Transformation in Pneumococcus: Nuclease Resistance of Deoxyribonucleic Acid in the Eclipse Complex

Transcription

1 JOUMRAL OF BACTERIOLOGY, Nov. 1979, p /79/11-655/11$2./ Vol. 14, No. 2 Trnsformtion in Pneumococcus: Nuclese Resistnce of Deoxyribonucleic Acid in the Eclipse Complex D. A. MORRISON* AND B. MANNARELLI Deprtment of Biologicl Sciences, University ofillinois t Chicgo Circle, Chicgo, Illinois 668 Received for publiction 23 My 1979 Donor deoxyribonucleic cid strnds in the eclipse phse of genetic trnsformtion of pneumococcus (Streptococcus pneumonie) re purified s complex with cell protein synthesized during the development of competence. The susceptibility of the deoxyribonucleic cid strnd in this complex to digestion by nucleses ws shown to be 5- to 1,-fold less thn tht of uncomplexed single strnds of deoxyribonucleic cid. Deoxyribonuclese I, micrococcl nuclese, Neurospor endonuclese, nuclese Pi, nd the mjor endogenous nuclese of cell-free extrcts were studied. Sensitivity to nuclese ttck ws not uniform long the deoxyribonucleic cid strnd; sequences of strongly protected bses were seprted by more sensitive regions. The minimum size of protected frgments ws bout 7 bses. A complex of protein with the protected deoxyribonucleic cid segments ws obtined fter prtil digestion. The sizes of these complexes, of the protected deoxyribonucleic cid segments, nd of the protein subunit relesed by complete nuclese digestion, re ll pproximtely identicl, s determined by gel exclusion chromtogrphy. Deoxyribonucleic cid strnds of eclipse complex were lso shown to be prticulrly well protected from ttck by the mjor pneumococcl endonuclese in cell extrcts. In genetic trnsformtion in pneumococci (Streptococcus pneumonie), the interction of donor DNA with competent recipient cells occurs in series of steps tht my be grouped into two mjor processes-uptke of the DNA into the cells nd integrtion or recombintion. During uptke, the DNA is bound to the cell surfce nd processed in such wy tht single-strnd segments, of sizes verging 6, kilobses, re brought inside the competent cell, wheres similr mss of the donor DNA is degrded to oligonucleotides found outside the cell (11, 21, 22). A recombinnt chromosome is then generted by the insertion of some of these singlestrnd segments into the recipient chromosome in plce of homologous resident requences (4). The kinetics of these steps re such tht it is possible to chieve prtil synchrony of uptke nd integrtion mong the cells of competent culture. Thus, DNA uptke t 25C for 5 to 1 min is ccompnied by very little integrtion (1, 18, 29), the remining DNA is digested by dded DNse, nd integrtion of the donor singlestrnd segments then proceeds to completion in bout 1 min t 37C. Donor lleles in the singlestrnd stte exhibit very little trnsforming ctivity in deproteinized lystes becuse of their single-strnd structure but recover from this eclipse of ctivity s they ssume doublestrnded form during integrtion. The kinetics of recovery from eclipse hve been studied in some detil (3, 6, 29), but the mechnism of insertion into the chromosome is not well understood. We hve erlier reported the results of studies of the properties of donor DNA in eclipse when isolted from cell lystes. Specificlly, the donor DNA strnds behved not s free polynucleotide chins, but s though complexed with other cell constituents (18). Using [5S]methionine nd [3H]leucine lbels, we hve developed procedure for purifiction of this complex from other cell proteins nd hve shown tht the complex contins protein synthesized by the recipient cells during the development of competence (19). Furthermore, the lbeled protein components of the eclipse complex pper to comprise just one smll polypeptide (2) tht is synthesized specificlly t competence (M. F. Bker, M.S. thesis, University of Illinois t Chicgo Circle, Chicgo, 1979). 655 In two other trnsformble species, S. snguis, nd Bcillus subtilis, DNA strnds in eclipse hve lso been reported to exhibit properties of complex with other cell constituents, some of which properties re similr to those we hve described for the pneumococcl eclipse complex (26, 27). Both of these reports included

