Download DNA conformation and restriction enzyme activity

348
BIOCHEMICAL SOCIETY TRANSACTIONS
The heterogeneity of DNA polymerase-a from mouse embryos and embryonal carcinoma cells
DUNCAN R. CLARK and
ALEXANDER G. McLENNAN
Department of Biochemistry, University of Liverpool, P.O. Box
147, Liverpool L69 JBX, U.K.
When chromatographed on anion-exchange resins, mammalian
DNA polymerase-a generally separates into at least three
species (Holmes et al., 1974; Lamothe et al., 1981). The
differences between these forms could be due to proteolysis
during isolation and/or to the differential association of subunits
of the presumed native holoenzyme with the polymerase-active
moiety. We have studied the heterogeneity of this enzyme in
whole 8- 17-day mouse embryos and teratocarcinomas induced
in strain- 129 mice by injection of PC 13 embryonal carcinoma
cells. These tumours consisted primarily of undifferentiated
embryonal carcinoma stem cells, and so differed from those
produced by the original isolate (Hooper & Slack, 1977).
To limit proteolysis of the D N A polymerases during isolation,
extractions were performed in the presence of 1OOpg of
soya-bean trypsin inhibitor/ml, 1mM-di-isopropyl fluorophosphate (freshly prepared) and 1 mM-EDTA, conditions
which inhibited all detectable proteinase activity. Selective
inhibitor studies showed that the detectable activity was due to
serine and metalloproteinases. These combined proteinase
activities were 10-fold higher in extracts of tumours than of
embryos. Despite lysosomal disruption during extraction, no
thiol proteinase activity could be measured.
Extracts of embryos and tumours were made by homogenization in 3 vol. (v/w) of 0.2 M-potassium phosphate, p H 7.4,
containing 5 m~-2-mercaptoethanol, 10% (v/v) glycerol and
inhibitors, followed by centrifugation at lOOOOOg for 1h.
Extracts were freed of D N A polymerase-fi and nucleic acids by
chromatography on DEAE-cellulose and then analysed on
5-20% (w/v) sucrose density gradients in 10% (v/v) glycerol
and by gradient sievorptive elution chromatography on DEAESephadex A-25 (Kirkegaard, 1973).
D N A polymerase-a activity from both tumours and embryos
of all gestational ages sedimented at 9.2s in O.~M-KCI.When
chromatographed on DEAE-Sephadex, the embryo enzyme
separated into two activities, a,E, eluted between 70 and
100mM-KC1, and a,E, between 130 and 1 6 0 m ~ - K C l When
.
similarly chromatographed, the tumour enzyme also yielded two
species, a,T, eluted between 70 and 100mM-KC1, and a,T,
between 200 and 2 3 0 m ~ - K C I .In terms of their sensitivities-to
aphidicolin, N-ethylmaleimide and dideoxy-TTP, all four activities appeared similar and were thus classified as a-polymerases. However, when their relative activities with activated
and heatdenatured D N A were compared, differences were
noted. DNA polymerase-a,T showed a high (20-fold) preference
for activated DNA, whereas polymerases a,E and a,T showed
only a 4-fold preference and polymerase a,E a 2-fold preference
for activated DNA. Thus D N A polymerase a,E showed a
marked ability to use a denatured D N A template.
These results are similar to those of Lamothe et al. (198 I),
who separated three activities, D N A polymerases a,, a, and a,,
from HeLa cells on DEAE-Bio-Gel. These enzymes show
similar preferences for activated and denatured D N A to those of
our a,E/a,T, a,E and a,T respectively. Those authors showed
that polymerase a2contains two accessory subunits, C 1 and C2,
which confer on it the ability to utilize single-stranded DNA.
Thus it appears that mouse embryos, which contain many
differentiated cell types, and the undifferentiated embryonal
carcinoma cells may express different components of the
a-polymerase pool.
A further difference was noted between the embryo and the
tumour enzymes. When the sedimentation coefficients of the
chromatographically separated enzymes were determined, a,T
and a,T still sedimented at 9.2S, but a,E and a,E sedimented at
6.7s. Furthermore, when the gradient profile of polymerase a2E
was assayed with denatured DNA, the 6.7s activity was found
to have lost its ability to use this template. Thus the embryo
enzymes must differ from the tumour enzymes in some way that
allows separation of certain subunits to occur after gradient
sievorptive chromatography and density-gradient centrifugation in 0 . 4 ~ - K C l . The relationship between the 6.7s
enzyme generated from polymerase a,E, the 9.2 S polymerase
a,T and the 7 S a3 enzyme of Lamothe et al. ( I 98 l), all of which
are unable to utilise denatured D N A efficiently as a template, is
unclear.
