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A denaturation map of polyoma virus DNA
242
OIOCHIM[CAET BIOPHY$ICAACT&
SHORT COMMUNICATIONS BBA 93330 A denaturation map of po|yomo virus O N A DNA partially denatured by heating in the presence of formaldehyde shows specific sites of strand separation, as seen with the electron microscope. A map of the denaturable regions can be constructed. Those regions which are denatured at the lowest temperature probably represent adenine-thymine-rich sequences. Such studie have been made on ~ DNA 1 and on human papilloma virus DNA 2. In this communication the partial denaturation of the circular form If of polyoma virus DNA will be described. Polyoma virus exists in two forms: 'supereoiled circular DNA I' (eovalently closed duplex rings) and 'open circular DNA If' (el' mlar duplex with at least one single-strand scission) which does not show supereoiling a. Only in DNA II, which comprises 5 - t o % of the DN'A prepared from purified virus, can visible regions of strand separation be demonstrated after heating in the presence of formaldehyde 4. Preparations containing 4o-6o % of DNA I[ (Fig. za) are obtained by a short treatment of purified DNA I (2o-4o #g/ml, o.z M NaCI, o.oi M EDTA) with low concentrations of hydroquinone 3.~ or pancreatic deoxyribonucleas#. In the first case, the reaction mixture contained o.ooi M hydroquinone and o.oi M Tris-HC1, pH 8.5 (z h at 2o°). The reaction is stopped by addition of E D T A , o.oo 5 M final concentration. The DNA is precipitated in 66 % ethanol at 4 ° and resuspended in o.r M NaCl, pH 7.o. In the other case, the final reaction mixture contained 5.IO -5 /~g/ml or I. zo -s pg/ml deoxyribonuclcase, o.oi M Tris-HCl, pH 7.8, o,oo5 M MgCIa, I itg[ml bovine serum albumin (3° rain at 37°). The reaction was stopped by addition of EDTA, o.o 3 M final concentration, and the solution was directly used for electron microscopy. DNA samples (in o.I M NaCI, pH 7,o, and IO % formaldehyde treated as described by FREIFELDER AND DAVISON7) are heated for Io rain and cooled in an ice bath. They are prepared for electron microscopy by the protein monolayer technique of KLEINSCHM~DTAND ZAHN8.
Fig, 1, Electron micrographs ot ~otyoma viru~ DN A I[ 1o.[ M NaCI, pH 7.0,1o% formaldehyde), a. At room temperature, b. After he~ting for to min at 5z ~, ffollowed by rapid cooling, Arrow~ indicate regions of strand sf.l~.r~tion (bar = u,5 #L Biovhim. Biophys. Avta, 166 (1968l ~42-245
SHORT COMMUNICATIONS
2~
At 46-48° , regions of strand separation are already present in some molecules. The fraction of molecules containing such regions, as well as their number and extent in each molecule, increase with the temperature. At 52 °, 6o~70 ~o of the DNA I I molecules have an average of three denatured regions, whose mean length is o.z2 p (Fig. xb). Most frequently, the denatured regions have two continuous single strands. Thus denaturation d o ~ not occur preferentially ' in regions of single-strand breaks, In agreement with this point, INMA~l obtains similar denaturation patterns of linear ], DNA which contain no single-strand breaks. Above 60 ° , most molecules are completely separated into two single strands. one circular, the other linear (Fig. a), both having about the same length (see below), This demonstrate~ that the treatment of DNA I with hydroqulnone or deoxyrihonuelease has generally produced one single-strand seission per molecule.