2 656 MORRISON AND MANNARELLI observtions of protection of DNA in the complex ginst degrdtion by some nucleses but not by others. In ddition, single-strnd DNAbinding protein reported to pper specificlly in B. subtilis competent cultures ws detected by using its DNA-protecting ctivity s n ssy (2). Since one hypotheticl role of the complex is protection ginst degrdtion by endogenous nucleses of competent cells, nd since nucleses could serve s probes of the structurl detils of the complex, we hve begun n exmintion of the effects of nucleses on the purified pneumococcl eclipse complex. In this pper we report initil results which both imply regulr orgniztion in the rchitecture of the complex nd indicte tht these enzymes will provide vluble nlyticl tools for purifiction nd study of the complex nd its constituents. They lso provide evidence in support of the specultion by Lcks (8) tht the DNA in the eclipse complex my be protected from cell nucleses. MATERIALS AND METHODS Mterils. Chemicls nd regents were obtined s follows: pncretic DNse I (EC ) nd Neurospor crss endonuclese (EC ) were from Boehringer-Mnnheim; nuclese P1 (EC ) nd micrococcl nuclese (EC ) were from Sigm Chemicl Co.; pneumococcl endonuclese ws prepred by the method of Lcks et l., (1); [3H]thymidine (62 Ci/mmol) ws obtined from Schwrz/Mnn, nd L-[3S]methionine (483 Ci/mmol) ws from New Englnd Nucler Corp. 3H-lbeled DNA. 3H-lbeled DNA ws prepred s described previously (19). Trnsformtion. Trnsformtion ws s described previoulsy (19), with the exception tht the trnsformed wshed cells were suspended in SSC (lx SSC is.15 M NCl plus.15 M sodium citrte) mde 1% in glycerol before freezing t -82 C. Eclipse complex purifiction. Frozen trnsformed cells (either [3H]thymidine lbeled or double lbeled with [3H]thymidine nd [3S]methionine) were thwed in n ice wter bth, centrifuged t 1, rpm for 1 min, nd suspended in.3 ml of lysing solution (.5 M NCl,.5 M Tris-hydrochloride [ph 7.5],.3 M EDTA, 1 lag of RNse per ml,.5% Srkosyl, nd.1% Triton-X-1). The mixture ws incubted 4 to 8 min t 37 C, then chilled nd frctionted on 5 to 2% sucrose grdient s described erlier (19). The grdient ws divided into nine sections, nd the middle three sections, which contined the lbeled eclipse complex, were combined nd lyered on 2% grose column. The first lbeled pek obtined ws pooled nd used immeditely for further studies. Nuclese digestion. Smples of single-strnded [3H]DNA were obtined by boiling ntive [3H]DNA for 5 min, followed by rpid cooling in n ice wter bth. Rection components were mixed in test tubes (13 by 1 mm) t C (totl volume, 1. ml) then trnsferred to wter bths t 37 C for specified lengths of time. The rection ws stopped by dding.5 ml of 18% trichlorocetic cid nd llowing precipitte to form for 15 min t room temperture. The mixture ws centrifuged t 7, rpm for 1 min t 4C, nd the superntnt, contining cid-soluble products, ws counted in liquid scintilltion counter. Acid-precipitble frgments were obtined by dding 1.5 ml of 6% trichlorocetic cid to the precipitte, boiling for 15 min, nd counting ll the resulting mixture. Rection mixes contined either lbeled single-strnded DNA t concentrtion of bout.1,ug/ml or lbeled purified eclipse complex plus pproprite concentrtions of enzyme s indicted in Tble 1 nd Fig. 1 through 7. Additionl individul components were s follows. (i) DNse L. The DNse I rection mixture contined MgCl2 t 5 pmol/ml; mercptoethnol t 6,umol/ml; bovine serum lbumin (BSA) t 4,ug/ml; NCl t 5 nmol/ml; nd Tris-hydrochloride (ph 8.) t 5,umol/ml. The mixture ws incubted for 1 min. (ii) Neurospor endonuclese. The Neurospor endonuclese rection mixture contined MgCl2 t 5 ltmol/ml; mercptoethnol t 6,umol/ml; BSA t 4,tg/ml; NCl t 5,mol/ml; nd Tris-hydrochloride (ph 8.) t 5,umol/ml. The mixture ws incubted for 1 min. (iii) Micrococcl nuclese. The micrococcl nuclese rection mixture contined CCl2 t ltmol/ 1 ml; BSA t 4 yg/ml; NCl t 5,umol/ml; nd Trishydrochloride (ph 8.) t 5 umol/ml. The mixture ws incubted for 15 min. (iv) Nuclese P1. The nuclese PI rection mixture contined, 2-[N-morpholino]ethnesulfonic cid buffer (ph 6.5) t 1,umol/ml. The mixture ws incubted for 15 min. (v) Pneumococcl endonuclese. The pneumococcl endonuclese rection mixture contined NCl t 4,umol/ml; MgCl2 t 1 pmol/ml; BSA t 4,ug/ ml; RNse t 2 ilg/ml; nd Tris-hydrochloride (ph 7.5) t 1,umol/ml. The mixture ws incubted for 2 min. The mount of bcteril protein dded to the ssy ws estimted from the culture opticl density ccording to Lcks et l. (1) (3,ug of protein per ml per 1. bsorbnce unit). Nuclese ctivity of the crude cell extrct, prepred s described by Lcks et l. (1), ws found to degrde single- nd double-strnded DNA t equl rtes. (vi) Hydroxylptite chromtogrphy. Hydroxylptite chromtogrphy ws performed s previously described (18). Agrose chromtogrphy. Agrose (2%) ws poured in columns 1.5 cm in dimeter nd with bed volumes of 3 to 5 ml. Ech ws clibrted by mesuring the elution volume for ntive DNA nd bromophenol blue. Elution buffer consisting of.1 M NCl,.1 M Tris-hydrochloride (ph 8.), nd.1 M EDTA ws pumped t rte of.32 ml/min t 4 C. Applied smples were from.5 to 2. ml mde 1% with sucrose. Agrose (8%) ws prepred in jcketed column 1.6 cm in dimeter with bed volume of 5 ml. Column clibrtion ws chieved by using blue dextrn nd urcil with stndrd grose elution buffer plus.5% Srkosyl t flow rte of.32 ml/min. Smple preprtion ws the sme s described bove. The opticl density of the effluent ws continuously monitored in Beckmn DB-G Spectrophotometer, using flow- *J. BACTEFRIOL.

