1977 Volume 4 Number Number 1111 November November 1977

Volume 4

Nucleic Acids Research

Nucleic Acids Research

Specific fragmentation of T7 phage DNA at low-melting sites

V.M.Pavlov, Yu.L.Lyubchenko, A.S.Borovik and Yu.S.Lazurkin

Department of Biology, I.V.Kurchatov Institute of Atomic Energy, Moscow, USSR

Received 18 October 1977

ABSTRACT A method has been developed for selective fragmentation of T7 DNA at AT-rich regions. The molecules have been subjected to complete digestion with single-strand-specific SI endonuclease after fixation of DNA AT-rich regions in the denatured state by glyoxal. The treatment resulted in three fragments having molecular weights of I3.6+0.4, 8.2+0.4 and 3.5+0.I6 megadaltons as determined by electron microscopy. The position of these fragments along the T7 DNA molecule has been determin by means of analysis of the intermediates during SI-

-cleavage.

INTRODUCTION In the last few years considerable progress has been achieved in the study of high resolution thermal denaturation profiles for viral DNAIs (I-7). Such profiles have much information about peculiarities of the nucleotides distribution in DNA. The comparison of lambda DNA and its deletion mutants lamda b2 and b2b5 melting profiles have been made to c.orrelate the peaks of derivative denaturation profiles with the deletion regions b2 and b5 (4). Such a correlation can be used for both qualitative and detailed quantitative characterization of DNA and can have important applications in the molecular genetics. In the present paper we have studied the location of AT-rich regions along the T7 DNA molecule. These regions being melted at lower temperatures than DNA as a whole can therefore be easily revealed in differential melting curves. Such regions have been fixed in the melted state by glyoxal. It's known (8) that the glyoxal-guanosiae reaction results in the stable adduct with the half lifetime 50 hrs at 200C and pH 7.0. The adC Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England

4053

Nucleic Acids Research duct block the formation of the perfect DNA helix (9) and glyoxal therefore is the suitable agent for fixation of the denatured regions of DNA. The glyoxal-fixed AT-rich regions were digested with SI endonuclease, specific for single-stranded DNA (IO). The electron mieroscopy and agarose gel electrophoresis data show the high homogeneity of the fragments.

MkTERIAS AND IMHODS DNA preparations. Phage T7 DNA was prepared from purified phage suspension by the hot phenol method (II). Protein content in the DNA preparations determined according the tecbnique of Lowry et al.(I2) was about T°.The number of single-stranded breakes determined from the denatured DNA sedimentation data (I3) were not more than one per molecule. EcoRI restriction fragments of lambda DNA was kindly provided by Dr. R.Sh.Beabealashvily. Eaz;m. The SI endonuclease from A.oryzae was isolated according the procedure of Vogt (I4) from taka-diastase powder, which was a gift from Dr. V.Z.Tarantul. The last stage of purification (gel-filtration by Sephadex G-I00) was omitted. The SI endonuclease preparation was stored in I0o glycerol at -20°C. DNA digestion was accomplished in buffer A which contained 0.02 M CH3COONa, I0 M ZnS04(pH 4.6). Gl 1. O.I M stock solution was prepared by dissolving the glyoxal monobydrate (B.D.H.Ingland) in deionized water. Meltg. The melting curves of DNA were recorded by spectrophotometers Hitachi EPS-20 and Opton PMAQ 3 equipped with a thermostated cell compartment. Differential melting curves were obtained by graphical differentiation of melting curves and by the previously reported method (15). Fix 2 ml of the DNA solution with O.I SSC as a solvent (O.OI5 M NaCl + O.OOI5 M trisodiuncitrate, pll 7) in the thermostated cell compartment of the spectrophotometer were heated up to the temperature which corresponds to the desired extent of DNA denaturation. On the first stage of fixation 4 Al of glyoxal stock solution was added. After 20 min of reaction the temperature of the solution was 4054

Nucleic Acids Research lowered, the decrement being 35C, and 50/si of glyoxal stock solution was added (the second stage of fixation). After 50 mm of the second stage the solution was cooled and dialised against buffer A. The extent of DNA denaturation was controlled optically during all the stages of fixation. T2 DNA. The GC-content of This method was developed us T2 DNA is equal to 35%, i.e. it is close to the GC-content of low-melting regions of T7 DNA (see below). T2 DNA preparations fixed by the above technique had two step melting curves. The first step of the curve corresponds to the melting of the glyoxal-fixed DNA regions with the melting temperature (T ) of 45 C. The second step corresponds to the melting of the nondisturbed

in T2

regions

Digestion

DNA

(Tm=670C).

