomolarrange and spontaneous release of zinc (II) ions from themetallothioneins system to target proteins does not occurs[12] In addition to damaging zinc finger motifs oxidantsmay be involved in zinc metabolism Current investigationssuggest that the effects of oxidants on zinc finger motifs arelinked to the release of zinc (II) ions [13] According to thishypothesis zinc fingers inmetalloproteinsmay serve as a zincpool Ionizing radiation differs from oxidative agents that arecommonly used for oxidation of zinc finger motifs becausethey induce formation of not only the oxidative radical butalso the reduced radical that is the hydrated electron Thepresence of the hydrated electron may reduce zinc II ions

Transcription factor IIIA (TFIIIA)was the first zinc fingerprotein to be identified in Xenopus and it consists of ninetandem repeat Cys

2His2-type zinc fingers [14 15] The three-

dimensional conformation of this type of zinc finger includesan 120572-helix and two 120573-sheets The internal control region(ICR) of the 5S rRNA gene is a 120-base sequence includingthree separate promoter regions of an A-box (+50 to +64)an intermediate element (+67 to +72) and a C-box (+82to +92) [16] The nine zinc fingers of TFIIIA specificallybind with the ICR through insertion of their 120572-helixesinto the major or minor grooves of these three promoterregions [17] TFIIIA was chosen to investigate damage tothe zinc finger motif because Cys

2His2-type zinc fingers are

relatively electrically neutral and possess the low conditionalassociation constant among that of all known zinc fingerproteins [18 19] In this experiment we explored the effectof ionizing radiation on the zinc finger motif of TFIIIA andthe corresponding mechanism of damage Specifically weinvestigated the binding capacity of the zinc finger in gamma-irradiated TFIIIA with the ICR and identified potential sitesof irradiation-induced damage To the best of our knowledgeonly one article has been published on the effects of ionizingradiation on a protein containing zinc-sulfur clusters that ismetallothionein with the aimof analyzing its radioprotectionfunction [20] Our results indicate that gamma radiationaffects specific binding of TFIIIA with the ICR throughthioldisulfide and zinc (II) ionzinc atom exchanges in thezinc finger motifs

2 Materials and Methods

21 Preparation of Plasmid pMD18T-ICR The full ICRsequence was synthesized on an automated DNA synthesizer(ABI 394 Applied Biosystems Inc Carlsbad CA USA)using phosphoramidite chemistry The ICR sequence wasthen ligated to a pMD18T vector (TaKaRa Co Dalian Liaon-ing China) which was transformed into Escherichia colicompetent cells (TaKaRa) The targeted DNA was amplifiedby polymerase chain reaction (PCR) analyzed by agaroseelectrophoresis and confirmed by DNA sequencing

22 Preparation of Recombinant TFIIIA Recombinant Xeno-pus laevis TFIIIA was produced by Escherichia coli BL21(DE3) cells harboring the plasmid pcoldII-TFIIIA [21]Briefly cells were cultured at 37∘C with shaking at 220 rpmand when the cell density (OD600) reached 06 TFIIIAexpression was induced by addition of 01mM isopropylthio-120573-d-galactoside (IPTG) and 10120583MZnCl

2 After 4 h of further

incubation at 37∘C cells were harvested and lysed with anEmulsiFlex-C3 homogenizer (Avestin Ottawa ON Canada)After cell lysis the inclusion bodies which contain most ofthe TFIIIA were first washed with buffer 1 (50mM Tris-Cl100mM NaCl 1mM EDTA and 01 Triton X-100 pH 80)before being dissolved in buffer 2 (50mM Tris-Cl 100mMNaCl 6M guanidine hydrochloride and 5mM DTT pH80) The whole dissolution process was performed at 4∘Cand 24 h was required to completely dissolve the TFIIIAThe supernatant was then slowly diluted in buffer 3 (50mMTris-Cl 50mM NaCl 100 120583M ZnCl

2 01M Arg 1mM GSH

01mM GSSG 10 glycerol and 01 Nonidet P-40 pH80) at a speed of one drop per second and the sample wasslowly stirred at 4∘C for 24 h to refold the TFIIIA After therefolding process was complete the sample was concentratedusing an ultrafiltration tube (Amicon Ultra Millipore CoBillerica MA USA) before being purified by fast proteinliquid chromatography using a Superdex 75 column (GE CoFairfield CT USA)

To detect zinc (II) ions in irradiated TFIIIA recombinantTFIIIA was also prepared in buffer without zinc (II) ionsupplementation Similar to the procedure described abovethe TFIIIA contained in inclusion bodies was refolded usingbuffers 1 2 and 3 The refolded TFIIIA was then precipitatedwith 40 ammonium sulfate at 4∘C for 30min The pelletwas again denatured with buffer 2 and refolded in buffer 3lacking the 100 120583MZnCl

2The rest of the purification process

was performed as described aboveThe protein concentrationwas measured using the Bradford method and the purifiedTFIIIA solution (025120583g120583L) was stored at minus80∘C until use

