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Biochimica et Biophysica Acta, 4 7 7 ( 1 9 7 7 ) 1 3 2 - - 1 4 3 © Elsevier/North-Holland
Biomedical Press
BBA 98959
P E C U L I A R I T E S OF THE S E C O N D A R Y S T R U C T U R E OF B A C T E R I O P H A G E DNA IN SITU VII. R E L E A S E OF DNA • P R O T E I N COMPLEX FROM S d PHAGE *
T.I. TIKCHONENKO,
E.N. DOBROV,
N.A. OGAREVA
and A.A. MANIKIN
Laboratory o f Bioorganic Chemistry, Lomonosov State University, and Institute of Virology, U.S.S.R. Academy o f Medical Sciences, Moscow (U.S.S.R.) (Received November 26th, 1976)
Summary In an a t t e m p t to elucidate the role of different factors in determining the specific secondary structure of DNA in particles of S~ phage, the complex of released Sd DNA with phage proteins was obtained and some properties of D N A in this complex were studied. The complex was prepared by heating the Sd phage preparations at 52°C in a low ionic strength medium (0.01 X SSC). By means of viscosimetry, gradient centrifugation and electron microscopy it was shown that the Sd complex consists of a released DNA molecule and a phage protein coat attached to it at one or several internal points. DNA in this complex loses the specific ultraviolet light absorption and circular dichroism spectral characteristics typical of intraphage S~ DNA and does not differ in these parameters from free Sd DNA in solution. But the melting temperature (Tin) of the complexed DNA in low (0.01 X SSC) and high (1 X SSC) ionic strength solutions with 1.5 per cent formaldehyde was significantly higher than the Tm of free Sd DNA in the same medium. Besides, the melting curves o f the DNA in the complex showed a partial loss of cooperativity resemble the melting behaviour of intraphage Sd DNA of the transition. Both the increased Tm and the loss of melting cooperativity in the presence of formaldehyde. On the basis of the data obtained it is supposed that the different specific properties of the intraphage Sd DNA are determined by different c o m p o n e n t s of the phage particle.
* Paper VI of this series a p p e a r e d in Arch. Biochem. Biophys. (1976) 175, 1--10. Abbreviations: SSC, standard saline-citrate solution 0.15 M NaCI/0.O15 M s o d i u m tricitrate, pH 7. HCHO, formaldehyde; SDS0 s o d i u m dodecyl sulfate; CD, circular diehroism.
133 Introduction In the previous communications of this series [1--5], the results of an investigation of intraphage Sd DNA structure by means of optical methods and chemical modification were presented. It was found that Sd DNA in situ differs from free Sd DNA in a number of properties: absorption and CDspectra, reactivity to formaldehyde and O-methylhydroxylamine and so on. But it remains u n k n o w n whether all these peculiarities of the intraphage DNA are caused by a single factor or different specific properties of intraphage DNA have, so to speak, different "substrates". One of the possible ways of solving this problem lies in the study of the changes in DNA properties during intermediate stages of its transition from the intraphage to the free state. To this end, we have developed a m e t h o d for obtaining a Sd DNA • protein complex, similar to the complexes described previously for T1 [6], T7 [7] and X [8--10] phages. Although the S d complex differs from these complexes in some important features, we had no intention to conduct a comparative study. Our main object was to compare some properties of Sd DNA in such a complex to the properties of free and intraphage Sd DNA. Materials and Methods