DNA samples with SI endonuclease. The digestion was carried out in buffer A at 50°C. The reaction mixture consisted. of O.I ml SI endonuclease and 2 ml of DNA solution (30 ,,g/ml). Such amount of SI endonuclease digest 40 ,g denatured DNA

ixL

20

of

min

as

determined

by following

the

increase

of

the

solution absorption at 260 nm which occurs during the digestion. The reaction was stopped by chilling to 250C and adding of the equal volume of the solution containing 0.I M NaHCO3 and. 0.2 mM EDTA (pH 7.5). The electrophoretic separation of _ g the DNA fragments was carried out in cylindrical gels (0.6 x x I0 cm) by procedure described elsewhere (I6). 0.7 and 0.39S agarose

(Serva)

was

used.

The

gels

were

stained

in

ethidiunm

bromide solution (Iy.g/ml). After 30 min the stained bands were visualized through fluorescence in long-wavelength ultraviolet. Gels were photographed through the red filter. Molecular

weight

determination

of

the

DNA

fra&ments.

a-) -Electrhoresis. The six endonuclease R.IEcoRI-generated fragments of lambda DNA were used as standards for the molecular weight estimations of other DNA species. The molecular weights of lambda fragments are I5.7, 4.y/, 3.7, 3.5, 3.0 and 2.I megadaltons (I6). b)_Analytical centrifugation. Sedimentation was performed at 20°C in Beckman E analytical ultracentrifuge. The molecular 4055

Nucleic Acids Research weights of DNA were evaluated by the equation of Freifelder(I7). c) Electron Microscopy. Samples for electron microscopy were prepared by the modified formamide method (I8). H;yperphase contained DNA and citochrome in 0.03 M phosphate buffer, pH 7 with 50% formamide. The drop of this solution was spreaded on the surface of deionized water. DIA-protein film was picked up by carbon-coated copper grid, rotatory shadowed with Pt and photographed with a JEM 7 electron microscope. The magification was calibrated by use of a grating carbon replica. Molecular weight of DNA was derived using the DNA linear density data (I9). The number of measured fragments was 600. RESULTS The differential melting curve of T7 DNA is shown in Fig.I. .&ach sharp peak on the curve corresponds to melting out of the definite region(s) in DNA (2). The arrow in Fig.I indicates the position of the first peak. The appearance of this peak shows that the phage T7 DNA contains the regions with low thermostability. The GC-content of these low-melting regions is 28±47% as determined from the ratio of the first peak areas at 260 and 280 nm accordingly (20). Thus, their GC-content is 20% lower than the mean GC-content of T7 DNA (48%). Now let us estimate the size and the number of AT-rich regions making use of the parameters of their melting. The melting temperature (Tm) of DNA having of 287% mole of GC pairs in O.I x SSC should be equal 65.I°C (2I-22). This value is 2.90C lower than that of the low-melting sites (see Fig.I). Such a difference in TM's results from the end effects (2). The length of the AT-rich sites is equal to I20-40 base pairs as estimated from Tm data in Appendix I. One more estimation of the length was obtained from the width of the first peak. The value I00+20 of v) was obtained in this case (see Appendix 2). Both estimation of a are in a good agreement. The area under the first peak is equal to about 1S of the total area under differential melting curve, i.e. the low-melting sites comprise about 400 base pairs (T7 DNA contis 38,000 base pairs (2)). Thus, the number of AT-rich sites in T7 DNA range from three to four. 4056