23 Radiation Exposure The TFIIIA solution was irradiatedwith 60Co sources in an atmosphere of air at 4∘C Theabsorbed doses were 0Gy 1 Gy 10Gy 100Gy 500Gy 800Gyand 1000Gy The low doses (1 Gy and 10Gy) were admin-istered with 60Co source (Gaotong Isotope Co ChengduSichuan China) with a radioactivity of approximately 222times 1012 Bq The dose rate was 020Gymin The high doses(100Gy 500Gy 800Gy and 1000Gy)were administeredwith60Co source (Nordion Inc Kanata ON Canada) with aradioactivity of approximately 148 times 1016 Bq The dose ratewas 800Gyh Dosimetry was performed on a regular basiswith a 06 cm3 Farmer Ionization Chamber (Type 30010)which was connected to a dosimeter (PTW-Freiburg CoFreiburg Breisgau Germany) The chamber was placed nextto the tubes for irradiation

24 Electrophoretic Mobility Shift Assay For preparation offluorescent FAM-labeled probes the ICRof the 5S rRNAgenewas amplified by PCR using a high-fidelity Dpx DNA Poly-merase (Tolo Biotech Shanghai China) from the plasmid

Bioinorganic Chemistry and Applications 3

pMD18T-ICR using the primers M13F-47(FAM) and M13R-48 The FAM-labeled probes were purified using the WizardSV Gel and PCR Clean-Up System (Promega Co MadisonWI USA) and were quantified with a NanoDrop 2000cSystem (Thermo Co Waltham MA USA) Electrophoreticmobility shift assay (EMSA) was performed in a 20 120583Lreaction volume containing 50 ng of probe and 25120583g ofirradiated TFIIIA in a reaction buffer of 50mM Tris-HCl(pH 80) 100mM KCl 25mM MgCl

2 005mM DTT and

10glycerol After incubation for 30min at 25∘C the reactionsystem was loaded onto a 2 agarose gel buffered with 05 timesTBE Gels were scanned with an ImageQuant LAS 4000 minisystem (GE) Sheared salmon sperm DNA (100 ng120583L) wasadded to prevent nonspecific binding

25 Quantification of Disulfide in Irradiated TFIIIA Theprocedures for disulfide detection were as follows TFI-IIA solutions (025 120583g120583L 200120583L) were pipetted into the551015840-dithiobis-2-nitrobenzoic acid (DTNB) and 2-nitro-5-thiosulfobenzoate (NTSB) solution (3mL) respectively TheDTNB solution (pH 80) was prepared with 8M urea200mM Tris 1 SDS 3mM EDTA and 10mM DTNB TheNTSB solution was prepared from the stock solution (100mgof DTNB was dissolved in 10mL of 1M Na

2SO3 with the

pH adjusted to 75 the bright red solution was brought to38∘C and oxygen was bubbled through it) by diluting it1 100 with a freshly prepared solution containing 8M urea200mM Tris 100mM sodium sulfite 1 SDS and 3mMEDTA The mixtures of protein and DTNBNTSB solutionwere incubated in the dark for 30min The absorbance at412 nmwas then recorded against a blank of 3mL ofDTNBorNTSB solution and 200120583L ofwaterThe absorbance at 412 nmfor theDTNBmixturewas found to increase linearlywith freethiol contents and that for the NTSB mixture with total thiolcontentsThus the concentration of disulfide can be acquiredby using the following formula

Disulfide (mmolg) = 119860412NTSB times 119863 times 10minus6

119862 times 119881 times 120576

minus119860412

DTNB times 119863 times 10minus6

119862 times 119881 times 120576

(1)

where 119863 is the dilution multiple 119862 is the concentration ofTFIIIA 119881 is the volume of TFIIIA and 120576 is the extinctioncoefficient (13900Mminus1 cmminus1)

26 Quantification of Zinc (II) Ions in Irradiated TFIIIAA zinc (II) ion assay kit (Jiancheng Co Nanjing JiangsuChina) which is based on a colorimetric method wasused to determine the concentration of zinc (II) ions ingamma-irradiated TFIIIA The following procedures wereused Reagent 1 (ascorbic acid and sodium citrate dissolvedin HEPES buffer (02M pH 60) with final concentrationsof 005M ascorbic acid and 02M sodium citrate) was mixedwith irradiated TFIIIA (150 120583L) and the standard solution(150 120583L 306 120583M ZnSO

4) The solution was incubated for

5min at 37∘C The optical density of the solution was thenread as 1198601 at 578 nm using an ultraviolet-visible spectropho-tometer (Shimadzu Co Kyoto Japan) After the reading

1 2 3 4 5 6 7 8

+ + + + + + + ++ + + + + + +minus

minus minus 1 10 100 500 800 1000

LaneTFIIIA (25 120583g)

DTT (005mM)

Dose (Gy)

ProbeDNA

DNA

(a)

Dose (Gy)

IOD

0 10 400 800

0

2000

4000

6000

8000

10000

12000

(b)

Figure 1 Binding of irradiated transcription factor IIIA (TFIIIA)with the internal control region (ICR) (a) Band of binding ofgamma-irradiated TFIIIA (025120583g120583L 10120583L) with the ICR (the finalconcentration 25 ng120583L) Gamma irradiation was administered atdoses of 1 Gy 10Gy 100Gy 500Gy 800Gy and 1000Gy (b) Integraloptical density (IOD) of the binding

600120583L of 5-Br-PAPS was added to the solution The mixedsolution was incubated for 5min at 37∘C The optical densityof the solution was read as 1198602 The concentration of zinc (II)ions was calculated using the following formula

CZn2+ (120583molL) = Cstandard times1198602sample minus 1198601sample

1198602standard minus 1198601standard (2)