Fig. z, Electron micrograph of polyoma virus DNA II Io.l M NaCI, pH 7.o, to % lormaldehydel alter heating fur io min at 7o~, showing (i) one molecule in which tile two strands are almost completely dissociated; (21 one circular single-stranded molecule; (3) one linear single-stranded molecule; 14) two double-stranded molecules (DNA I) (bar = o.sp j. Representative maps of the localisation of denatured regions at 52 °, as well as a histogram showing the frequency of strand separation along the DNA molecules are presented in Figs. 3a and 3b. The method employed by FOLLnT AND CRAWFORD~ for human papilloma virus DNA l I has been used. Briefly, a map of the denatured regions of each molecule is constructed by first normalizing the length of each molecule to 1.5/t. the length of undenatured DNA, and then indicating on a disc the relative positions and lengths of the denatured regions. The discs are aligned to obtain maximum correspondance. At 5z ° most molecules (more than 5o %) have one significantly shorter undenatured region. These molecules are aligned at the midpoint of this region. The remainder permits several equivalent alignments; however, this ambiguity makes no qualitative difference in the histograms. To obtain linear maps. the discs are opened between two denatured regions. The histograms obtained are the same, independently of the metllod of preparation of DIqA II. It can be seen that there are three regions (A, B and C) of local denaturation. These three regions correspond almost exactly, both in length and relative position, to the three regions A, B and 12 defined by the denaturation map at 50 ° of human papilloma virus DNA 2. The latter Bio~him. Biophys. Aola, 16fi Ix968) ~42-245
2~
SHORT COM'uUNICATIO~S
.
t! . . . 1,0
°
I
tungPb 1~)
1,S
~o
60
$0
/
i 4
tengtb t~-I
tenetb (~,)
Fig. 3. a. Typical linear maps of denatured regions of polyoma DNA heated to 5~°. b. Histo. gram constructed trom linear maps, showing the freqnencyof sites of strand separation as a function of distance along the molecule. Fig. 4. Length distribution of: a, Molecules at ~f~ minus formaldehyde, b. Molecules after heating at 52~ for Io rain in presence of tormaldehyde, c. Highly denatured molecules after betting at 70*; - - -, circular strand; , linear strand (Both strands are not yet completely separsted). d. Circular single.stranded DNA after heating at 7o°. DNA, which is longer, contains an additional region D. There thus seems to be a similarity in t h a D N A o f these two viruses which have m a n y other points i n Commone. Measurements of the length of about ~00 molecules a t each t e mpe rat ure h a s shown t h a t strand separation is accompanied b y a shortening of the DN'A molecules which indicates a change in configuration (Table I, Fig. 4). Only a relatively small n u m b e r of measurements have been m a d e tur the circular a n d Hnear strands of highly denatured D N A because of the r a r i t y of sufficiently disentangled molecules. Isolated circular singla-stranded polyoma D N A have a ~:~5 % shortening compsred to t h e 4 ° % observed in single-stranded circular h u m a n papilloma virus D N A . Biochim. Biop~ys. Acta, i66 (z968) *42-245
~5
SHORT COMMONICATIONS
TABLE I EFFECTOF TI~Mp]~RATUREOI4 LENGTH0]~ :POLYOMADNA II MOLKCHLES V~llues from DNA obtained by both method~ of prepar~tlon axe not ~ignifica~tly different and have been l~ooled.
Mean langth (~)* ~ , piss or m i ~ formaldehyde 3~°, pins formaldehyde 4 o°, ~lus torn~Idehyde 5~ °, plus ~ormaldehyde 7o°, ~lus Iormaldehyde Highly denatured DNA circular strand "• linear strand -• Circular ~ingle-st~anded DNA
L53:~o.06 (Pig. 4~) L5O4-O-O6 t.554-o.o6 x.~O-4-o.o7 (Fig. 4b) ~,3O~:o.x (Fig. 4C) l.~3~:o.i (Fig. 4c) L~5~-0.I ~Fig. 4d)
• & probable error. ~• Not completely dissoci~d from each other. W e are grateful to Professor M. E R e ~ for his advice and encouragement in this work, W e are also indebted to Dr. P. BOUR~AVX for providing polyoma virus suspensions and purified D N A solutions, These studies were carried out under the contract ]~uratom-ULB co7-6x-~o A B I B and supported b y the N~tional Institutes of Health, U.S.A. (Research Grant-62x4) and b y the "Centre National de Biochimie e t de Biologie Mol~culaire". The author is an "aspirant an Fends National de la Recherche Sclentifiqne".