1. Preparation o f phage Sd and its DNA. Sd phage was grown on Escherichia coli SK. The methods of preparation and purification of unlabelled and asSlabelled Sd phage were descibred previously [4,11]. The preprations were additionally purified b y Biogel P-300 chromatogrpahy and centrifugation on CsC1 cushions [ 12]. Sd DNA was prepared by phenol deproteinisation [ 13]. 2. Phage suspensions in a low ionic strength medium. Throughout this work phage Sd preparations in 1 × SSC and 0.01 × SSC solutions were used. This phage suspensions in 0.01 X SSC were obtained by centrifugation at 2 0 0 0 0 g for 40 min, resuspension in 0.01 × SSC and 24 h dialysis against a large volume of the same solution. Solutions of low ionic strength were prepared with deionised water obtained on a "Millipore" apparatus. 3. Preparation o f formaldehyde. Formaldehyde solutions were prepared by distillation of dry paraformaldehyde into deionised water with the use of a specially constructed device. Formaldehyde solutions were neutralised with a small a m o u n t of dilute NaOH prepared in deionised water. Concentration of formaldehyde was determined as described by Walker [ 14]. 4. Viscosity measurements. The temperature dependence of specific viscosity of the Sd preparations was determined in a spiral capillar viscosimeter with gradient values (~) o f 70--100 s-'. Phage suspensions containing 35--45 #g of DNA per ml were used. The suspensions were heated for 30 min at different temperatures and their viscosity was measured at 30 ° C. 5. Determination of DNA melting curves in phage suspensions. Melting curves o f DNA in phage suspensions were determined as described previously [15]. True values of absorption of light-scattering suspensions were estimated b y the standard extrapolation m e t h o d [1]. 6. CD spectra in the 230--300 nm region were measured in a Roussel~louan II Dichrographe at r o o m temperature using 1 cm cells. Ae values were calcu-
134
lated for DNA in suspensions. DNA concentration was determined from the phosphorus c o n t e n t [16] or using extinction coefficients of 0.020 absorbance units at 260 nm for 1 pg/ml of free DNA and 0.023 absorbance units for intraphage DNA. 7. Centrifugation in the Cs2S04 density gradients was performed in a 3 X 5 swing-out rotor of the MSE-50 centrifuge at 3 5 0 0 0 rev./min for 14 h at 5°C. The preformed linear Cs2SO4 gradients were used. The a m o u n t of DNA was 100 #g per tube. The 10--12
Biochimica et Biophysica Acta, 4 7 7 ( 1 9 7 7 ) 1 3 2 - - 1 4 3 © Elsevier/North-Holland
Biomedical Press
BBA 98959
P E C U L I A R I T E S OF THE S E C O N D A R Y S T R U C T U R E OF B A C T E R I O P H A G E DNA IN SITU VII. R E L E A S E OF DNA • P R O T E I N COMPLEX FROM S d PHAGE *
T.I. TIKCHONENKO,
E.N. DOBROV,
N.A. OGAREVA
and A.A. MANIKIN
Laboratory o f Bioorganic Chemistry, Lomonosov State University, and Institute of Virology, U.S.S.R. Academy o f Medical Sciences, Moscow (U.S.S.R.) (Received November 26th, 1976)
Summary In an a t t e m p t to elucidate the role of different factors in determining the specific secondary structure of DNA in particles of S~ phage, the complex of released Sd DNA with phage proteins was obtained and some properties of D N A in this complex were studied. The complex was prepared by heating the Sd phage preparations at 52°C in a low ionic strength medium (0.01 X SSC). By means of viscosimetry, gradient centrifugation and electron microscopy it was shown that the Sd complex consists of a released DNA molecule and a phage protein coat attached to it at one or several internal points. DNA in this complex loses the specific ultraviolet light absorption and circular dichroism spectral characteristics typical of intraphage S~ DNA and does not differ in these parameters from free Sd DNA in solution. But the melting temperature (Tin) of the complexed DNA in low (0.01 X SSC) and high (1 X SSC) ionic strength solutions with 1.5 per cent formaldehyde was significantly higher than the Tm of free Sd DNA in the same medium. Besides, the melting curves o f the DNA in the complex showed a partial loss of cooperativity resemble the melting behaviour of intraphage Sd DNA of the transition. Both the increased Tm and the loss of melting cooperativity in the presence of formaldehyde. On the basis of the data obtained it is supposed that the different specific properties of the intraphage Sd DNA are determined by different c o m p o n e n t s of the phage particle.