3 Results and Discussion

31 Binding of Gamma-Irradiated TFIIIA with the ICR Theeffect of gamma radiation on the binding of TFIIIA withthe ICR was studied using EMSA Different doses of gammarays ranging from 1Gy to 1000Gy were used to irradiatethe TFIIIA solution Figure 1(a) shows the EMSA band forbinding of irradiated TFIIIA with the ICR and Figure 1(b)shows the integral optical density (IOD) of each band Asshown in Figure 1(a) gamma doses equal to or below 800Gyexerted no significant effect on the binding of TFIIIA withthe ICR and TFIIIA irradiated with these doses could stillbindwith the ICR (lanes 3ndash7)The results are concordantwiththe corresponding IODs (Figure 1(b)) However 1000Gy ofgamma irradiation eliminated this binding capacity and atthis dose binding of TFIIIA with the ICR was significantlydecreased (lane 8) The corresponding IOD for 1000Gywas 34570 These results indicate that 1000Gy of gamma


radiation impairs the function of zinc finger motifs in TFIIIAand decreases specific binding of TFIIIA with the ICRBinding of TFIIIA with the ICR is the first step in expressionof 5S rRNA the decrease in the specific binding of TFIIIAwith the ICR may therefore affect the expression of 5S rRNA

32 Damage Sites in the Zinc Finger Motif of Gamma-Irradiated TFIIIA The redox potential of the sulfur atom inproteins ranges from minus027 to minus0125V [22] The low redoxpotential indicates that the thiol group in zinc finger motifsis readily oxidized Gamma radiation-induced impairment ofthe binding capacity of TFIIIAmight involve oxidation of thethiol groups in zinc finger motifs dl-dithiothreitol (DTT)an agent that reduces the reversibly oxidative thiol groupwas used to explore whether the thiol groups in the zincfinger motifs of irradiated TFIIIA were oxidized Figure 2(a)presents the EMSA bands for binding of irradiated TFIIIAwith the ICR after irradiated TFIIIA was incubated withdifferent concentrations of DTT and Figure 2(b) shows theIOD of each band The concentrations of DTT used were005mM 01mM 02mM 05mM and 10mM respectivelyThe radiation dose was 1000Gy As shown in Figure 2(a)DTT at 01mM resulted in no significant recovery in thebinding capacity of irradiated TFIIIA (lane 2) The corre-sponding IOD was only 39844 (Figure 2(b)) Application ofDTT at 02mMproduced a slight recovery (lane 3) Howeverthe recovery induced by DTT at 05mM was significant(lane 4) with IOD of 737814 Furthermore application ofDTT at 10mM produced a similar recovery to that inducedby 05mM of DTT (lane 5) with IOD of 796355 Theproducts of oxidation of the thiol group can be reversibleor irreversible The reversible end product is disulfide [23]and the irreversible products are sulfinic acid and sulfonicacid The recovery of zinc finger binding capacity observedfollowing incubation of irradiated TFIIIAwithDTT indicatesthat the water radiolysis products of gamma radiation oxidizethe thiol groups of zinc finger motifs to generate disulfideproducts

In order to further demonstrate the result obtained inFigure 2 the concentration of disulfide in irradiated TFIIIAwas detected with the use of the colorimetric method Thegamma doses used were from 1Gy to 1000Gy Figure 3 showsthe concentrations of disulfide in TFIIIA irradiated with dif-ferent gamma doses The concentration of disulfide increaseswith increasing dose and disulfide in 1000Gy irradiatedTFIIIA is significantly higher than that in the control groupThe result supports the indication that the water radiolysisproducts of 1000Gy of gamma radiation oxidize the thiolgroups of zinc finger motifs to form the disulfide productsThe water radiolysis products are hydroxyl free radicalhydrogen peroxide hydrated electron and hydrogen freeradical Their 119866 values are 27 055 27 and 07 respectivelyThe corresponding reactions of formation of disulfide are asfollows

NH2ndashCH(COOH)ndashCH2ndashSH +OH∙

997888rarr NH2ndashCH(COOH)ndashCH2ndashS∙ +H2O

1 2 3 4 5

+ + + + ++ + + ++

005 01 02 05 10


DTT (mM)Dose (Gy)

ProbeDNA

DNA

(a)

DTT (mM)00 02 04 06 08 10

0

2000

4000

6000

8000

IOD

(b)

Figure 2 Restoration of the binding capacity of irradiated transcrip-tion factor IIIA (TFIIIA) with the internal control region (ICR) byDL-dithiothreitol (DTT) solution (a) Band of binding of gamma-irradiated TFIIIA (1000Gy)with the ICR after irradiated TFIIIAwasincubated with different concentrations of DTT (005mM 01mM02mM 05mM and 10mM) (b) Integral optical density (IOD) ofthe corresponding binding

Dose (Gy)

Disu

lfide

(mm

olg

)

12

09

06

03

0 40 80 120 400 800

lowast

Figure 3 Concentration of disulfide in irradiated transcriptionfactor IIIA (TFIIIA) Gamma irradiation was administered atdoses of 1 Gy 10Gy 100Gy 500Gy 800Gy and 1000Gy Asterisksrepresent the significance level compared to the control group (0Gy)(lowast119875 lt 005)