Whether the differences between the tumour and embryo
enzymes reflect a real difference between undifferentiated and
differentiated cells and their abilities to modify the structure of
DNA polymerase-a, or whether the propensity of the embryo
enzymes to dissociate is caused by undetected proteolysis or
some other modification in vitro, remains to be determined.
However these two related cell systems should prove useful in
the evaluation of the factors responsible for the experimentally
observed heterogeneity of D N A polymerase-a.
This work was supported by Grant No. G9771799lC from the
Medical Research Council to A. G. McL.
Holmes, A. M., Hesslewood, I. P. & Johnston, I. R. (1974) Eur. J .
Biochem. 43, 487-499
Hooper, M. L. Slack, c. (1977) Dev. Biol. 55, 271-284
Kirkegaard, L. H. (1973) ~
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Lamothe, P., Bad, B., Chi, A., Lee, L. & Baril, E. (1981) Proc. Natl.
Acad. Sci.U.SA. 78,4723-4727
DNA conformation and restriction enzyme activity
GEORGES SNOUNOU and ALAN D. B. MALCOLM
Department of Biochemistry, St. Mary’s Hospital Medical
School, Paddington, London W2 IPG, U.K.
Although the major use of restriction enzymes is in the analysis
and manipulation of DNA, they are also excellent models for
DNA-protein interactions (Halford et al., 1979).
Our present knowledge of the static structure of D N A is
derived mainly from X-ray studies on well-defined synthetic
D N A polymers. It is generally accepted that in aqueous
solution, at low ionic strength, D N A adopts the B-conformation. However, such a state can be altered by modifying
the physical environment or by the addition of certain proteins.
It is clear that D N A and/or protein conformational changes
in DNA-protein interactions are not rare events and may
possibly be required for maximal emciency. In a restriction
enzyme-DNA system the specificity of the reaction allows the
effects of D N A conformation on the reaction to be determined
precisely. Such changes can be effected by a variety of
means :ionic strength of the solution, temperature, the use of
topoisomerases, binding of non-catalytic proteins or of DNAspecific ligands. An increasing number of such ligands are being
studied and their detailed effect on D N A conformation
characterized; these include such antibiotics as quinomycin (Lee
& Waring, 1978) and intercalating molecules as ethidium
bromide (Wang, 1974).
1982
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600th MEETING. OXFORD
349
With JW 102 at 0.25 pM-netropsin, 35% inhibition was
The restriction enzyme EcoRI was reacted with linear and
supercoiled plasmid DNA in the presence and absence of observed with the supercoiled form and 24% with the linear one.
The rate differences seen in both plasmids, in the absence of
antibiotic, the effect of which was ascertained by measuring the
antibiotic, cannot be accounted for by the 15% difference in size
rate of EcoRI cleavage.
EcoRI was purified as described previously (Woodhead & of the two DNA species. These differences might be due either
Malcolm, 1980). The plasmids used were pMB9 and JW102. to different superhelical densities in the two plasmids or to the
JW102 is made by inserting a b-globin cDNA in the single adjacent sequences, around the EcoRI site, which are different in
EcoRI site of pMB9 by poly(dA-dT) tailing. Only the single pMB9 and JW 102.
EcoRI site of the b-globin insert is present in JW 102.
The observed differential inhibition between the linear and
Linear plasmids were generated by HsuI digestion followed supercoiled forms of a given plasmid gives rise to two
by phenol extraction and ethanol precipitation.
conclusions.
Assays were performed in a 125pl volume at 37'C, in
(i) The sequences surrounding the EcoRI site are important,
lOOmM-Tris/HCI (PH 7.3)/50m~-NaC1/5mM-MgCI,. Since the since pMB9 is inhibited more by the AT-specific netropsin than
enzyme added was in storage buffer negligible concentrations of is JW 102.
other components such as glycerol were also added (Woodhead
(ii) The above effect is more pronounced in the superhelical
forms than in the linear ones. This is presumably due to changes
et al., 198 1).