3 VOL. 14, 1979 through cell. Blue dextrn nd urcil were monitored t 54 nd 26 nm, respectively. Unless otherwise indicted, 8% grose chromtogrphy ws performed t room temperture. Polycrylmide gel electrophoresis. Polycrylmide gel electrophoresis ws performed by the method of Mnitis et l., (14). Tube gels contined Tris-borte-EDTA buffer (.9 M Tris-borte, ph 8.3, nd 2.5 mm EDTA). Electrode chmbers (Bio-Rd model 155) contined the sme buffer without ure. The gels were prepred by mixing 31.5 g of ure, 7.5 ml of 1Ox Tris-borte-EDTA buffer, 3 ml of 3% crylmide (1 g of N,N'-methylenebiscrylmide plus 29 g of crylmide/1 ml), 3. ml of 1.6% mmonium persulfte, nd enough wter to bring the finl volume to 75 ml. After degssing, 25 Ai of N,N,N,'N'- tetrmethylethylenedimine ws dded, nd 2.5-ml gels were cst in glss electrophoresis tubes (7 by 125 mm) where the gels polymerized. Smples were ethnol precipitted before electrophoresis to remove slt nd buffer nd suspended in 25 pl of formmide contining.25% xylene cynol FF nd.25% bromophenol blue. Electrophoresis ws performed t room temperture t 1 V/cm until the leding dye (bromophenol blue) ws bout 4 cm from the bottom. The gels were then removed from the tubes, frozen, nd cut into 1-mm slices with gel slicer. Individul slices were trnsferred to glss scintilltion vils, dissolved overnight with.2 ml of 3% H22 t 6 C, cooled, nd dissolved in 3. ml scintilltion fluid for determintion of rdioctivity. The reltive mobility of poly(dt)jo ws determined by scnning the gels t 26 nm in Gilford model 24 spectrophotometer. Ethnol precipittion. One-tenth volume of 2% sodium cette, 5,ug of RNA crrier, nd 2 volumes of 95% ethnol were dded to ech smple. The mixture ws plced in n ethnol-dry Ice bth for 15 min nd centrifuged in Beckmn Microfuge for 5 min t room temperture. The precipitte ws rinsed twice by suspending the precipitte in 1. ml of 7% ethnol nd centrifuging for 5 min. Determintion of rdioctivity. Aqueous smples were mixed with 9 volumes of scintilltion fluid contining 5 ml of Triton X-1 nd 5 g of 2,5- diphenyloxzole per liter of toluene, plus sufficient wter to prevent formtion of precipitte or visible seprtion of phses. RESULTS Digestion of eclipse complex by nucleses. To evlute the sensitivity to nuclese ttck of the DNA in the eclipse complex, prllel digestions were crried out with eclipse complex contining [3H]DNA nd with dentured purified [3H]DNA. The relese of cidsoluble products ws determined fter fixed incubtion time, nd over rnge of nuclese concentrtions (Fig. 1). The enzyme levels extended from the highest prcticble down to levels without detectble effects on the control dentured DNA. In ll cses shown, the mount of enzyme required to relese given frction of eclipse ECLIPSE COMPLEX: NUCLEASE RESISTANCE 657 I z Q z / Oft. so. o D.,= = q 1 1 lo o le Reltive Enznn. Concentrtion FIG. 1. Nuclese resistnce ofeclipse complex. Purified 3H-lbeled eclipse complex () ws incubted t 37 C under conditions described in the text with the indicted mounts of ech nuclese. The relese of trichlorocetic cid-soluble products ws then determined. The mximum levels of enzyme exmined were: () DNse I, 1,OXpg/ml; (b) nuclese P1, 1lg/ ml; (c) micrococcl nuclese, 1. pg/ml; (d) N. crss endonuclese, 25 jg/ml; nd (e) pneumococcl endonuclese, 1 pg of cell extrct protein per ml. Dentured DNA ws digested in prllel control smples (). complex DNA, in the rnge of 15 to 9%, ws 5 to 1, times tht required to relese the sme frction of control single-strnded DNA. Clerly, residence in eclipse complex fforded significnt protection from digestion by these four purified nucleses nd by endogenous nuclese. However, this protection is not bsolute; in those cses where very high enzyme levels could be tested, the DNA in eclipse complex ws shown to be digestible. For micrococcl nuclese nd nuclese Pl, 9% solubiliztion ws observed t enzyme levels 3- nd 1,-fold, respectively, greter thn those required for similr digestion of free DNA. Although the shpes of the digestion curves re not uniform, in the cse of micrococcl nuclese, detectble solubiliztion (bout 5%) of DNA in eclipse complex did not occur t n enzyme level 8-fold bove tht required for 1% solubiliztion of single strnds nd sufficient for complete solubiliztion of single strnds. Thus, we cn conclude tht t lest 95% of the DNA in eclipse complex is protected from these endonucleses.

