Indian Journal of Biochemi stry & Biophysics Vo l. 39, October 2002, pp. 31 2-3 17

Contrasting effects of mutating active site residues, Aspartic acid 64 and Histidine 187 of Escherichia coli uracil-DNA glycosylase on uracil excision and

interaction with an inhibitor protein

Priya Handa, Narottam Acharya, Ramappa K Talawar, Sudipta Roy and Umesh Varshney;'

Department of Mi crobiology and Ce ll Biology, Indian In stitute of Science, Bangal ore 560 01 2, India

Received 29 May 2002 ; revised 22 Jllly 2002

Uracil , a promu tageni c base, ari ses in DNA by spont aneous deaminati on of cytosine or by the malfunctioning of DNA polymerases. To maintain the genomi c integrity, ce ll s possess a hi ghl y conserved base excision repair enzy me. urac il -DNA glycosy lase (U DG). UDGs have a notab ly hi gh turnover num be r and strict spec ificity for uracil in DNA . UOGs are inhi bi ted by a small protei naceous inhibitor. Ug i, whi ch ac ts as a transiti on state substrate mimic. Crystal structure studies have ick n­tified the residues crucial in catal ys is, and in their interac tion with Ugi. Here, we report on the mutational analyses o f 064 (0 64H and D64 N) and H 187 (H 187C, H 187L and H 187R) in the ac tive site pocket of Escherichia coli UDG. The mutant s were compromi sed in uracil exc ision by - 200-25,000 fold when compared to the nati ve protein . In cont rast. our Jna lys is of the ill vivo formed UDG-Ug i co mplexes on urea ge ls shows th at D64 and H 187 contri bute minimall y to the interac ti on of the two protein s. Thus, our findin gs prov ide further ev idence to the primary fun cti on of D64 Jnd H 187 in cJtalysis.

Uracil , a natural component of RNA, ari ses in DNA either due to deamination of cytosine or by occasional incorporation of dU MP by DNA polymerases. Occur­rence of uracil in DNA is promutagenic and if left unrepaired, leads to GC~A T mutations. However, to safeguard the informational content of the genome, cell s have evolved with uracil-DNA glycosylase (U DG)1.2 UDGs have been identified from a vari ety of sources of prokaryotic and eukaryoti c origin and these enzymes show a hi gh degree of conservation2

.

The UDGs al so interact with a number of proteins such as the Bacillus sub/ilis phage encoded !!racil DNA glycosylase inhibitor, Ugi, and the cellular fac­tors such as single stranded DNA binding protein '.4 . Ugi (9.4 kDa), is an early gene product of the Bacillus sub/ilis bacteri ophage (PBS-I/-2), which forms a hi ghl y specific and ex tremely stable complex with the conserved class of UDGs in I: I stoi chi ometr/ . The complex of Escherichia coli UDG (EcoUDG) and Ugi is irreversibl e under phys iological conditions. How­ever, it can be di ssociated by treatment with SDS, or 8 M urea at 70 °C. Thus, UDGs constitute a remarka­bl y interesting model system to understand the basis

"ALIthor for correspondence Pho ne: 9 1-80-394 2686 Fax: L) 1-80-360 2697; 9 1-80-360 0683 E-mai l: varshney @mcb l. iisc .ernet.in

~

of catalytic prowess and specificity associ ated with protein-DNA and protein-protein interactions.

The crystal structures of several UDGs or their complexes with DN A or the Ugi, have suggested a prime role for the hi gh ly conserved D64 and H 187 in catalyzing (numbering according to E. coli UDG) the glycosidic bond cleavage (see Discussion )s- II . Inter­estingly , these res idues are differentl y oriented in Eco UDG and warrant independent mutational analy­SCS

IO.II . The UDG-Ugi crystal structures have shown that the hi ghl y conserved sequence mot ifs in UDGs, such as the water activating loop, 62-GQDPYH-67 ; the Pro-Ser loop, 84-AIPPS-88; and the DNA inter­calation loop, 187-HPSPLS-I92 which li e at the inter­face with Ugi contribute to the stability of the com­plex through the interactions at the hydrophobic cav­ity (between the a2 helix and the anti parallel ~- s hee t )

or the ~ I-edge res idues (Q 19, E20, S2 1, L23 and E28) of Ugi. The key UDG res idues such as Q63, D64, Y66, H67, and H 187 in the acti ve site pocket of UDG make direct or water mediated H-bonds with the Ugi ~1-edgelo. ll .