Laboratoire de Bic p~ys~qne d R~diobiologle, U~iversil~ Libre, Brusseb (Belgium)
~ARIE-~?RANCE]~OURGUIGNON
x R, B, Is~Av., J, Mol, Biol,, 28 0967) xo3. E. A. C. FOLr,ZT A~V L. V, CnZ,WFO~ZD,]. Mol. Biol.. 28 (x967) 46x3 J, VmC~RAV. J. L~BOWrrZ,R. RAwove, R. W^Tso~ ^NO P. LAtPSIS, PJ'o¢. Natl..4cad. $ci. U,S,, 53 (x~.~) *Io4, . M. F, BOUn~UX¢~ON,Arab. Inlet., PhysioL Biochem., 76 {i968) 4+ D- BOUP.GAffX-RAMOISY, N, V A N TtEGHEM AND P. BOURGAE TM, f. Gen. VigoiogY, ] (I957) 589 . 6 R. DuL~ZcCO ANJOM, VolT, Pr~. Natl. A~ad, SCL U.S., 5° (~963) ±36. D, F ~ L O l ¢ ~ ^an P. F. DAWSOS,Riop?~ys. J., 3 (1963) 49. A. K, KI,~INSCR~XDT ASP R. K. ZAH~, Z. Nat~/o~h., x4b (1959) 77o9 L. V, CI~WVOZD^~D E. M. Cs~wvo~.v, ViroloBy, ~ (x963) z58. ~OE. A. C. FOLLETAND L, V, C~WI~OZD,f . MOl. Biol., 35 (I968) 565. Received April I6th, I968
No¢¢ addad in proo[ (Received July Io th, ~968) At the t t ~ of proc~f reading a paper by Feller and Crawford1° gives a denaturation map of polyoma DNA, Five regions were obtained by the~v a,uthors, the discrepancy with the present r~ults ntsy however only he aFpaxent, the denaturation conditions being slightly different, Bioddm. Riophys. H~fa. i66 (i968) 24~-2~
OIOCHIM[CAET BIOPHY$ICAACT&
SHORT COMMUNICATIONS BBA 93330 A denaturation map of po|yomo virus O N A DNA partially denatured by heating in the presence of formaldehyde shows specific sites of strand separation, as seen with the electron microscope. A map of the denaturable regions can be constructed. Those regions which are denatured at the lowest temperature probably represent adenine-thymine-rich sequences. Such studie have been made on ~ DNA 1 and on human papilloma virus DNA 2. In this communication the partial denaturation of the circular form If of polyoma virus DNA will be described. Polyoma virus exists in two forms: 'supereoiled circular DNA I' (eovalently closed duplex rings) and 'open circular DNA If' (el' mlar duplex with at least one single-strand scission) which does not show supereoiling a. Only in DNA II, which comprises 5 - t o % of the DN'A prepared from purified virus, can visible regions of strand separation be demonstrated after heating in the presence of formaldehyde 4. Preparations containing 4o-6o % of DNA I[ (Fig. za) are obtained by a short treatment of purified DNA I (2o-4o #g/ml, o.z M NaCI, o.oi M EDTA) with low concentrations of hydroquinone 3.~ or pancreatic deoxyribonucleas#. In the first case, the reaction mixture contained o.ooi M hydroquinone and o.oi M Tris-HC1, pH 8.5 (z h at 2o°). The reaction is stopped by addition of E D T A , o.oo 5 M final concentration. The DNA is precipitated in 66 % ethanol at 4 ° and resuspended in o.r M NaCl, pH 7.o. In the other case, the final reaction mixture contained 5.IO -5 /~g/ml or I. zo -s pg/ml deoxyribonuclcase, o.oi M Tris-HCl, pH 7.8, o,oo5 M MgCIa, I itg[ml bovine serum albumin (3° rain at 37°). The reaction was stopped by addition of EDTA, o.o 3 M final concentration, and the solution was directly used for electron microscopy. DNA samples (in o.I M NaCI, pH 7,o, and IO % formaldehyde treated as described by FREIFELDER AND DAVISON7) are heated for Io rain and cooled in an ice bath. They are prepared for electron microscopy by the protein monolayer technique of KLEINSCHM~DTAND ZAHN8.