4 658 MORRISON AND MANNARELLI The nucleses were still fully ctive in the presence of the complex protein or other proteins tht elute with eclipse complex on 2% grose (Tble 1). Single-strnd [3H]DNA ws incubted with DNse I or N. crss endonuclese in the presence or bsence of purified eclipse complex. It cn be seen tht no inhibition ws observed; prllel runs of nuclese nd eclipse complex showed substntil protection of DNA in eclipse complex. Thus, the mechnism of protection ppers to be steric, nd the protein does not ct s nuclese inhibitor. Products of complete nuclese digestion of eclipse complex. Digestion with high levels of micrococcl nuclese provides n opportunity to seprte the protein component of eclipse complex from the DNA, without drstic or denturing tretments previously used (18, 19). In Fig. 2 is shown comprison of ntive eclipse complex (Fig. 2) with the products of exhustive digestion by micrococcl nuclese (Fig. 2b nd c). The [3S]methionine-lbeled protein digestion product ppered, s described elsewhere (2), s single size species on grose gel chromtogrphy, distinct from the nucleotide or oligonucleotide DNA products. Comprison with ctlse on the sme column showed tht this protein 'subunit' elutes t pproximtely the position of globulr protein of moleculr weight 2,. Hydroxylptite chromtogrphy lso demonstrtes complete seprtion of DNA nd protein components: the DNA products eluted very erly from hydroxylptite, s expected for cid-soluble frgments; the protein product ws tightly bound, eluting only t step from.2 to.4 M phosphte buffer. Protein-bound protected frgments in prtil digest of eclipse complex. To explore TABLE 1. J. BACTERIOL. the nture of the protection ginst nuclese ttck described bove, the DNA products from vrious levels of prtil digestion by DNse I were exmined on 8% grose columns fter dissocition with NOH. Both protein nd DNA lbels of intct eclipse complex re excluded from this gel, nd the dissocition chieved by NOH tretment seprtes the two (Fig. 3). The effects of vrying the extent of DNse I digestion re shown in the grose elution profiles of Fig. 3b, c, nd d. Smll nuclese "doses" reduced the verge chin length of the DNA in the eclipse complex; t higher levels, two clsses of product ccumulted. Up to 4% of the DNA ws broken down to frgments identicl in size to the limit products of digestion of free single strnds (Fig. 3d); the reminder is in distinctively lrger frgments, pprently more thoroughly protected from enzyme ttck. Control digestions (Fig. 3e) of denturted DNA showed no similr ccumultion of the two distinct frgment size clsses. Since the existence of preferentilly protected frgments of specific size could rise from locl protection by protein subunits, [ 'S]methionine- [3H]DNA-lbeled eclipse complex ws prtilly digested, nd the products were exmined without dissocition to look for such n ssocition mong the products. Figure 4c shows the 4% of DNA lbel relesed s oligonucleotides eluting from grose ner urcil, nd in the first frctions of hydroxylptite column (Fig. 4b), s expected for smll oligonucleotides. The reminder of the [3H]DNA lbel coeluted with single pek of 'S on both columns nd presumbly represents the protected DNA frgments bound to protein subunits. The protected frgmentprotein complex migrted with Kd of bout Effect of eclipse complex protein on nuclese ctivity Enzyme concn (ug/ml)' Substrte(s) Neurospor Precipitble cpm Soluble cpm % Soluble DNse I endonuclese DNA 1-42,484 19, DNA + eclipse complex 1-44,688 14,272 7 Eclipse complex 1-2, DNA 5-17, , DNA + eclipse complex 5-22, ,75 85 Eclipse complex 5-2, DNA ,75 114, DNA + eclipse complex , , Eclipse complex , Single-strnded DNA ws 3H-lbeled s previously described (19). 3H-lbeled trnsformed cells were lysed s described in the text. The lyste ws then frctionted on 2% grose column, nd the first lbeled pek obtined ws pooled nd used s eclipse complex;.6 ml of this pooled frction ws used for the ssys. b Enzyme ssys were performed s described in the text. Totl rection mixture ws 1. ml.-, No enzyme.

5 VOL. 14, 1979 ECLIPSE COMPLEX: NUCLEASE RESISTANCE f l b S1 I A I n.1 1. Z s.1 ' A.2 >.1x Io -C IL C Downloded from FIG. 2. Complete digestion of eclipse complex by micrococcl nuclese. Purified [3H]DNA-[36Slmethionine-lbeled eclipse complex ws digested with 1 pg of micrococcl nuclese per ml for 15 min. The products were nlyzed (b) on n 8% grose column with opticl mrkers of DNA nd urcil (*), nd (c) on hydroxylptite column, using sodium phosphte grdient followed by.1 M NOH. For comprison, chromtogrphy of undigested eclipse complex on 8% grose is shown in. Symbols: 35S (); 'H (). In b, elution position of ctlse is indicted by rrow t A..38 on grose nd eluted from hydroxylptite t nominl phosphte buffer concentrtion of.7 M. Direct comprison of eclipse complex with such prtil digestion products on two prllel hydroxylptite columns is shown in Fig. 4 nd b, to demonstrte tht the protected frgment-protein complex is eluted by lower concentrtion of phosphte buffer thn the intct complex. Prtil digestion of eclipse complex by micrococcl nuclese or by nuclese Pi lso gen- FIG. 3. DNA products of prtil digestion of eclipse complex. After incubtion t 37C for 1 min 3 z) with DNse I, ech rection ws terminted with EDTA, nd the DNA ws relesed with.1 N NOH nd nlyzed on column of 8% grose. Elution profiles re shown for digestions by the following, enzyme levels: (b).1, 1., nd 5. pg/ml; (c) 2 nd 5 pg/ml; nd (d) 25 nd 1, pg/ml. Pnel shows dissocition ofundigested 3S- nd 3H-lbeled eclipse complex by.1 NNOH. Digestion ofdentured DNA by DNse I t.5, 2., nd 1 pg/ml is shown in e for comprison. Opticl mrkers of DNA nd urcil re indicted in ech digrm by rrows ta nd B, respectively. on November 23, 218 by guest