Mutati onal analysis of 064 (D64N) and H 187 (0 , G) in Eco UDG as well as human and viral counter­parts has earlier shown that these mutant s are hi ghl y

. d· ·1 . . P 11 T ' I compromise In uraCi exclston -. ' . 0 tun ler assess the significance of these residues in catalys is, in thi s study , we have included a di fferent set of mutations at

..

HAN DA el al. : MUTATIONAL ANALYSIS OF D64 AN D H 187 IN E. COLI UDG 3 13

D64 (H, N) and H 187 (c, L, R). In additi on, we ex ­ploited these mutants to gain insights into the ir inter­action with Ugi, a trans iti on state substrate mimic.

Materials and Methods

O/igodeoxyribol1l1c1eotides and radio isotopes Oligodeoxyribonucleotides were obtained from

Ranso m Hill Biosc ience, USA . [y 2p] ATP was ob­tained from DuPont NEN, USA .

Generation 0/ 0 64 IIllItonlS The UDG gene fragment encompassing D64 was

released from pTZUng4S '.J as an Eco RI-Hindlll fragment and subcloned into pTZI 8R between the same sites (pTZU ng4S '). The sing le stranded DNA

from pTZUng4S ' and the o li gomer, 5'­ctcggccag(alc)a tccttatcac-3' , were used to generate D64 N and D64 H mutants by a modified Kunkel's mutagenes is protocol 15

.16 and confirmed by DNA se­

quencing ' 7 The mutati on containing reg ions from pTZU ng4S ' constructs (50 ng) were PCR ampl ifi ed

by a gene speci fi c (5' -cggaattcco/ggctaacgaattaacc-3 ') and a vector spec ific (5' -gttttcccagtgacgac-3' ) primers (20 pmol each) ll sing Pji{ DNA polymerase. The PC R

was carri ed out for 25 cycles of incubati ons at 95°C for 45 s, 50°C for 30 s and n oc fo r I min after initi al

denaturation at 95°C fo r I min . The final ex tension was allowed at n oc for 10 min . The resulting PC R products were di gested with Ncol and Nrul and ll sed to replace the Ncol-N m I (-450 bp) fragment fro m

pTrcEcoU DG (Fig. I). The mutati ons in pTrcE­coUDG were confirmed by DNA sequencing.

Generation of the H1 87 Eco UOG lIlulants The single stranded DNA template deri ved fro m

pTZU ng2B,I6 and the mutageni c 01 igomers 5' ­

cgacggaagcggtgctttc-3' ; 5'-cggcgacggacgcggtgctttc-3' ;

and 5' -gcgacggacacggtgc tttc-3 ' were used to obtain H 187L, H 187R and H1 87C mutants, respecti vely' 6 and confirmed by complete nucleotide sequencing. The Nml-HiHdiII fragments (- 550 bp) were exc ised from the pTZ constructs and subcloned into simil arl y di gested pTrcEcoUDG to generate express ion con­structs.

Purification and estimation of the 064 and H 187 lIIutal/ts

The wild type and the various UDG mutants (D64N , D64H , H 187L, H 187C and H 187R) were pu­rifi ed to apparent homogeneity by chromatograph y on Superdex G-200, MonoS and heparin Sepharose col­umns I6

.I S

. The purity o f proteins was checked by el ectrophores is on SDS (0.1 %) po lyacrylamide ( 15%) gel17 and estimated by Bradford 's reagent using BS A as standard ' 9 .

Assay of IIracil excision acti vity

The DN A 01 igomer, SS U4 (5' -agcucatagt tt ac­c tgaagaatat-3' ) was 5' e2P]-end labelled and puri fied on Sephadex G 50 mini columns2o

. Reacti on mi xtures

(70 Ill ) containing 35 pmol of the o li gomer in UDG

Nrul Sphl

~p~~~re:-N--lr=O=' ==D=6= ••• ~=a~:~HU[]I~~~G8=7=\~(=ZJ_==-r---------~i~~111 (A)

(8)

(e)