Fig, 1, Electron micrographs ot ~otyoma viru~ DN A I[ 1o.[ M NaCI, pH 7.0,1o% formaldehyde), a. At room temperature, b. After he~ting for to min at 5z ~, ffollowed by rapid cooling, Arrow~ indicate regions of strand sf.l~.r~tion (bar = u,5 #L Biovhim. Biophys. Avta, 166 (1968l ~42-245
SHORT COMMUNICATIONS
2~
At 46-48° , regions of strand separation are already present in some molecules. The fraction of molecules containing such regions, as well as their number and extent in each molecule, increase with the temperature. At 52 °, 6o~70 ~o of the DNA I I molecules have an average of three denatured regions, whose mean length is o.z2 p (Fig. xb). Most frequently, the denatured regions have two continuous single strands. Thus denaturation d o ~ not occur preferentially ' in regions of single-strand breaks, In agreement with this point, INMA~l obtains similar denaturation patterns of linear ], DNA which contain no single-strand breaks. Above 60 ° , most molecules are completely separated into two single strands. one circular, the other linear (Fig. a), both having about the same length (see below), This demonstrate~ that the treatment of DNA I with hydroqulnone or deoxyrihonuelease has generally produced one single-strand seission per molecule.
Fig. z, Electron micrograph of polyoma virus DNA II Io.l M NaCI, pH 7.o, to % lormaldehydel alter heating fur io min at 7o~, showing (i) one molecule in which tile two strands are almost completely dissociated; (21 one circular single-stranded molecule; (3) one linear single-stranded molecule; 14) two double-stranded molecules (DNA I) (bar = o.sp j. Representative maps of the localisation of denatured regions at 52 °, as well as a histogram showing the frequency of strand separation along the DNA molecules are presented in Figs. 3a and 3b. The method employed by FOLLnT AND CRAWFORD~ for human papilloma virus DNA l I has been used. Briefly, a map of the denatured regions of each molecule is constructed by first normalizing the length of each molecule to 1.5/t. the length of undenatured DNA, and then indicating on a disc the relative positions and lengths of the denatured regions. The discs are aligned to obtain maximum correspondance. At 5z ° most molecules (more than 5o %) have one significantly shorter undenatured region. These molecules are aligned at the midpoint of this region. The remainder permits several equivalent alignments; however, this ambiguity makes no qualitative difference in the histograms. To obtain linear maps. the discs are opened between two denatured regions. The histograms obtained are the same, independently of the metllod of preparation of DIqA II. It can be seen that there are three regions (A, B and C) of local denaturation. These three regions correspond almost exactly, both in length and relative position, to the three regions A, B and 12 defined by the denaturation map at 50 ° of human papilloma virus DNA 2. The latter Bio~him. Biophys. Aola, 16fi Ix968) ~42-245
2~
SHORT COM'uUNICATIO~S
.
t! . . . 1,0
°
I
tungPb 1~)
1,S
~o
60
$0
/
i 4
tengtb t~-I
tenetb (~,)
Fig. 3. a. Typical linear maps of denatured regions of polyoma DNA heated to 5~°. b. Histo. gram constructed trom linear maps, showing the freqnencyof sites of strand separation as a function of distance along the molecule. Fig. 4. Length distribution of: a, Molecules at ~f~ minus formaldehyde, b. Molecules after heating at 52~ for Io rain in presence of tormaldehyde, c. Highly denatured molecules after betting at 70*; - - -, circular strand; , linear strand (Both strands are not yet completely separsted). d. Circular single.stranded DNA after heating at 7o°. DNA, which is longer, contains an additional region D. There thus seems to be a similarity in t h a D N A o f these two viruses which have m a n y other points i n Commone. Measurements of the length of about ~00 molecules a t each t e mpe rat ure h a s shown t h a t strand separation is accompanied b y a shortening of the DN'A molecules which indicates a change in configuration (Table I, Fig. 4). Only a relatively small n u m b e r of measurements have been m a d e tur the circular a n d Hnear strands of highly denatured D N A because of the r a r i t y of sufficiently disentangled molecules. Isolated circular singla-stranded polyoma D N A have a ~:~5 % shortening compsred to t h e 4 ° % observed in single-stranded circular h u m a n papilloma virus D N A . Biochim. Biop~ys. Acta, i66 (z968) *42-245
~5
SHORT COMMONICATIONS
TABLE I EFFECTOF TI~Mp]~RATUREOI4 LENGTH0]~ :POLYOMADNA II MOLKCHLES V~llues from DNA obtained by both method~ of prepar~tlon axe not ~ignifica~tly different and have been l~ooled.