6 66 MORRISON AND MANNARELLI nd for DNA strnds in the eclipse complex. A ifa Virtully complete protection ws provided by eclipse complex ginst the pneumococcl en- =, zyme, even enzyme in 1,-fold excess over the, 3 1 / t/; \ level required to begin libertion of cid-soluble ' 3 I 1/\ ; s products from dentured DNA The more sensitive ssy for prtil digestion U 2 provided by grose gel chromtogrphy is z2 _ j - _ 2_ shown in Fig. 5c. Comprison with the digestion C b I results shown bove (Fig. 1 nd 3) revels tht A Z the DNA in eclipse complex is protected from 5 X the pneumococcl endonuclese so effectively s +2 to reduceeven therte ofbrekgebetween t3- protected frgments to t lest 1,-fold below 3 - tht observed with DNse I..1 J Electrophoresis of protected DNA frg- X= 11 ty ments. Digestion of eclipse complex by high V " 1 \ DNse I concentrtions (1 mg/ml) resulted in ir c /~~~~~~~~~~~~!6 3. 2l C ~~~~~~~~~~~~~~~~~~A ~~~~~~~~ FIG. 4. Ntive products ofprtil DNse I diges-i tion of eclipse complex. [36S]methionine-[3H]DNAlbeled eclipse complex digested with DNse I t 1,OOO pg/ml for 1 min ws nlyzed, without disso- z A B cition, on hydroxylptite (b) nd t 5C on n 8% grose column (c). Prllel controls of dentured DNA (), undigested eclipse complex (), nd dentured DNA digested with DNse I t 1, jg/mlm for 1 min (*) re shown (). b X.f.1. BACTERIOL. erted protected DNA frgments of similr size, (Fig. 5 nd b). These hve not been investigted further. Effect of pneumococcl nuclese. Lcks A~~~~~~~~ hs shown tht most (-8%) of the nuclese 1 ctivity (7) exhibited by crude lystes pretreted with RNse is due to the ction of one enzyme, the mjor endonuclese of pneumococcus. To explore the sensitivity of the eclipse complex to FIG. 5. Limited dige8tion of eclipse complex by this nuclese, then, RNse-treted lystes of dditionl nucleses. The DNA ws relesed with pneumococci were prepred s described (1),.1 N NOH fter enzyme tretment, nd the [IHInd their effects on DNA nd eclipse complex DNA products were nlyzed on column of 8% grose. Enzymes: () micrococcl nuclese (.1 were mesured s described bove for commer- pg/ml); (b) nuclese P1 (.1 pg/ml); nd (c) pneumocil enzymes. In Fig. 1 re shown the mounts coccl endonuclese (5 nd 1 pg of cell extrct of cid-soluble products relesed by vrious protein per ml). Elution volumes of DNA nd urcil mounts of such lyste for single-strnd DNA, re indicted by rrows t A nd B, respectively.

7 VOL. 14, 1979 ECLIPSE COMPLEX: NUCLEASE RESISTANCE 661 -u c f-u v I I z I- D z 2C 1 so 1 b FRACTION RELATIVE A A NUMBER MOBILITY 71 SLICE NUMBER FIG. 6. Anlysis ofdnse I digestion products by electrophoresis on 12% crylmide gels in 7 M ure. 3Hlbeled eclipse complex ws digested with DNse I t 1,OOO or 2.5 pg/ml for 1 min t 37 C. Proteins were removed by chloroform tretment (29), nd the cler superntnts were frctionted on columns of8% grose (). Frctions were pooled s shown, lyophilized, dissolved in 1. ml of elution buffer, nd prepred for electrophoresis s described in the text. The electrophoretic profiles for frctions I, II, nd III re shown in c, d, nd e, respectively. Theposition ofxylene cynol FF (A) nd bromophenol blue (B), which coelectrophorese with single-strnd DNA segments of 58 nd 13 nucleotides, respectively, re indicted. The mobility versus pprent nucleotide length of the DNA frgments obtined from frctions I (A), II (-), nd III () is plotted in b. The two mrker dyes, xylene cynol FF (A) nd bromophenol blue (B), nd the oligonucleotide poly(dt)lo (C) re indicted s (A). The mobility is plotted reltive to tht of bromophenol blue. the production of two clsses of DNA frgments-one clss composed of smll DNA segments few nucleotides long, nd nother clss composed of longer DNA segments (Fig. 3d). The longer DNA segments re pprently derived from regions of the eclipse complex which re more resistnt to digestion nd might represent the length of bsic protein unit which shields the DNA t these regions from nuclese ttck. Digestion with smller mounts of DNse I produces brod distribution of frgments lrger thn those produced by extensive digestion (Fig. 3b). These frgments might represent multiples of the bsic clss produced in 1,-,ug/ml digestion, indicting repeting structure in the complex. The length of protected frgments ws reexmined t the much higher resolution fforded by electrophoresis in 12% polycrylmide gels contining 7 M ure (14). The reltive mobilities of protected frgments were determined in gels clibrted by internl mrkers of the two dyes, xylene cynol FF nd bromophenol blue, which coelectrophorese in 12% gels with single strnd DNA segments of 58 nd 13 nucleotides, respectively, nd single-strnd DNA segment of known length [poly(dt)lo]. The pooled lrge frgments from DNse I digestion (4% cid soluble) purified by grose column chromtogrphy (frction I in Fig. 6) electrophoresed s two distinct but brod size clsses; mjor nd minor clss centered t CL