Nrul Sphl Neal 8amHI S (

~pt.t-:;::e--tl ===D=6= •• 4=i!1un;DgG['=' ~~=EJX:!mn1'Ugij>l.fit~ 1 Nrul Sphl

8amHI \ ( I H187 EeoRI

..•..•..•... UDG ' ~ ..... r-Y)1h;n{i;:~tU1(t~l:q;iifi;1

.-----Pstl .-----Hindlll

.-----Pstl .-----Hind II I

Fi g. I- Diagralll lllnt ic sketches o f vnri ous constructs used for ill vilro production of UDG. (A): Expression constructs for wi Id type and the UDG Illutants. (B): Bicistl"O ni c constructs used for simultaneous ex press ion of UDG mutants (D64) and Ugi. (C): Constructs used for simu ltaneous expression of UDG mutants (1-11 87) and Ugi.

3 14 INDI AN 1. BIOCHEM. BIOPHYS .. VOL 39. OCTOBER 2002

buffer (50 mM Tri s-HCI, pH 7.4, I mM Na2EOTA, I mM OTT and 25 ~lg/ml BSA) were suppl emented wi th 5 pi of appropri ate dilution of UOG (25 pg of wild type enzy me, 25 ng of H 187L, 250 ng of H 187 R, 25 ng of H 187C, 250 ng of 064H and 2.5 ng of 064N) and incubated at 37"C for 10 min . The percent urac il excision activity of the mutants was calculated with respect to wild type UOG I6.

ilicistrollic COll structs fo r ave /p rodll ctioll of UDC­Ug i cO/llp lexes

The bici stroni c constructs of Ug i wi th the 064 mutant U OGs in pTrc99C ex press ion vector, were

d I 'b d l6 IS TI b" . ge nerate as c cscn e ' . le ICl stronlc co nstructs of the UOG mutants (H 187) were also the same ex­cept that pET II d expression vector was used (Fig. 1). O I · f hi ' I 161 8 ve rproc uctlon 0 t e comp exes was cam ec out . in E. coli TG I and E. coli BL21 (OE3) for the 064 and H 187 UOG mutants, res pectively.

!\ lIo lvsis a/ill vivo /anlled UDC-Ug i cO/llplexes The cell free extracts of the reco mbinants harbor­

ing bici stroni c constructs of Ugi with 064H , 064N , H 187 L, H 187R and H 187C mutants, were made afte r inducti on of mid log phase cultures with 0.5 mM IPTG and analysed on 15% pol yacrylamide ( 19: 1 cross linking) gels without or with different concen­trati ons of urea l6 and stained with CoomJss ie brilliant b l ue~l .

Resu lts

Gelleratioll (~f expressioll COllstruCts for UDG, alld UDG-Ugi

Fig. I shows diagrammatic sketches of the various ex press ion constructs used in overproduction of the wild type and the 064H, 064N, H 187C, H 187L and HI 87 R mutants of EcoU OG . The bicistronic con­structs used in co-express ion of various UOGs and Ugi, for the format ion of the UOG-Ugi complexes in ce llular milieu are also shown.

Overexpressioll alld pllr~ficatio1/ of UDG

The pTrc99C based express ion constructs (Fi g. I) were trans formed into an illig-strain of E. coli BW3 10, and the UOG purified as described in Mate­rial s and Methods. Analysis of the pooled fractions from the heparin Sepharose column, on SOS-PAGE (Fig. 2) showed that the proteins were puri ficd to ap­paren t homogenei ty.

Uracil excision by HIB7 and D64 mlltallts Urac il excision activ iti es of the various mutants

were determined using an o li godeoxy ri bonucleotide, SSU4 containing uracil at the fourth posi ti on from the Sf-end, as substrate. As shown in Table I, relati ve to the wi ld type protein , all the mutants at the 064 and H 187 positions were compromised -200 to 25 ,000 fold in their uraci l exc ision acti vity. Among the mu­tants, the 064N with an activity of -·0.5% was the most act ive whereas the H 187 R with an acti vity of -0.004% was the least active.