Nucleic Acids Research 0 -4

>4

H

E-4

00 66

68

70

72

74

76

78

TMPBRATURE ( t OC) Fig.I The differenial melting curve of T7 DNA in O.IxSSC. The arrow indicates the first pick which corresponds to the melting out AT-rich sites. We used the following scheme to determine the number of AT-rich sites and to map them along T7 DNA molecule. First of all, the low-nelting sites were fixed in thle denatured state by glyoxal (the procedure is described in Methods). Then such fixed DNA samples were digested with SI endonuclease. The differential melting curve of DNA resulted from the extensive SI-treatment is shown in Fig. 2. This curve is similar to the differential melting curve of the untreated T7 DNA (Fig.I), but the curve in Fig.2 have no the first peak. This peak disappears due to digestion of the low-melting sites with SI endonuclease. The fragments after digestion were analysed by means of agarose gel electrophoresis. Fig.3a shows the electrophoretic separation of these fragments. For comparison an electrophoresis pattern of EcoRI fraGments of lambda DNA is shown in Fig.3b. The bands have approximately the same width in both patterns. The electrophoretic pattern in Fig.3a demonstrates that tbree fragments are resulted from SI-digestion of the fixed AT-rich sites. Their molecular weights determined by different methods are given in Table I. All methods give practically coinciding results. The dispersion of molecular weights of the fragments

determined by the electron microscopy is about 5% in all cases. The same dispersion is obtained for the value of ColEI plasmid 4057

Nucleic Acids Research

0.3

0.1

66

68

70

72

74

76

78

TEMPERATURE (t 0C) Fig. 2 The differential melting curve of a mixture of the T7 DNA fragments. DNA: 4.2+0.2 megadaltons. This fact points to a high homogeneity of the fragments obtained by the proposed method of specific fragmentation. The most accurate molecular weight data of the fragments are ob-tained by electron microscopy. The electrophoresis molecular weight data of fragments A and C coincide with the electron microscopy measurements. The discrepancy between the values of the fragment B molecular weight obtained from the electrophoresis and el.ectron microscopy data is due to rather rough calibration of electrophoresis in this molecular weight range. Thus, the procedure used enables us to detect two AT-rich regions in T7 DNA which divide the molecule into three fragments. In order to locate their positions along the DNA molecule we analysed the DNA intermediates at the early stages of the digestion with SI endonuclease. A photograph of the gel containing the intermediates is shown in Fig.4. It is clearly seen that among intermeadiate products there are the fragments

A+B, B+C, and there is no fragment A+C. It means that the arrangement of the fragments in the T7 DNA molecule is as follows * A B $C The arrows in the figure showrthe position of the low-melting sites. 4058

Nucleic Acids Research

T7 DNA A+B - T7 DNA A

N B+C

- B

B

-- c

C

Fig 3a

Fig. 3b

Fig.4

Fig.3 a) electrophoretic separation of the T7 DNA fragments obtained by digestion the DNA with Si endonuclease at the fixed low melting sites b) electrophoretic separation of EcoRl digested lambda DNA fragments.Upper band corresponds co-electrophoresed T7 DNA and the next band appeared due to renaturation lambda DNA end fragments(13.7 and 2.19 megadaltons) at the cohesive ends of the whole DNA. One microgram of DNA was electrophoresed in 0.7% agarose gels for 7 hr at 2 V/cm. Fig.4

Electrophoretic separation in 0.3% agarose gels of DINA fragments obtained by incomplecte digestion of T7 DNA with Si endonuclease at the fixed low melting sites,

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Nucleic Acids Research Table- I Molecular weights of the DNA fragments obtained after SI-digestion of T7 DNA at fixed low-melting sites (in

megadaltons) F r a g m e n t a

Method A

B

C

Electron

microscopy Electrophoresis

I3.6-+0.4 I3.7+I

Sedimentation

I4. 2+2

8.2+0.35

3.5+0.16

7.55+0. 7

3.7+0.3

+I 5 7±I

DISCUSSION We detected two AT-rich regions in T7 DNA molecule. The estimations given above show that their number is ranged from three to four. It is possible that T7 DNA contains one or two AT-rich sites in addition to two sites discovered. If the additional sites were located near the ends of the molecule or very close to another sites, short fragments should be formed after SI-digestion and could not be detected by the techniques used. It should be emphasized that the resulting fragments are comparatively homogeneously distributed over their length. Indeed the lengths in a fraction cannot differ more than 400 base pairs because the size of AT-rich site is about 200 base pairs. Thus, our specific fragmentation method permits to map the low-melting sites in DNA and can serve as a method of obtaining comparatively homogeneous fragments of DNA, so it can be applied for the DNA fragmentation in addition to DNA cleavage by restriction endonucleases.

ACKNOWLEDG

TS

We thank Dr. E.D.Sverdlov for advise to use glyoxal as a fixation agent and Dr. M.D.Frank-Kamenetskii for useful discussi-ons.