8 662 MORRISON AND/MANNARELLI lengths of bout 7 nd 17 nucleotides, respectively (Fig. 6c). Digestion with smller mount of DNse I (2.5,ug/ml) produced only bout 1% cid-soluble products nd brod pek on grose of lrger frgments including some t the position of limit products. The two sets of pooled frctions from this column (pools II nd III in Fig. 6) were similrly nlyzed by electrophoresis (Fig. 6d nd e) nd found to contin distinct clsses of frgments with pprent lengths of: 85, 125, 148, 165, nd 18 nucleotides. The pprent lengths from the two DNse I digestions re both plotted in Fig. 6b. It ppers tht they re seprted by regulr intervl of bout 15 to 25 nucleotides. Recovery of the pplied smple in the gels ws t lest 9%. Since frgments smller thn 7 bses might hve been lost during the preprtion procedure, or the pek seen in 8% grose chromtogrphy might not represent n oligonucleotide 7 bses long, further experiment ws performed to exmine the resolution nd to clibrte the grose column. Dentured [3H]DNA ws prtilly digested by DNse I so s to produce segments of vrious lengths. The digest ws prepred nd electrophoresed s in the previous experiment. Sections of the gel corresponding to 5-, 8-, nd 1-nucleotide segments were cut out (2-mm slices), nd the DNA ws eluted into buffer nd lyered on 8% grose. The pek observed in previous 8% grose chromtogrphy runs does T _1 3 6 U- FIG. 7. Stndrdiztion of 8% grose column by using electrophoreticlly purified frctions. Dentured [3HJDNA ws digested by DNse I t 1 pg/mi, nd electrophoresed on 12% crylmide gels contining 7 M ure. Slices (2 mm) corresponding to nucleotide lengths of5, 8, nd 1 wereplced in seprte test tubes contining 1. ml of stndrd grose buffer, pulverized nd llowed to stnd overnight to extrct the polynucleotides. The gel frgments were removed by centrifugtion, nd the extrcts were nlyzed on 8% grose columns. (A) 1-nucleotide segments; (B) 8-nucleotide segments; nd (C) 5- nucleotide segments. Opticl stndrds (.) of DNA nd urcil. The position of the protected DNA frgmentpek obtined fter extensive DNse I digestion of eclipse complex is indicted by rrow t D. indeed correspond to length of bout 7 nucleotides (Fig. 7). DNA eluted from these slices proved to be completely precipitble by the method used. Although the precise frgment lengths involved re not certin with this clibrtion, the resolution nd reproducibility of these gels re clerly sufficient to llow the following conclusions. The products of limited nuclese digestion (to 1% cid-soluble products) fll minly in the rnge of 5 to 2 bses, not s rndomly cut popultion, but s set of six specific size clsses. Clerly, the sites of initil nuclese ttck re limited nd distributed in regulr wy long ll DNA in the eclipse complex. DISCUSSION In compring the effects of vrious nucleses on DNA in the eclipse complex, we considered it pproprite to mesure digestion by equivlent levels of enzyme ctivity. Although the unit of ctivity to be used here is clerly somewht rbitrry, we believe tht resonble first step is to compre digestion of eclipse complex DNA with the digestion of single-strnded DNA over wide rnge of enzyme levels. This llows, in effect, stndrdiztion of enzyme levels in terms ofthe mount of enzyme required to effect given degree of digestion ofuncomplexed single strnds. Specific interctions of eclipse complex with prticulr nucleses my then be reveled s differences in the reltive protection fforded by the complex. Such comprison for four commercilly prepred nucleses s well s for the mjor pneumococcl endonuclese reveled tht most or ll of the DNA bound in the eclipse complex ws protected from nuclese ttck. The extent of the protection, given s the increse in concentrtion of n enzyme required to effect given level of digestion over tht required with free single strnds, vried from 5- to 1,-fold, depending on the mesure of digestion considered. The rnge of possible mesures includes vrious degrees of solubiliztion, s well s detectble reduction in verge strnd length. Since subregions of the DNA in the eclipse complex vry considerbly in their susceptibility to digestion, detiled interprettion of the shpe ofthe curves describing solubiliztion versus enzyme concentrtion ws not ttempted. However, the overll properties of the complex re cler: the entire nuclese susceptibility curve is shifted to higher enzyme levels by fctor of 5 to 1, with respect to tht for single-strnd DNA. Essentilly ll DNA in the complex is protected to significnt degree. Only three quntittive differences were detected mong the nucleses.j. BACTFRIOI,.