Allalysis of the stability of the ill vivo formed UDG­Ugi complexes

We have earli er described an assa/(', wherein the cell free extracts of the transforman ts containing ill

H187 064 M L R C H N

.-,

~--., ~- UOG

..- (25 6 kDa)

2 4

Fig. 2- A nal ysis of the purifi ed UDC preparations IThe protei ns (I 11 87L. H1 87R. H1 87C, D64 H and D64N. lall":s 2-6, r..:spcc­ti ve ly) from the heparin Sepharose col umn were resolved on 15rk SDS- PACE, and detected by Commassie hrill ian t blue staining. As determined from the estimati ons, the protei n contents vari ed about 2 Jl g to 4 ~t g. M olecular weight markers (Ian..: I ) correspond to bovine serum albumin (66 kDa). ova lbumi n (-+) kDa) and ca r­bonic anhydrase (29 kDa) I

Tab le I - The relat i ve acti vity of uraei I exc ision by the w i Id type. D64 and HI 87 UDCs

UDC pmols of uraci I released per ~t g R..: lati ve acti vity o f UDC per min (x 10~ ) ( '70 )

WT 530 100

DMH OA 0.0075

D64N 25.7 0.-1850

1-1 187C XA 0. 1584

H I X7 L 4 O.D7.'),)

HI 8R 0.2 0.0037

HANDA e{(d.: M UT ATION A L ANALYSIS OF D64 AND HI 87 1N E. COLI UDG 3 15

1' I VO fo rmed co mplexes of the two pro teins are elec­trophoresed on polyacrylam ide gels contain ing diffe r­ent concentrati ons of urea (0 to 8 M) to determine the stabil it y of the UDG-Ugi complexes. In such gels, the UDG-Ugi comp lex (35 kDa) with a pI of 4.9 mi grates in the middle of the gel as a di stinct band .

Ana lys is of the cell free ex tracts containing ill vivo fo rmed complexes of Ug i with the UDG mutants is shown in Fig. 3. Ex pectedly, the co mplexes of Ugi with the D64H and D64N (Fig. 3 A) and H 187 L, H 187C, and H 187 R (Fig. 3 B) mutants co-migrate with the co mp lex of wil d type proteins (co mpare lanes 2 and 3 with I, Fig. 3 A: or lanes 3 to 5 with 2, Fig. 3 B). Interes tingly, the com plexes of Ug i with the mutant UDGs were observed to be co mpletely stable in 6 M urea (co mpare panels i and ii , Figs . 3 A and 3 B). The complexes of Ug i with the wil d type, D64H (lanes I and 2, Fig. 3 A, panel iii ) or the H 187C and H 187 R (l anes 4 and 5, Fi g. 3 B, panel iii ) UDGs were stabl e to even 8 M urea. However, as seen fro m the dimini shing intensity of the UDG-Ugi band , some dissociation of the co mplexes of D64N and H 187 L UDGs occurred at 8 M urea. Dissoc iati on of these

(A) . 064 __ VlfT H N Uc i WT

I m. ...... ,.t<. '" -~

Complex~ _ - =. Complex '" •

Ugi" Ugi ...

2 3 4

(i)O M lJrea

complexes in 8 M urea was also supported by the presence of a smear below the UDG-Ugi complexes. The smear corresponds to Ugi that results from disso­ciati on of the comp lex. Because of its ac idic pI (4.2) and low molecul ar weight (9.4 kDa), Ug i migrates fas ter than the complex (l ane 6, Fig. 3 B). While the pure U gi mi grates as a sharp band, its presence in the form of a smear as well as its retarded mobili ty is in­di cati ve of the fact that the co mplexes dissociated whi le they electrophoresed th rough the ge l. Further, as described earli er i li

, the presence of a sharp band corresponding to free Ug i in lanes 2 and 3 of the na­ti ve as well as 6 M and 8 M urea gels (Fig. 3 A) is due to the ex pression of Ug i in molar excess to the UDG from the pTrc99C based bicistroni c ex pression con­structs. I t may also be menti oned that a band or smear representi ng UDG whi ch woul d have ari sen from the dissoc iat ion of the co mplexes in these lanes is not ex pected to be detec tabl e. Because of it. near neut ral pI (6.6) it mi grates near the well (lane I, Fig. 3 B) where a large number of cellul ar pro teins also frac ­tionate. The analys is of the purifi ed complex of the wi Id type protei ns (Fig. 3 A, lane I) prov ided a

H N Ugi WT H N Ugi

" " .. • Complex " ,. • • Ugi" l " - .... .... ..,

2 3 4 2 3 4

(i i) 6 M urea (i ii) 8 M urea

(B) H1 87 WT L C R UCji WT L C R Ugi WT L C R Ug!