The reaction was stopped by the addition of 0.33 vol. of 40% in the binding affinity or mechanism of the antibiotic at two
(w/v) sucrose/0.2% Bromophenol Blue/0.4% sodium dodecyl different conformational states of the D N A species.
sulphate and cooling in ice. Each reaction mixture contained
One can therefore conclude that, although the rate of
3 nM-DNA and was digested with 0.5 units of EcoRI.
digestion of pMB9 and JW 102 by EcoRI does not appear to be
The samples were electrophoresed through a 1% agarose gel affected by the superhelicity of the DNA, the surrounding
made up with electrophoresis buffer ( 4 0 m ~ - T r i s / 2 0 m ~ - s o d i u msequences of the EcoRI site seem to be important in the kinetics
acetate/2m~-sodium EDTA, adjusted to pH 7.7 with acetic of the cleavage. The observed dependence of netropsin inhiacid). Electrophoresis was carried out on 20cm x 20cm hori- bition on superhelicity shows that a conformational change in
zontal slabs a 1.25V.cm-' for 15h. The gel was stained in the DNA can affect its reactive and binding properties.
electrophoresis buffer containing 0.5 pg of ethidium bromide/ml
for 30min and photographed under U.V. light. The negatives
We thank J. L. Woodhead, J. R. Moffatt and P. Stambrook for help,
were scanned and the peak heights measured. The latter were
processed to give the rates of appearance and disappearance of advice and encouragement.
the various bands in pmol min-'.
In the absence of any antibiotic, there is little difference
between the rates of digestion of linear and supercoiled plasmids; Halford, S. E., Johnson, N. P. & Grinsted, J. (1979) Biochem. J. 179,
353-365
however the rate of digestion of JW102 is approx. 3.5 times
Lee, J. S . & Waring, M. J. (1978)Biochern.J. 173, 115-128
slower than that of pMB9.
Wang, J. C. (1974)J. Mol. Biol. 89,783-801
In the case of supercoiled pMB9, the digestion rate was Woodhead, J. L. & Malcolm, A. D. B. (1980) Nucleic Acids. Res. 8,
inhibited by 53% by 0.25p~-netropsin,whereas with the linear
389-402
plasmid a 30% inhibition was observed at the same netropsin Woodhead, J. L., Bhave, N. & Malcolm, A. D. B. (1981) Eur. J .
Biochem. 115,293-296
concentration.
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Interaction of core histones with DNA
BEATRICE M. DIAZ and IAN 0. WALKER
Department of Biochemistry, University of Oxford, South Parks
Road, Oxford OX1 JQU, U.K.
The tryptic digestion of core chromatin (DNA bound to the core
histone H2A, H2B, H3 and H4) in low salt produces five large
limit peptides with apparent molecular weights in the range
7000- 10000 on sodium dodecyl sulphate/polyacrylamide gels.
Amino acid analysis of these peptides shows that 35-40 amino
acids from the basic N-terminal tails of each histone have been
cleaved from the rest of the histone molecule. The N-terminal
fragments have an average size of six amino acids, as measured
by gel-exclusion chromatography. Both the large and small
peptide fragments remain bound to the DNA after trypsin
digestion.
After trypsin digestion of core chromatin in low salt, the c.d.
spectrum in the near-u.v. region (260-300nm) increased to a
value slightly less than that observed for DNA, whereas the
spectrum in the range 260-200nm remained unchanged. These
spectral changes imply that trypsin digestion produces a change
in the conformation of the DNA, possibly a relaxing of the
DNA supercoil around the core histone, whereas there is no
change in the secondary structure of the histone.
However, when trypsin-digested core chromatin is made 2 M
in NaCI, conditions that dissociate the histone fragments from
VOl. 10
the DNA, there is a decrease in the c.d. spectrum in the range
260-200nm, which suggests that the histone peptides lose 50%
of their structure on dissociation. Dissociation of intact histones
from DNA in 2M-NaCI produces no change in structure. This
shows that the DNA maintains the secondary structure of the
histone fragments in the complex. Since the peptide secondary
structure of the core histones is predominantly a-helix
(Beaudette et al., 1981), DNA must interact, either directly or
indirectly, with a-helical peptide regions.
Trypsin digestion of core chromatin produces a dramatic
change in the 'H n.m.r. spectrum of the complex. The spectrum
of the native complex shows a broad, rather featureless, band in
the range 0-4p.p.m., and no aromatic resonances are observed
in the range 6-l0p.p.m. On trypsin digestion the aliphatic
region (0-4 p.p.m.) shows very sharp resonances characteristic
of a denatured protein. There is no evidence of the ringcurrent-shifted methyl resonances at < 0.9 p.p.m. which are
characteristic of tertiary structure formation in histones (Moss
et al., 1976). Furthermore, aromatic resonances of tyrosine and
phenylalanine become visible. These two observations, taken
together, suggest that the tertiary structure of the core histones
is largely disrupted on trypsin digestion.
The sedimentation coefficient of core chromatin decreases
from 2 1 s to 1 6 s on trypsin digestion. Since no peptides
dissociate from the complex on digestion, this may be