9 VOL. 14, 1979 tested: (i) reltive protection vried rther subtly mong enzymes nd mong levels of digestion, (ii) the shpe of the sensitivity curve t low digestion levels lso vried significntly, (iii) protection ginst endogenous enzyme(s) ws clerly superior to tht ginst others. Studies on other single-strnd binding proteins (5, 16, 17, 23-25) hve concluded tht protection ginst nuclese ction ppers to be generl property of these proteins nd suggest tht the mechnism of protection is probbly steric, the protein covering the DNA templte nd preventing the nuclese from reching the site of scission. Severl of these reports hve shown tht protection vries with the mount of protein ssocited with the DNA nd tht "complete protection" (defined s zero detectble relese of cid-soluble frgments) cn be chieved with excess binding proteins. Comprison of our observtions with published dt is difficult in the bsence of control mesurements with free dentured DNA in some of those reports. However, we observe tht, in generl, the dt in Fig. 1 suggest tht chrcteriztion of the DNA in complex s "sensitive" or "resistnt" to given nuclese is more likely to depend on the level of enzyme used, in single-level experiment, thn on ny bsolute property of the complex. Although mesurement of relese of cid-soluble products provides convenient ssy for extensive nuclese digestion, it does not detect the first steps in nuclese ttck, which consist, in the cse of the enzymes used here, of the introduction of widely seprted breks nd which thus yield no cid-soluble products. Such result ws reported by McKy nd Linn (13); n Escherichi coli-binding protein-dna complex exposed to exonuclese V digestion ws not degrded to cid-soluble products but ws degrded to internedite-sized products. The method of chromtogrphy on 8% grose columns is of course much more sensitive to such breks, since it detects relese of frgments below bout 3 bses long. By this ssy, nuclese levels relesing little cid-soluble mteril from the eclipse complex DNA were nevertheless shown to introduce nicks in the most sensitive portions of the DNA. At the sme time, high levels of the pneumococcl endonuclese were shown to be prevented from nicking eclipse complex DNA. Exmintion of eclipse complex fter prtil nuclese digestion reveled bimodl distribution of DNA product sizes: oligonucleotides similr in size to those produced by digestion of free single-strnded DNA, nd clss of lrger frgments. The lrger frgments evidently represent stretches of DNA enjoying superior protection ECLIPSE COMPLEX: NUCLEASE RESISTANCE 663 ginst nuclese ttck. These well-protected frgments were in fct found bound to eclipse complex protein in the form of smll prticle with unique chromtogrphic properties on hydroxylptite, nd with the size of the protein subunit discussed below. Clerly, certin portions of the DNA strnd in the eclipse complex re especilly well protected, nd they remin bound to protein subunits s protected frgments during the erly stges of nuclese digestion. At the point of digestion yielding 4% in smll frgments, the remining 6% of the DNA lbel ppered in uniform popultion of such protected frgment-protein complexes. The length of the DNA segment protected from nucleses ws found by electrophoresis to be bout 7 nucleotides. Since uniform popultion of these frgments ws produced, we infer tht bsic protein unit must repet long the length of the complex. However, further interprettion of the dt is difficult due to the pprently nonuniform differences between successive frgment sizes nd the fct tht these differences re clerly smller thn the length of the smllest protected frgment. A cler description of these products will require their reexmintion with gels well clibrted in the 5- to 3-bse rnge. Digestion with high levels of nuclese mde it possible to remove the DNA portion of the eclipse complex without protein-denturing tretments nd to exmine the intct protein subunit of the complex. The [3H]DNA lbel ws completely seprted from the protein subunits, nd the protein chromtogrphed on 8% grose s n pprently homogenous species t position corresponding to globulr protein of moleculr weight 2,. Some very high-moleculr-weight protein complex lso remined in some digestions. Since the constituent polypeptide moleculr weight is only 19,5 (M. Bker, M. S. Thesis, 1979), this implies tht the protein subunit must be composed of either very symmetric monomer or complex of severl (s1) monomers. The possibility tht the subunit is very symmetric monomer seems less likely thn tht it ssocites into distinctly sized ggregte. Other binding proteins hve been found to form specific oligomeric complexes. E. coli binding protein under nondenturing conditions exists s tetrmer (15). Recent X-ry diffrction nlysis hs shown tht in the DNA-gene 5 protein complex there exists fundmentl unit composed of 12 gene 5 monomers (A. McPherson, F. Jurnk, I. Molineux, nd A. Wng, Fed. Proc. 37:134, 1978). The effects of prtil nuclese digestion on the chromtogrphic properties of the eclipse complex, both on grose nd on hydroxylp-

10 664 MORRISON AND MANNARELLI tite, should provide vluble nlyticl nd preprtive tools, since very few other cell components, if ny, would be expected to respond in the sme wy to such specific tretment. The effects of nuclese digestion on purified eclipse complex lso serve s nother bsis for estimting the proportion of the 3S-lbeled mteril purified with the complex tht is ctully DNA bound. All lbel initilly excluded from 2% grose tht shifts to lower moleculr weight fter nuclese digestion must, we ssume, be prt of structure dependent on nucleic cid for its size. The results presented here (Fig. 2c) set n upper limit of 4%, then, on the proportion of contminting, non-dna-bound 'S-lbeled prticles fter the two-step frctiontion procedure used. Combined with our previous demonstrtion of only single, smll 'S-lbeled polypeptide in such preprtions, this lends substntil confidence to the view tht the purifiction procedure described previously (19) yields eclipse complex tht is essentilly free of contminting [3S]methionine-lbeled mteril. Considering the possibility of role of the