UDG ~ - '·I'~ -!!I! UDG ~ -t'" ~ . Complex ~ ;;, ~-~ UDG~

Complex ..., Complex ...,

U9i .... \ • Ugi,.. Ugi .... '...., 2 3 4 5 6 2 3 4 5 6 2 3 4 5 6

(i}O M urea (ii }6 M urea (iii) 8 M urea

Fig. 3- Analys is or the ill vivo formed cOlllp lexes of Ugi with UDG [The ce ll free ex tracts (- I 0 ~t g of total prote ins) from the transror­man ts harboring the bici stron ic const ruct for Ugi and the var ious UDG mu tants were analyzed on 15% polyacry lamide gels con taining no urea (pu lle! i) . 6 M (pulle! ii) or 8 M (]Ju li e! iii) urea.J (A): Ana lys is of the comp lexes of Ugi wi th D64 Illutants. Lanes: I , EcoU DG-Ugi complex marker; 2 and 3. ce ll rree ex trac ts harboring bicistronic constructs or Ugi with D64H and D64N UDGs, respectively: 4, Ugi marker. ( 13 ): Analysis of the complexes of Ugi with H 187 Illutants. Lanes: I . UDG marker. lanes 2 to 5. ce ll free ex tract s harboring bicis­tronic l'OnSlrllcts of Ugi with wild type, 1-1 187 L , H 187e and 1-11 R7R UDGs. respecti vely ; 6, Ugi marker.

316 INDIAN J. BIOC HEM . BIOPH YS., VOL. 39, OCTOBER 2002

marker for the UDG-Ugi complex. On the other hand, the analys is of total cell ex tract containing the com­plex of the wild type proteins (Fig. 3 B, lane 2) served as a control to rule out di ssociati on of the Ugi com­pl ex with H 187L in 8 M urea due to some unknown fac tors in the cell extracts. [t may also be noted that with, the increasing urea concentration in the gels, the relati ve mobility of the UDG- Ugi and Ugi bands changes . Thi s is because in the nati ve gels the Ugi is in its native confo rmati on, whi ch mi grates faster on the gel, whereas in the presence of urea the Ugi un­fo lds and mi grates slowerl6. On the other hand, the UDG-Ugi complex is recalcitrant to urea, and retains . . . 16 I ts terti ary structure .

Discussion

Crystal structures of several UDGs and their co mpl exes with the substrate DNA or the substrate mimic, Ugi have prov ided maj or clues to the mechan isms that regul ate ex treme substrate spec ificity and catal ys is by the conserved class of UDG 0· 11 "7, d ' I d h ' s .--.-. . Accor mg to t le propose mec al1l sms a water molecule is acti vated by 0 64 to launch an attack on 10 C I ' of the sugar leading to formation of oxocarbenium ca ti on and urac il ate ani on (assoc iati ve

d . 1' 1,7, S mo e 01 bond cleavage) -. "-.. ubsequentl y, the departure of urac il ate is fac ilitated by a hydrogen bond donated to 0 2 by Hi s 187. More recentl y, a dissoc iati ve mode of catalys is has been proposed wherein the glycos idi c bond cleavage occurs by an autocatalytic step facilitated by the interacti ons bet ween the Ser-Pro loops of the enzy me with the backbone phosphates in the substrate, and the attack by the water molecule occurs subsequent to the bond cleavage24

. However, in both the proposed mechani sms, the 0 64 and H 187 pl ay a crucial rol e in catalyzing the cleavage of the N-glycos idi c bond. Consistent with proposed mechani sm and earli er mutational anal ys is, our results suggested that 0 64 and H 187 are essential fo r efficient catalys is.