NH2ndashCH(COOH)ndashCH2ndashSH + eminus

aq

997888rarr NH2ndashCH(COOH)ndashCH3∙ + SH∙

NH2ndashCH(COOH)ndashCH2ndashSH +H∙

997888rarr NH2ndashCH(COOH)ndashCH2ndashS∙ +H2


997888rarr NH2ndashCH(COOH)ndashCH3∙ +H2S

NH2ndashCH(COOH)ndashCH2ndashSH +NH2ndashCH(COOH)ndashCH3∙

997888rarr NH2ndashCH(COOH)ndashCH2ndashS∙ + CH3CH(NH2)COOH

2NH2ndashCH(COOH)ndashCH2ndashS∙

997888rarr NH2ndashCH(COOH)ndashCH2ndashSndashSndashCH2ndashCH(COOH)ndashNH2

2NH2ndashCH(COOH)ndashCH2ndashSH +H2O2


+ 2H2O

(3)

Several studies have demonstrated that formation of thedisulfide bond in zinc finger motif leads directly to release ofthe zinc (II) ion [13 24] This release promotes final collapseof the structure of the zinc finger motif which may be thereason for the decrease in the binding of irradiated TFIIIAwith ICR

Although binding of TFIIIA with the ICR was affected bythioldisulfide exchange the modulation was partial becauseDTT at 05 or 10mM did not fully recover the bindingcapacity of irradiated TFIIIA (Figure 2(a) lanes 4 and 5)Thecysteine residues and zinc ions are essential determinants ofthe structure of various types of zinc finger motifs such asCys8and Cys

4 which indicates that the thiol group and the

zinc binding site are very important for the structure andfunction of the zinc finger The finding that DTT partiallyrestored the binding capacity of irradiated TFIIIA suggeststhat the zinc site in addition to the thiol group might beinvolved in the damaging of zinc finger motifs Figure 4(a)presents the EMSA band results for binding of irradiatedTFIIIA with the ICR after irradiated TFIIIA was incubatedwith different concentrations of ZnCl

2 Figure 4(b) shows

the IOD of each band The concentrations of ZnCl2used

were 20120583M 50120583M and 100 120583M respectively As shownin Figure 4(a) ZnCl

2at 20120583M produced a slight recovery

of the binding capacity of irradiated TFIIIA (lane 2) Thecorresponding IOD was 92992 However ZnCl

2at 50120583M

resulted in a relatively significant recovery of the bindingcapacity (lane 3) and the IOD was 378467 Treatmentwith 100120583M ZnCl

2produced IOD of 333812 (lane 4 and

Figure 4(b))These results suggest that the zinc site is involvedin gamma radiation-induced damage in zinc finger motifs

The binding capacity of irradiated TFIIIA was fullyrestored after it was incubated with both 50120583M ZnCl

2and

05mM DTT (Figure 5(a)) The corresponding IOD was1015685 (Figure 5(b)) The full recovery produced by thecombination of these two agents further demonstrates that

ZnCl2 (120583M)

+ + + +

+ + + ++ + ++

minus 20 50 100

1 2 3 4LaneTFIIIA (25 120583g)

DTT (005mM)

ProbeDNA

DNA

Dose (1000Gy)

(a)

IOD

0 30 60 90 1200

1000

2000

3000

4000

ZnCl2 (120583M)

(b)

Figure 4 Restoration of the binding capacity of irradiated tran-scription factor IIIA (TFIIIA) with the internal control region (ICR)by ZnCl

2solution (a) Band of binding of gamma-irradiated TFIIIA

(1000Gy) with the ICR after irradiated TFIIIA was incubated withdifferent concentrations of ZnCl

2(20 120583M 50 120583M and 100120583M) (b)

Integral optical density (IOD) of the corresponding binding

damage to the zinc finger motifs in TFIIIA by 1000Gy ofgamma radiation involves both the thiol group and the zincsite

33 Damage to the Zinc Site of Zinc Finger Motifs in Gamma-Irradiated TFIIIA Although the thiol group reduced byDTTcould recapture the existing zinc (II) ion to reconstruct thefunction of the zinc finger a full recovery of function onlyoccurred after the reduced irradiated TFIIIA was incubatedwith a fresh ZnCl

2solution (Figure 5(a)) This result suggests

that some of the zinc (II) ions in irradiated TFIIIA are alteredAs zinc is a metal ion such an alteration would occur in theform of changed valence state To date no method has beendeveloped to directly detect changes in valence induced byionizing radiation in metal ions in solutionThe colorimetricmethod for determination of the concentration of the zinc(II) ion was therefore used to indirectly assess whether thevalence of the zinc ion changed in irradiated TFIIIA Thebuffer of the irradiated TFIIIA did not contain zinc ionsTable 1 shows the content of zinc (II) ions in TFIIIA irradiated


Table 1 Contents of the zinc (II) ion (119909 plusmn SD) in gamma-irradiatedTFIIIA (119899 = 4)

Dose (Gy) Zinc (II) ion (120583molL)0 1872 plusmn 028

1000 1215 plusmn 149lowast

lowastCompared to 0Gy group 119875 lt 005

+

05+

50

1LaneTFIIIA (25 120583g)

DTT (mM)

ProbeDNA

DNA

Dose (1000Gy)

ZnCl2 (120583M)

(a)

IOD

0

3000

6000

9000

12000

Z D Z and DIncubation factor

(b)