eclipse complex in protecting the single-strnd donor DNA from digestion fter uptke, we exmined the interction of the complex with the mjor endonuclese of pneumococci. This enzyme, isolted nd studied by Lcks nd Greenberg (9), digests ntive DNA, nd dentured DNA, nd is loclized in the cell membrne (12). About 1% of norml enzyme ctivity ppers to be required for uptke of DNA (1), nd Lcks (7) hs shown tht bout 8% of the nuclese ctivity in crude cell extrct is contributed by this one enzyme. Thus, our results showing little degrdtion of eclipse complex by such extrcts certinly demonstrte strong protection ginst this enzyme nd my lso reflect protection ginst other cell nucleses s well. Proof of significnt protection in vivo must, however, wit method to determine the fte of uncomplexed DNA in competent cells. In view of the protection fforded ginst hydrolysis by the mjor pneumococcl endonuclese, however, eclipse complex protein would pper to be good cndidte for the role proposed by Lcks (8) for DNA-binding protein in DNA uptke in this species. Specificlly, s one strnd of the donor-bound molecule is degrded by the endonuclese during uptke, this protein could immeditely cot the other strnd, protecting it from further ttck by the membrne-bound endonuclese. Subsequent or concurrent involvement in moving the DNA through the cell membrne is lso conceivble. The results obtined with high levels of DNse I serve to emphsize potentil dnger J. BACTERIOI,. in the interprettion of n observed protection ginst degrdtion when digestion is mesured s relese of cid-soluble products. High levels of DNse I relesing only 4% of the DNA in cid-soluble form might be construed to show substntil (6%) protection of strnds in the complex. Closer exmintion of the products, however, shows tht the 6% remining precipitble is not unffected; it survives s short frgments bound to protein subunits. The trnsforming ctivity of such products in vivo would be nil (21). Biologiclly significnt protection must preserve much longer stretches of DNA from ttck. ACKNOWLEDGMENT This mteril is bsed upon work supported in prt by the Ntionl Science Foundtion under grnt NSF LITERATURE CITED 1. Coflins, C. J., nd W. R. Guild Events occurring ner the time of synpsis dunng trnsformtion in Diplococcuspneumonie. J. Bcteriol. 19: Eisenstdt, E., R. Lnge, nd K. Willecke Competent Bcilus subtilis cultures synthesize dentured DNA binding ctivity. Proc. Nti. Acd. Sci. U.S.A. 72: Ghei,. K., nd S. A. Lcks Recovery of donor deoxynbonucleic cid mrker ctivity from eclipse in pneumococcl trnsformtion. J. Bcteriol. 93: Gurney, T., nd M. S. Fox Physicl nd genetic hybrids formed in bcteril trnsformtion. J. Mol. Biol. 32: Hung, W. H., nd L R. Lehmn On the exonuclese ctivity of phge T4 deoxyribonucleic cid polymerse. J. Biol. Chem. 247: Lcks, S Moleculr fte of DNA in genetic trnsformtion of pneumococcus. J. Mol. Biol. 5: Lck, S Mutnts of Diplococcuspneumonie tht lck deoxyribonucleses nd other ctivities possibly pertinent to genetic bnsformtion. J. Bcteriol. 11: Lcks, S Binding nd entry of DNA in bcteril trnsformtion, p In J. Reissig (ed.), Microbil interctions. Chpmn & Hll, London. 9. Lcks, S., nd B. Greenberg Deoxyribonucleses of pneumococcus. J. Biol. Chem. 242: Lcks, S., B. Greenberg, nd M. Neuberger Identifiction of deoxyribonuclese implicted in genetic trnsformtion of Diplococcus pneumonie. J. Bcteriol. 123: Lcks, S., nd B. Greenberg Single-strnd brekge on binding of DNA to cells in genetic trnsformtion of Diplococcuspneumonie. J. Mol. Biol. 11: Lcks, S., nd M. Neuberger Membrne loction of deoxyribonuclese implicted in the genetic trnsformtion of Diplococcus pneumonie. J. Bcteriol. 124: McKy, V., nd S. Linn Selective inhibition of the DNse ctivity of the recbc enzyme by the DNA binding protein from E. coli. J. Biol. Chem. 251: Mnitis, T., A. Jeffrey, nd H. Vn DeSnde Chin length determintion of smll double nd singlestrnded DNA molecules by polycrylmide gel electrophoresis. Biochemistry 14: Mollneux, L J., S. Friedmn, nd M L. Gefter Purifiction nd properties of the E. coli deoxyribonu-

11 VOL. 14, 1979 ECLIPSE COMPLEX: NUCLEASE RESISTANCE 665 cleic cid unwinding protein. J. Biol. Chem. 249: Molineux, L. J., nd M. L Gefter Properties of the E. coli DNA binding (unwinding) protein: interction with DNA polymerse nd DNA. Proc. Ntl. Acd. Sci. U.S.A. 71: Molineux, I. J., nd M. L Gefter Properties of the E. coli DNA binding (unwinding) protein interction with nucleolytic enzymes nd DNA. J. Mol. Biol. 98: Morrison, D. A Trnsformtion in pneumococcus: existence nd properties of complex involving donor deoxyribonuclete single strnds in eclipse. J. Bcteriol. 132: Morrison, D. A Trnsformtion in pneumococcus: protein content of eclipse complex. J. Bcteriol. 136: Morrison, D. A., M. Bker, nd B. Mnnrelli A protein component of the pneumococcl eclipse complex In S. W. Glover nd L.. Butler (ed.), Trnsformtion, Cotswold Press Limited, Oxford. 21. Morrison, D. A., nd W. R. Guild Trnsformtion nd deoxyribonucleic cid size: extent of degrdtion on entry vries with size of donor. J. Bcteriol. 112: Morrison, D. A., nd W. R. Guild Brekge prior to entry of donor DNA in pneumococcus trnsformtion. Biochim. Biophys. Act 299: Nss, K, nd G. D. Frenkel Adenovirus induced inhibition of cellulr DNse. J. Virol. 26: Oey, J. L., nd R. Knippers Properties of the isolted gene 5 protein of bcteriophge fd. J. Mol. Biol. 68: Pcumbb, R., nd M. S. Center Prtil purifiction nd properties of bcteriophge T7 inhibitor of the host exonuclese V ctivity. J. Virol. 16: Pienizek, D., M. Piechowsk, nd G. Venem Chrcteriztion of complex formed by nonintegrted frction of trnsforming DNA in Bcillus subtilis with recipient cell constituents. Mol. Gen. Genet. 156: Rin, J. L, nd A. W. Rvin Fte of homospecific trndorming DNA bound to Streptococcus snguis. J. Bcteriol. 133: Sevg, M. G., D. B. Lckmn, nd J. Smolens The isoltion of the components of Streptococcl nucleoproteins in serologiclly ctive form. J. Biol. Chem. 124: Shoemker, N. B., nd W. R. Guild Kinetics of integrtion of trnsforming DNA in pneumococcus. Proc. Ntl. Acd. Sci. U.S.A. 69: Downloded from on November 23, 218 by guest