Recentl y, the mi smatch specifi c urac il DNA glyco­sy lase (MUG) which bears stri king architectural ho­mology to the UDGs has been characteri zed22.2' . Al­though the phys iological substrate for thi s enzy me might be ethenocytosines in DNA, it does exc ise ura­cil from G:U mi smatches at an ex tremely sluggish rate"5. Interes tingly, the equi valent of the catal yti c 0 64 of the UDGs in the water activating motif (GQDPY), in MUG is represented by an Asn in the topologicall y equi valen! moti f, G[NPG. It has been

suggested that Asn can also positi on the water mole­cule and activate it but extremely poorly as compared to the conserved class of UDGs. Although, the D64N mutant is compromised about 200 fo ld in its ac ti vity, the observation th at it is far more ac ti ve than the D64H mutant supports the proposed ro le of Asn in MUG class of urac il DNA glycosy iasesC6

On the basis of the studies carried out for the HS V I and 2 UDGs, it was beli eved that the protonati on of the 0-2 of the urac il is carried out by the conserved hi stidine present in HPSPLS , acting as a general ac idn . Mutati onal studies with EcoU DG, however, have shown that thi s conserved hi stidine does not act as general acid , but is instead neutral and ac ts as an electrophile I2.1J

. On thi s bas is, it was proposed that the electrophilic interac tion stabili zes the developing enolate on the uracil 0-2 in the course of its excision. We ex pected that such a ro le could be fulfilled by Cys in the H 187C mutant. However, the observation that H 187C mutant was more than 600 fo ld compromised in uracil exc ision, suggests that the interacti on of HI 87 with the 0 2 or urac il of the urac ilate an ion is hi ghly directi onal. And, therefore it is no t surpri sing that the H 187R and H 187 L mutants, wherein the side chains of Arg and Leu will fail altogether to substitute fo r such a fun ction, were more than a 1000 fol d worse in their urac il exc ision acti vity. Interes tingly, the topological equi valent of His of HPSPLS moti f of UDG in MUG is an Asn in the PNPGL motif. Al­though, none of our mutations contained Asn at H 187, the observation that all three mutants tes ted were ex­tremely compro mised in uracil excision, su pports that lack of the equi val ent of H 187 in MUG also contri b­utes to its extremely inefficient acti vity in urac il exc i-

. 26 slon .

In thi s study, we have also assessed the effect of mutati ons in the acti ve site residues of UDG on their interacti on wi th U gi. Crys tal structure showed th at H 187 of the DNA interca lation loop, makes hydro­phobic contact with [22 and MS6 of Ugi. Al so, the H 187 main chain N es tabli shes a water-medi ated hy­drogen bond with the E28 OEI of Ugi ID.li. However. thi s interacti on does not appear to be hi ghl y con­served as the HSVU DG- Ug i co-crys tal does not revea l thi s interaction. Our observati on that the su bstituti ons at H 187 with Arg, Cys or Leu do not su bstan ti all y alter the stability of the UDG-Ugi complexes sug­ges ts, a minimal ro le fo r the Hi s at thi s positi on. Fur­ther, the crys tal structure suggests that the 064 i nter­acts via the side chain Oli"-mediated hydrogen bond

•

HAN DA et 01.: MUTATIONAL ANA LYSIS OF D64 AND HI 87 1N £. COLI UDG 3 17

with main chain S21 0 of Ugi 10.1 1. Thus, our observa­tion that the complexes of Ugi with D64H as well as D64N are also hi ghl y stable suggests that even thi s interacti on is not critical for the stability of the com­plex. While the poss ibility of compensatory interac­tions with the side chains of Asn or Hi s at thi s posi­ti on has not been ruled out, in the li ght of the obser­vation that S21 side chain oy is fixed with an addi­tional hydrogen bond with the side chain of H67 of UDG, such a poss ibility appears unlikely .

In conclusion, our observations with the mutati ons at the D64 and H 187 pos iti ons of UDG are in agree­ment wi th the predictions from the crystal structure studies and provide additional ev idence for the slug­gish nature of the MUG enzy me. More importantl y, in thi s study, for the first time, we ex ploited the UDG mutants at D64 and H 187 to gain insights into their interacti on with Ugi. The mutati ons severely hamper the catalys is of uraci I exc ision from DNA . On the contrary, even though Ugi is a substrate mimic of UDG, the mutations have minimal effect on the sta­bility of their complexes with Ugi.

Acknowledgement

We th ank our laboratory co lleagues fo r their sug­ges ti on on the manuscript. This work was supported in part by research grants from the Counci I of Scien­tific and Industrial Research (CS IR), and the Depart­ment or Bi otechnology, and the Department of Sci­ence and Technology, New Delhi , India . The use of phosphor imaging central facility supported by the Departmenr of Biotechnology is acknowledged. P. H. and N. A. are supported by a Senior Research Fellow­ship of CS IR.

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