J. gen. Virol. (1967), 1, 387-390
387
Printed in Great Britain
The Supercoiling of Papilloma Virus D N A
(Accepted 20 March I967)
DNA from viruses of the papilloma group is circular and supercoiled with a molecular weight of 5"3 million daltons (i). At present little is known of the mechanism by
which supercoiling arises or of the biological functions which may be performed by
this configuration. To guide speculation on these points it would be desirable to know
the sense of the supercoiling turns, whether right- or left-handed, and the number of
such turns per molecule of DNA. In the case of polyoma virus DNA, a smaller supercoiled molecule, the interaction of ethidium bromide with the DNA has been used to
investigate the twisting of the molecule (z). This communication presents the results
of a similar study with DNA from Shope rabbit papilloma virus and human papilloma
virus.
Supercoiling appears to result from the number of turns in the Watson-Crick
double helix of these circular DNA molecules being either greater or less than the
number found in linear DNA of the same size. Since each of the two strands of the
DNA is continuous the number of turns in the molecule is fixed: thus the excess or
deficiency is compensated by twisting of the molecule as a whole. It is possible to
distinguish between excess and deficiency of turns in the basic double helix by observing the effect on supercoiling of gradually untwisting this helix. This may be done
by several different methods and the one used here is the intercalation of the drug
ethidium bromide into the DNA. The molecules of this drug are inserted into the
DNA between the adjacent nucleotide pairs. The nucleotide pairs are thus moved
apart and the amount of rotation between them reduced. In the B form of DNA the
rotation between successive nucleotide pairs is 360 (3). Intercalation of ethidium reduces
this value to 24°, an untwisting of I2°(4), although much higher estimates of the
amount of untwisting due to intercalation of acridines have been made (5). If supercoiling results from a deficiency of turns in the basic double helix of papilloma virus
DNA insertion of increasing amounts of ethidium into the DNA should at some stage
result in the loss of supercoiling. At this point the amount of untwisting due to insertion of the drug would be equal to the deficiency of turns in the original molecule. Had
the supercoiling been in the opposite sense, due to an excess of turns, insertion of drug
would not result in the loss of supercoiling but in progressively tighter supercoiling.
Shope rabbit papilloma virus and human papilloma virus were extracted and purified as described previously and the DNA extracted from the virus suspensions by
treatment with sodium dodecyl sulphate (6). The preparations used here contained the
following amounts of component I (supercoiled molecules) and component II (open
circular molecules with a single strand scission): Shope papilloma virus DNA: 7o %
component I, 3o% component II: human papilloma virus DNA: 75 % component I,
25 % component II. DNA samples were suspended in 2-amino-2-hydroxymethylpropane-x,3-diol (tris) buffer (o'o5 M, pH 8-o) at a concentration of 3° to 5o/zg./ml.
Solutions of ethidium bromide were made up in the same buffer and kept in the dark.
Mixtures of drug+DNA were kept in the dark at room temperature for io rain.
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388
before centrifugation. Samples were e x a m i n e d b y analytical centrifugation using a
S p i n c o m o d e l E ultracentrifuge a n d the f o l l o w i n g c o n d i t i o n s : I2 m m . cell with 4 ° K e l - F
centrepiece, s a m p l e v o l u m e o f 0"5 ml., centrifuged a t 31,4Io rev./min. 20 °. T w o cells
were r u n simultaneously, one being m a d e u p with a side wedge window, a n d u l t r a v i o l e t
a d s o r p t i o n p h o t o g r a p h s t a k e n o f the cells alternately at 8 min. intervals. T h e p h o t o g r a p h s were scanned with a Joyce L o e b l m i c r o d e n s i t o m e t e r to d e t e r m i n e the n u m b e r
a n d p o s i t i o n o f the boundaries.
181
0
I
0.05
I
I
0-1
0
0.05
Drug molecules bound per nucleotide
I
0.10
).15
Fig. I. The effect of ethidium bromide on the sedimentation behaviour of papilloma virus
DNA. Samples of DNA were mixed with increasing amounts of ethidium bromide and
then analysed by boundary sedimentation. - - O - - , Fast component (supercoiled molecules); - - / ~ - - , slow component (open ring molecules); - - • - - , single boundary. (a) Result
with Shope papilloma virus DNA, (b) result with human papilloma virus DNA.