Figure 5 Restoration of the binding capacity of irradiated transcrip-tion factor IIIA (TFIIIA) with the internal control region (ICR) byDL-dithiothreitol (DTT) and ZnCl

2solutions (a) Band of binding

of gamma-irradiated TFIIIA (1000Gy) with the ICR after irradiatedTFIIIA was incubated with 05mM of DTT and 50 120583M of ZnCl

2

(b) Integral optical density (IOD) of the binding after irradiatedTFIIIA was incubated with ZnCl

2(50120583M) DTT (05mM) and

DTT (05mM) and ZnCl2(50 120583M) respectively Z 50 120583Mof ZnCl

2

D 05mM of DTT

with 1000Gy the concentration of zinc (II) ions was lower inirradiated TFIIIA than in nonirradiated TFIIIA The valenceof zinc atoms was 0 and II respectively

The decrease in the concentration of zinc (II) ionsindirectly indicates that zinc (II) ions underwent reductionand zinc atoms were generated in irradiated TFIIIA Gammaradiation-induced hydrated electrons are a strong reductionagent with a redox potential of 277V The zinc atoms in

irradiated TFIIIA were generated by hydrated electrons Thecorresponding reaction is as follows

Zn2+ + 2eminusaq 997888rarr Zn (4)

Organisms protect themselves against metal elements byusing various proteins to sequester the metal elementsGenerated zinc atoms function as free radicals and mightbe an important source of radiation damage Importantlythe formation of zinc atoms in irradiated TFIIIA suggeststhat valence electrons outside of the atomic nuclei of metalions are susceptible to ionizing radiation Valence electronsoutside of the atomic nuclei of metal ions may be a new radi-ation sensitive site Metalloproteins are involved in variousphysiological processes and their susceptibility to radiationeffects based on this sensitive site therefore is significant inradiation biology

4 Conclusion

TFIIIA is a transcription factor that specifically binds tothe 5S rRNA gene through formation of zinc finger-ICRcomplexes Gamma radiation (1000Gy) can impair bindingof zinc finger motifs with the ICR The sites of radiation-induced damage in the zinc finger motif are the thiol groupsof the cysteine residues and zinc (II) ions The thiol groupswere oxidized to form disulfide bonds and the zinc (II)ions may be reduced to zinc atoms In this experiment weobtained in vitro data on gamma radiation-induced damageto the zinc finger motifs of TFIIIA Further work shouldfocus on gamma radiation-induced impairment of the zincfinger motif in vivo The effects of radiation on proteinscontaining metal ions andor thiol groups are significant andmerit further investigation

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the National Key Basic ResearchProgram of China (973 Program) Grant 2013CB933904the National Nature Science Foundation of China Grants31400721 and 11575086 and the Priority Academic ProgramDevelopment of Jiangsu Higher Education Institutions

References

[1] A Witkiewicz-Kucharczyk andW Bal ldquoDamage of zinc fingersin DNA repair proteins a novel molecular mechanism incarcinogenesisrdquo Toxicology Letters vol 162 no 1 pp 29ndash422006

[2] J L Larabee J R Hocker and J S Hanas ldquoCys redox reactionsand metal binding of a Cys

2His2zinc fingerrdquo Archives of

Biochemistry and Biophysics vol 434 no 1 pp 139ndash149 2005[3] C-M Fan and TManiatis ldquoA DNA-binding protein containing

two widely separated zinc finger motifs that recognize the same


DNA sequencerdquoGenes andDevelopment vol 4 no 1 pp 29ndash421990

[4] R RWeichselbaumD EHallahan V Sukhatme ADritschiloM L Sherman and D W Kufe ldquoBiological consequences ofgene regulation after ionizing radiation exposurerdquo Journal of theNational Cancer Institute vol 83 no 7 pp 480ndash484 1991

[5] K-D Kroncke ldquoZinc finger proteins as molecular targets fornitric oxide-mediated gene regulationrdquo Antioxidants amp RedoxSignaling vol 3 no 4 pp 565ndash575 2001

[6] S-H Lee and W Maret ldquoRedox control of zinc finger proteinsmechanisms and role in gene regulationrdquo Antioxidants andRedox Signaling vol 3 no 4 pp 531ndash534 2001

[7] X L Yu Y Hathout C Fenselau et al ldquoSpecific disulfide forma-tion in the oxidation of HIV-1 zinc finger protein nucleocapsidp7rdquo Chemical Research in Toxicology vol 8 no 4 pp 586ndash5901995

[8] M A Baldwin and C C Benz ldquoRedox control of zinc fingerproteinsrdquoMethods in Enzymology vol 353 pp 54ndash69 2002

[9] K-D Kroncke ldquoCysteine-Zn2+ complexes unique molecularswitches for inducible nitric oxide synthase-derived NOrdquo TheFASEB Journal vol 15 no 13 pp 2503ndash2507 2001

[10] O Sidorkina M G Espey K M Miranda D A Wink and JLaval ldquoInhibition of poly(ADP-ribose) polymerase (PARP) bynitric oxide and reactive nitrogen oxide speciesrdquo Free RadicalBiology and Medicine vol 35 no 11 pp 1431ndash1438 2003

[11] X Wu N H Bishopric D J Bischer B J Murphy and KA Webster ldquoPhysical and functional sensitivity of zinc fingertranscription factors to redox changerdquo Molecular and CellularBiology vol 16 no 3 pp 1035ndash1046 1996