Mean langth (~)* ~ , piss or m i ~ formaldehyde 3~°, pins formaldehyde 4 o°, ~lus torn~Idehyde 5~ °, plus ~ormaldehyde 7o°, ~lus Iormaldehyde Highly denatured DNA circular strand "• linear strand -• Circular ~ingle-st~anded DNA
L53:~o.06 (Pig. 4~) L5O4-O-O6 t.554-o.o6 x.~O-4-o.o7 (Fig. 4b) ~,3O~:o.x (Fig. 4C) l.~3~:o.i (Fig. 4c) L~5~-0.I ~Fig. 4d)
• & probable error. ~• Not completely dissoci~d from each other. W e are grateful to Professor M. E R e ~ for his advice and encouragement in this work, W e are also indebted to Dr. P. BOUR~AVX for providing polyoma virus suspensions and purified D N A solutions, These studies were carried out under the contract ]~uratom-ULB co7-6x-~o A B I B and supported b y the N~tional Institutes of Health, U.S.A. (Research Grant-62x4) and b y the "Centre National de Biochimie e t de Biologie Mol~culaire". The author is an "aspirant an Fends National de la Recherche Sclentifiqne".
Laboratoire de Bic p~ys~qne d R~diobiologle, U~iversil~ Libre, Brusseb (Belgium)
~ARIE-~?RANCE]~OURGUIGNON
x R, B, Is~Av., J, Mol, Biol,, 28 0967) xo3. E. A. C. FOLr,ZT A~V L. V, CnZ,WFO~ZD,]. Mol. Biol.. 28 (x967) 46x3 J, VmC~RAV. J. L~BOWrrZ,R. RAwove, R. W^Tso~ ^NO P. LAtPSIS, PJ'o¢. Natl..4cad. $ci. U,S,, 53 (x~.~) *Io4, . M. F, BOUn~UX¢~ON,Arab. Inlet., PhysioL Biochem., 76 {i968) 4+ D- BOUP.GAffX-RAMOISY, N, V A N TtEGHEM AND P. BOURGAE TM, f. Gen. VigoiogY, ] (I957) 589 . 6 R. DuL~ZcCO ANJOM, VolT, Pr~. Natl. A~ad, SCL U.S., 5° (~963) ±36. D, F ~ L O l ¢ ~ ^an P. F. DAWSOS,Riop?~ys. J., 3 (1963) 49. A. K, KI,~INSCR~XDT ASP R. K. ZAH~, Z. Nat~/o~h., x4b (1959) 77o9 L. V, CI~WVOZD^~D E. M. Cs~wvo~.v, ViroloBy, ~ (x963) z58. ~OE. A. C. FOLLETAND L, V, C~WI~OZD,f . MOl. Biol., 35 (I968) 565. Received April I6th, I968
No¢¢ addad in proo[ (Received July Io th, ~968) At the t t ~ of proc~f reading a paper by Feller and Crawford1° gives a denaturation map of polyoma DNA, Five regions were obtained by the~v a,uthors, the discrepancy with the present r~ults ntsy however only he aFpaxent, the denaturation conditions being slightly different, Bioddm. Riophys. H~fa. i66 (i968) 24~-2~