Ca)
C~
(~
(~
(e)
Fig. 2. Diagrammatic representation of the removal and reversal of supercoiling turns. The
Watson-Crick helix is here represented as a single continuous line. The number of supercoiling turns in the original molecule (a) decreases as drug molecules (represented by bars
perpendicular to the helix axis) bind to the DNA (b). The drug molecules are inserted at
random. At equivalence the accumulated untwisting due to the number of drug molecules
bound just balances the initial number of supercoiling turns (c). The untwisting caused by
the binding of further drug molecules leads to the introduction of supercoiling turns now
in the opposite, left-handed, sense (d, e). This figure is taken from (2) by permission of
Academic Press.
T h e effect o f increasing a m o u n t s o f e t h i d i u m o n the s e d i m e n t a t i o n o f S h o p e
p a p i l l o m a virus D N A is s h o w n in Fig. I a. T h e fast c o m p o n e n t c o m p r i s e s the supercoiled f o r m o f the D N A a n d the slow c o m p o n e n t the o p e n circular molecules. A t l o w
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389
concentrations of drug two boundaries were observed, corresponding to the two
components. At a ratio of 0"037 drug molecules per nucleotide (I #g. ethidium bromide
per 21 #g. DNA) only one boundary was observed. Apparently the supercoiled molecules had been converted to a form sedimenting with the open circular molecules.
Addition of further drug caused the reappearance of the fast component, corresponding
to molecules now supercoiled in the opposite sense. The sequence of changes postulated
to explain these results is shown diagrammatically in Fig. z.
One intriguing point arises from consideration of a possible connexion between the
supercoiling of the DNA and the structure of the virus particle. Shope papilloma virus
and human papilloma virus particles probably both correspond to a T = 7 structure
with 72 capsomeres (7, 8). Since this is a skew structure left- and right-handed forms
exist and Shope papilloma virus appears to correspond to the left-handed version
while human papilloma virus corresponds to the right-handed version (7, 8). If there
were a connexion between the supercoiling of the DNA and the structure of the virus
particle it might be predicted that human papilloma virus DNA would be supercoiled
in the opposite sense to Shope papilloma virus DNA. The results presented in Fig. I b
show that this is not the case. The sense of the supercoiling in the two papilloma virus
DNAs is the same.
If it is accepted that these experiments prove that the supercoiling of papilloma virus
DNA is in the sense due to deficiency of turns in the basic double helix, we may calculate the number of turns deficient and thus the number of supercoiling turns in the
original molecule. This calculation depends upon knowing the number of drug molecules bound to the DNA at equivalence, i.e. the point at which the fast and slow components sediment together. Experience with polyoma virus DNA had shown that
binding of ethidium to supercoiled DNA was very nearly complete at levels in the
region of equivalence and that at higher drug: DNA ratios the binding curve was
similar to that seen with linear DNA (2). Since there is no reason to believe that
papilloma virus DNAs differ from polyoma virus DNA in respect of their interaction
with ethidium bromide, the binding levels shown in Fig. I were calculated from the
measurements with polyoma virus DNA. Thus, taking the molecular weight of
papilloma virus DNA to be 5"3 million daltons, the ratio of bound drug to DNA at
equivalence represents approximately 6oo molecules of drug per molecule of DNA.
If each molecule of drug inserted causes the untwisting of 12° then the insertion of
6oo molecules causes the untwisting of about 2o turns. This then is equal to the
number of supercoiling turns in the untreated molecule of papilloma virus DNA.
We are indebted to the Wellcome Foundation for the analytical ultracentrifuge and
microdensitometer used in this work, to Dr A. Klug for his comments on supercoiling
and virus structure, and to Mrs M. Scott for her expert technical assistance.
Institute of Virology
Church Street, Glasgow
Scotland
Subdepartment of Chemical Microbiology
Department of Biochemistry
University of Cambridge
L.V. CRAWrORD*
M . J . WARING
* Member of the Medical Research Council ExperimentalVirus Research Unit.
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390
Short communications
REFERENCES
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(Received I4 February 1967)
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