[12] W Maret ldquoZinc and sulfur a critical biological partnershiprdquoBiochemistry vol 43 no 12 pp 3301ndash3309 2004

[13] W Feng J Cai W M Pierce et al ldquoMetallothionein transferszinc to mitochondrial aconitase through a direct interaction inmouse heartsrdquo Biochemical and Biophysical Research Communi-cations vol 332 no 3 pp 853ndash858 2005

[14] M Huang D Krepkiy W Hu and D H Petering ldquoZn- Cd-and Pb-transcription factor IIIA properties DNA binding andcomparison with TFIIIA-finger 3 metal complexesrdquo Journal ofInorganic Biochemistry vol 98 no 5 pp 775ndash785 2004

[15] K L Brady S N Ponnampalam M J Bumbulis and DR Setzer ldquoMutations in TFIIIA that increase stability of theTFIIIA-5 S rRNA gene complexrdquo The Journal of BiologicalChemistry vol 280 no 29 pp 26743ndash26750 2005

[16] Z Yang and J J Hayes ldquoXenopus transcription factor IIIA andthe 5S nucleosome development of a useful in vitro systemrdquoBiochemistry and Cell Biology vol 81 no 3 pp 177ndash184 2003

[17] D J McColl C D Honchell and A D Frankel ldquoStructure-based design of an RNA-binding zinc fingerrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 17 pp 9521ndash9526 1999

[18] J M Berg and H A Godwin ldquoLessons from zinc-binding pep-tidesrdquo Annual Review of Biophysics and Biomolecular Structurevol 26 pp 357ndash371 1997

[19] A T Maynard and D G Covell ldquoReactivity of zinc finger coresanalysis of protein packing and electrostatic screeningrdquo Journalof the American Chemical Society vol 123 no 6 pp 1047ndash10582001

[20] P JThornalley andM Vasak ldquoPossible role formetallothioneinin protection against radiation-induced oxidative stress Kinet-ics andmechanism of its reaction with superoxide and hydroxylradicalsrdquo Biochimica et Biophysica Acta (BBA)mdashProtein Struc-ture and Molecular vol 827 no 1 pp 36ndash44 1985

[21] Y H Kwon and M J Smerdon ldquoBinding of zinc finger proteintranscription factor IIIA to its cognate DNA sequence withsingle uv photoproducts at specific sites and its effect on DNArepairrdquoThe Journal of Biological Chemistry vol 278 no 46 pp45451ndash45459 2003

[22] K G Reddie andK S Carroll ldquoExpanding the functional diver-sity of proteins through cysteine oxidationrdquo Current Opinion inChemical Biology vol 12 no 6 pp 746ndash754 2008

[23] H F Gilbert ldquoThioldisulfide exchange equilibria and disulfidebond stabilityrdquoMethods in Enzymology vol 251 pp 8ndash28 1995

[24] H Blessing S Kraus P Heindl W Bal and A HartwigldquoInteraction of selenium compounds with zinc finger proteinsinvolved in DNA repairrdquo European Journal of Biochemistry vol271 no 15 pp 3190ndash3199 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

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International Journal ofPhotoenergy


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International Journal of


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Chemistry


Advances in

Physical Chemistry

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Analytical Methods in Chemistry

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Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


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Applied ChemistryJournal of



Theoretical ChemistryJournal of


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CatalystsJournal of


A zinc atom is a relatively redox-inert element with anoxidation state of zinc (II) In animal cells zinc (II) ions arestored in cytosolic cysteine-rich proteins ormetallothioneinsin the form of zinc-sulfur clustersThe level of ldquofreerdquo intracel-lular zinc (II) ions is very low that is in the nanopicomolarrange and spontaneous release of zinc (II) ions from themetallothioneins system to target proteins does not occurs[12] In addition to damaging zinc finger motifs oxidantsmay be involved in zinc metabolism Current investigationssuggest that the effects of oxidants on zinc finger motifs arelinked to the release of zinc (II) ions [13] According to thishypothesis zinc fingers inmetalloproteinsmay serve as a zincpool Ionizing radiation differs from oxidative agents that arecommonly used for oxidation of zinc finger motifs becausethey induce formation of not only the oxidative radical butalso the reduced radical that is the hydrated electron Thepresence of the hydrated electron may reduce zinc II ions

Transcription factor IIIA (TFIIIA)was the first zinc fingerprotein to be identified in Xenopus and it consists of ninetandem repeat Cys

2His2-type zinc fingers [14 15] The three-

dimensional conformation of this type of zinc finger includesan 120572-helix and two 120573-sheets The internal control region(ICR) of the 5S rRNA gene is a 120-base sequence includingthree separate promoter regions of an A-box (+50 to +64)an intermediate element (+67 to +72) and a C-box (+82to +92) [16] The nine zinc fingers of TFIIIA specificallybind with the ICR through insertion of their 120572-helixesinto the major or minor grooves of these three promoterregions [17] TFIIIA was chosen to investigate damage tothe zinc finger motif because Cys