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Nucleic Acids Research APPENDIX I For the estimation of the length ) of a meltin in DNA we used the following equation (23): PS 0

region

-I

TEO 0

where,

To

- melting temperature of this region in

OK

of infinite DNA chain with the melting temperature T'3°o 0 same GC-content in K Fs5 stacking energy ff- melting enthalpy. K, T~~~0=34I Kg In our experimental conditions 0 = these values Putting (2). 7 8 keal/mol Fs= kealVmol, &H into (I.I), we obtain VJ=I20+40 base pairs.

Too=338+I.5

APPENDIX 2 The second estimation of ig forla (23): 4R T 0 &T &H'

)

was made by using the follow-

(2.I)

where, &T - the width of the peak R - gas constaat To- melting temperature of the region. Putting in (2.I) aT=I0C, To=34IK, we obtain ) = I00 base pairs and taking into account the uncertainty in A T determination ( T=I+0.2°C) -)=IOO+20 base pairs.

REFERENCES I

2

Gomez,B. and Lang,D. (1972) J.Mol.Biol. 70, 239-251 Ioyubchenko,Yu.L., Frank-Kamenetskii,M.D., Vologodskii,A.V., Lazurkin,Yu.S. and Gause,G.G.,Jr. (1976) Biopolymers 15,

1019-1036 3 Ansevin,A.T., Vizard,D.L., Brown,B.W. and McConthy,J. (1976) Biopolymers 15, 153-174 4 Yabuki,S., Gotoh,0. and Wada,A. (1975) Biochim.Biophys.Acta 395, 258-273 4061

Nucleic Acids Research 5 6

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I3 I4 I5 I6 I7 IS I9 20

2I 22

23

4062

VVada,A. Tachibana,H., Takanamii,M. and Gotoh,O. (1976) Nature 263, 439-440 Gotoh, 0., Husumi,Y., Yabuki,S. and Wada,A. (1976) Biopolymers 15, 655-670 Vizard,D.L. and Ansevin,A.T. (1976) Biochemistry 15, 741-750 Broude,N.E. and Budowsky,E.I. (1971) Biochim.Biophys.Acta 254, 380-388 Broude,N.E. and Budowsky,E.I. (1973) Biochim.Biopbys.Acta 294, 378-384 Ando,T. (1966) Biochim.Biopbys.Acta 114, 158-168 M1assie,IH.R. and Zimm,B.H. (1965) Proc.Nat.Acad.Sci. USA 54, 1641-1643 Lowry,0.H.., Rosenbrough,N.J., Fore,A.L. and Randal,R.J. (1951) J.Biol.Chem. 193, 265-275 Hagen,U. and Coquerelle,T. (1974) Biochim.Biopbys.Acta 374, 271-282

Vogt,V.M. (1973) Eur.J.Biochem. 33, 192-200 Pavlov,V.M. and Iyubehenko,Yu.L. (1977) Biopolymers, in press

Eelling,R.B., Goodman,H.M. and Boyer,H.W. (1974) J. of Virology 14, 1235-1244 Freifelder,D., Crothers,D.M. and Zimm,,B.H. (1970) J.Mol. Biol. 54, 567-577 Davis,R.W., Simon,M. and Davidson,N. (1971) in Method in 1nyumo1ogy,, vol.21,iD, pp.413-428, Academic press,New York Lang,D. (1970) J.Mlol.Biol. 54, 557-565 Felsenfeld,G. and Hirschman,S.Z. (1965) J.Mol.Biol. 13, 407-427 Gruenwedel,D.W., Hsu,C.H. and Lu,D.S. (1971) Biopolymers 10, 47-68 LazurkinYu.S., fLyubehenko,Yu.L., Pavlov,V.M., Frank-Kamenetskii,M.D. and Berestetskaja,I. (1975) Biopolymers 14, 1551-1552 I1yubchenko,Yu. L., Trifonov,,E.N., lazurkin,Yu.S., FrankIK.,amenetskii,,M.D. (1972) Mol.Biol. (in Russian) 2, 772-774

Specific fragmentation of T7 phage DNA at low-melting sites.

1977 Volume 4 Number Number 1111 November November 1977 Volume 4 Nucleic Acids Research Nucleic Acids Research Specific fragmentation of T7 phage...
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