* Paper VI of this series a p p e a r e d in Arch. Biochem. Biophys. (1976) 175, 1--10. Abbreviations: SSC, standard saline-citrate solution 0.15 M NaCI/0.O15 M s o d i u m tricitrate, pH 7. HCHO, formaldehyde; SDS0 s o d i u m dodecyl sulfate; CD, circular diehroism.
133 Introduction In the previous communications of this series [1--5], the results of an investigation of intraphage Sd DNA structure by means of optical methods and chemical modification were presented. It was found that Sd DNA in situ differs from free Sd DNA in a number of properties: absorption and CDspectra, reactivity to formaldehyde and O-methylhydroxylamine and so on. But it remains u n k n o w n whether all these peculiarities of the intraphage DNA are caused by a single factor or different specific properties of intraphage DNA have, so to speak, different "substrates". One of the possible ways of solving this problem lies in the study of the changes in DNA properties during intermediate stages of its transition from the intraphage to the free state. To this end, we have developed a m e t h o d for obtaining a Sd DNA • protein complex, similar to the complexes described previously for T1 [6], T7 [7] and X [8--10] phages. Although the S d complex differs from these complexes in some important features, we had no intention to conduct a comparative study. Our main object was to compare some properties of Sd DNA in such a complex to the properties of free and intraphage Sd DNA. Materials and Methods
1. Preparation o f phage Sd and its DNA. Sd phage was grown on Escherichia coli SK. The methods of preparation and purification of unlabelled and asSlabelled Sd phage were descibred previously [4,11]. The preprations were additionally purified b y Biogel P-300 chromatogrpahy and centrifugation on CsC1 cushions [ 12]. Sd DNA was prepared by phenol deproteinisation [ 13]. 2. Phage suspensions in a low ionic strength medium. Throughout this work phage Sd preparations in 1 × SSC and 0.01 × SSC solutions were used. This phage suspensions in 0.01 X SSC were obtained by centrifugation at 2 0 0 0 0 g for 40 min, resuspension in 0.01 × SSC and 24 h dialysis against a large volume of the same solution. Solutions of low ionic strength were prepared with deionised water obtained on a "Millipore" apparatus. 3. Preparation o f formaldehyde. Formaldehyde solutions were prepared by distillation of dry paraformaldehyde into deionised water with the use of a specially constructed device. Formaldehyde solutions were neutralised with a small a m o u n t of dilute NaOH prepared in deionised water. Concentration of formaldehyde was determined as described by Walker [ 14]. 4. Viscosity measurements. The temperature dependence of specific viscosity of the Sd preparations was determined in a spiral capillar viscosimeter with gradient values (~) o f 70--100 s-'. Phage suspensions containing 35--45 #g of DNA per ml were used. The suspensions were heated for 30 min at different temperatures and their viscosity was measured at 30 ° C. 5. Determination of DNA melting curves in phage suspensions. Melting curves o f DNA in phage suspensions were determined as described previously [15]. True values of absorption of light-scattering suspensions were estimated b y the standard extrapolation m e t h o d [1]. 6. CD spectra in the 230--300 nm region were measured in a Roussel~louan II Dichrographe at r o o m temperature using 1 cm cells. Ae values were calcu-
134
lated for DNA in suspensions. DNA concentration was determined from the phosphorus c o n t e n t [16] or using extinction coefficients of 0.020 absorbance units at 260 nm for 1 pg/ml of free DNA and 0.023 absorbance units for intraphage DNA. 7. Centrifugation in the Cs2S04 density gradients was performed in a 3 X 5 swing-out rotor of the MSE-50 centrifuge at 3 5 0 0 0 rev./min for 14 h at 5°C. The preformed linear Cs2SO4 gradients were used. The a m o u n t of DNA was 100 #g per tube. The 10--12