2His2-type zinc fingers are

relatively electrically neutral and possess the low conditionalassociation constant among that of all known zinc fingerproteins [18 19] In this experiment we explored the effectof ionizing radiation on the zinc finger motif of TFIIIA andthe corresponding mechanism of damage Specifically weinvestigated the binding capacity of the zinc finger in gamma-irradiated TFIIIA with the ICR and identified potential sitesof irradiation-induced damage To the best of our knowledgeonly one article has been published on the effects of ionizingradiation on a protein containing zinc-sulfur clusters that ismetallothionein with the aimof analyzing its radioprotectionfunction [20] Our results indicate that gamma radiationaffects specific binding of TFIIIA with the ICR throughthioldisulfide and zinc (II) ionzinc atom exchanges in thezinc finger motifs

2 Materials and Methods

21 Preparation of Plasmid pMD18T-ICR The full ICRsequence was synthesized on an automated DNA synthesizer(ABI 394 Applied Biosystems Inc Carlsbad CA USA)using phosphoramidite chemistry The ICR sequence wasthen ligated to a pMD18T vector (TaKaRa Co Dalian Liaon-ing China) which was transformed into Escherichia colicompetent cells (TaKaRa) The targeted DNA was amplifiedby polymerase chain reaction (PCR) analyzed by agaroseelectrophoresis and confirmed by DNA sequencing

22 Preparation of Recombinant TFIIIA Recombinant Xeno-pus laevis TFIIIA was produced by Escherichia coli BL21(DE3) cells harboring the plasmid pcoldII-TFIIIA [21]Briefly cells were cultured at 37∘C with shaking at 220 rpmand when the cell density (OD600) reached 06 TFIIIAexpression was induced by addition of 01mM isopropylthio-120573-d-galactoside (IPTG) and 10120583MZnCl

2 After 4 h of further

incubation at 37∘C cells were harvested and lysed with anEmulsiFlex-C3 homogenizer (Avestin Ottawa ON Canada)After cell lysis the inclusion bodies which contain most ofthe TFIIIA were first washed with buffer 1 (50mM Tris-Cl100mM NaCl 1mM EDTA and 01 Triton X-100 pH 80)before being dissolved in buffer 2 (50mM Tris-Cl 100mMNaCl 6M guanidine hydrochloride and 5mM DTT pH80) The whole dissolution process was performed at 4∘Cand 24 h was required to completely dissolve the TFIIIAThe supernatant was then slowly diluted in buffer 3 (50mMTris-Cl 50mM NaCl 100 120583M ZnCl

2 01M Arg 1mM GSH

01mM GSSG 10 glycerol and 01 Nonidet P-40 pH80) at a speed of one drop per second and the sample wasslowly stirred at 4∘C for 24 h to refold the TFIIIA After therefolding process was complete the sample was concentratedusing an ultrafiltration tube (Amicon Ultra Millipore CoBillerica MA USA) before being purified by fast proteinliquid chromatography using a Superdex 75 column (GE CoFairfield CT USA)

To detect zinc (II) ions in irradiated TFIIIA recombinantTFIIIA was also prepared in buffer without zinc (II) ionsupplementation Similar to the procedure described abovethe TFIIIA contained in inclusion bodies was refolded usingbuffers 1 2 and 3 The refolded TFIIIA was then precipitatedwith 40 ammonium sulfate at 4∘C for 30min The pelletwas again denatured with buffer 2 and refolded in buffer 3lacking the 100 120583MZnCl

2The rest of the purification process

was performed as described aboveThe protein concentrationwas measured using the Bradford method and the purifiedTFIIIA solution (025120583g120583L) was stored at minus80∘C until use

23 Radiation Exposure The TFIIIA solution was irradiatedwith 60Co sources in an atmosphere of air at 4∘C Theabsorbed doses were 0Gy 1 Gy 10Gy 100Gy 500Gy 800Gyand 1000Gy The low doses (1 Gy and 10Gy) were admin-istered with 60Co source (Gaotong Isotope Co ChengduSichuan China) with a radioactivity of approximately 222times 1012 Bq The dose rate was 020Gymin The high doses(100Gy 500Gy 800Gy and 1000Gy)were administeredwith60Co source (Nordion Inc Kanata ON Canada) with aradioactivity of approximately 148 times 1016 Bq The dose ratewas 800Gyh Dosimetry was performed on a regular basiswith a 06 cm3 Farmer Ionization Chamber (Type 30010)which was connected to a dosimeter (PTW-Freiburg CoFreiburg Breisgau Germany) The chamber was placed nextto the tubes for irradiation

24 Electrophoretic Mobility Shift Assay For preparation offluorescent FAM-labeled probes the ICRof the 5S rRNAgenewas amplified by PCR using a high-fidelity Dpx DNA Poly-merase (Tolo Biotech Shanghai China) from the plasmid



2 005mM DTT and



2SO3 with the



119862 times 119881 times 120576

minus119860412


119862 times 119881 times 120576

(1)





1 2 3 4 5 6 7 8

+ + + + + + + ++ + + + + + +minus

minus minus 1 10 100 500 800 1000


DTT (005mM)

Dose (Gy)

ProbeDNA

DNA

(a)

Dose (Gy)

IOD

0 10 400 800

0

2000

4000

6000

8000

10000

12000

(b)













1 2 3 4 5

+ + + + ++ + + ++

005 01 02 05 10


DTT (mM)Dose (Gy)

ProbeDNA

DNA

(a)

DTT (mM)00 02 04 06 08 10

0

2000

4000

6000

8000

IOD

(b)


Dose (Gy)

Disu

lfide

(mm

olg

)

12

09

06

03

0 40 80 120 400 800

lowast




aq












+ 2H2O

(3)





2 Figure 4(b) shows





2at 50120583M





2and


ZnCl2 (120583M)

+ + + +

+ + + ++ + ++

minus 20 50 100

1 2 3 4LaneTFIIIA (25 120583g)

DTT (005mM)

ProbeDNA

DNA

Dose (1000Gy)

(a)

IOD

0 30 60 90 1200

1000

2000

3000

4000

ZnCl2 (120583M)

(b)




2(20 120583M 50 120583M and 100120583M) (b)











+

05+

50


DTT (mM)

ProbeDNA

DNA

Dose (1000Gy)

ZnCl2 (120583M)

(a)

IOD

0

3000

6000

9000

12000


(b)




2


2(50120583M) DTT (05mM) and


2

D 05mM of DTT






4 Conclusion


Competing Interests


Acknowledgments


References






































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



2 005mM DTT and



2SO3 with the



119862 times 119881 times 120576

minus119860412


119862 times 119881 times 120576

(1)





1 2 3 4 5 6 7 8

+ + + + + + + ++ + + + + + +minus

minus minus 1 10 100 500 800 1000


DTT (005mM)

Dose (Gy)

ProbeDNA

DNA

(a)

Dose (Gy)

IOD

0 10 400 800

0

2000

4000

6000

8000

10000

12000

(b)













1 2 3 4 5

+ + + + ++ + + ++

005 01 02 05 10


DTT (mM)Dose (Gy)

ProbeDNA

DNA

(a)

DTT (mM)00 02 04 06 08 10

0

2000

4000

6000

8000

IOD

(b)


Dose (Gy)

Disu

lfide

(mm

olg

)

12

09

06

03

0 40 80 120 400 800

lowast




aq












+ 2H2O

(3)





2 Figure 4(b) shows





2at 50120583M





2and


ZnCl2 (120583M)

+ + + +

+ + + ++ + ++

minus 20 50 100

1 2 3 4LaneTFIIIA (25 120583g)

DTT (005mM)

ProbeDNA

DNA

Dose (1000Gy)

(a)

IOD

0 30 60 90 1200

1000

2000

3000

4000

ZnCl2 (120583M)

(b)




2(20 120583M 50 120583M and 100120583M) (b)











+

05+

50


DTT (mM)

ProbeDNA

DNA

Dose (1000Gy)

ZnCl2 (120583M)

(a)

IOD

0

3000

6000

9000

12000


(b)




2


2(50120583M) DTT (05mM) and


2

D 05mM of DTT






4 Conclusion


Competing Interests


Acknowledgments


References






































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of







1 2 3 4 5

+ + + + ++ + + ++

005 01 02 05 10


DTT (mM)Dose (Gy)

ProbeDNA

DNA

(a)

DTT (mM)00 02 04 06 08 10

0

2000

4000

6000

8000

IOD

(b)


Dose (Gy)

Disu

lfide

(mm

olg

)

12

09

06

03

0 40 80 120 400 800

lowast




aq












+ 2H2O

(3)





2 Figure 4(b) shows





2at 50120583M





2and


ZnCl2 (120583M)

+ + + +

+ + + ++ + ++

minus 20 50 100

1 2 3 4LaneTFIIIA (25 120583g)

DTT (005mM)

ProbeDNA

DNA

Dose (1000Gy)

(a)

IOD

0 30 60 90 1200

1000

2000

3000

4000

ZnCl2 (120583M)

(b)




2(20 120583M 50 120583M and 100120583M) (b)











+

05+

50


DTT (mM)

ProbeDNA

DNA

Dose (1000Gy)

ZnCl2 (120583M)

(a)

IOD

0

3000

6000

9000

12000


(b)




2


2(50120583M) DTT (05mM) and


2

D 05mM of DTT






4 Conclusion


Competing Interests


Acknowledgments


References






































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



aq












+ 2H2O

(3)





2 Figure 4(b) shows





2at 50120583M





2and


ZnCl2 (120583M)

+ + + +

+ + + ++ + ++

minus 20 50 100

1 2 3 4LaneTFIIIA (25 120583g)

DTT (005mM)

ProbeDNA

DNA

Dose (1000Gy)

(a)

IOD

0 30 60 90 1200

1000

2000

3000

4000

ZnCl2 (120583M)

(b)




2(20 120583M 50 120583M and 100120583M) (b)











+

05+

50


DTT (mM)

ProbeDNA

DNA

Dose (1000Gy)

ZnCl2 (120583M)

(a)

IOD

0

3000

6000

9000

12000


(b)




2


2(50120583M) DTT (05mM) and


2

D 05mM of DTT






4 Conclusion


Competing Interests


Acknowledgments


References






































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






+

05+

50


DTT (mM)

ProbeDNA

DNA

Dose (1000Gy)

ZnCl2 (120583M)

(a)

IOD

0

3000

6000

9000

12000


(b)




2


2(50120583M) DTT (05mM) and


2

D 05mM of DTT






4 Conclusion


Competing Interests


Acknowledgments


References






































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

Documents

Research Article Gamma Radiation-Induced Damage in the Zinc …downloads.hindawi.com/journals/bca/2016/1642064.pdf · oxidation state of zinc (II). In animal cells, zinc (II) ions