Download Role of Deoxyribonucleic Acid Polymerase beta in Nuclear

563rd MEETING, LONDON
807
Exonuclease activity has been measured in preparations of both enzymes. The activity
associated with DNA polymerase a requires denatured DNA, Mg2+and is ATP-dependent. The polymerase b-associated nuclease requires denatured DNA and Mg2+or Caz+.
Studies of the distribution of these enzymes during the cell cycle have shown that
extracts of cells harvested at the time of nuclear DNA synthesis (Chiang & Sueoka,
1967) have DNA polymerase b as the predominant DNA-synthesizing activity, whereas
DNA polymerase a is the major activity in cells harvested at a resting phase of growth.
The two major DNA polymerases of Chlamydomonas reinhardii therefore seem to
differ in physical and enzyme properties and in the time of appearance during the
vegetative cell cycle.
We thank the Medical Research Council for the award of a studentship to C. A. R.
Banks, G. R. & Yarranton, G. T. (1976) Eur. J. Biochem. 62, 143-150
Banks, G. R., Holloman, W. K., Kairis, M. V., Spanos, A. & Yarranton, G. T. (1976) Eur. J.
Biochcm. 62, I3 1- I42
Chang, L. M. S. (1971) Biochem. Biophys. Res. Commun. 44, 124-131
Chiang, K. S. & Sueoka, N. (1967) J. Cell. Physiol. 70, Suppl. 1, 89-112
Crerar, M. & Pearlman, R. (1974) J. Biol. Chem. 249, 3123-3131
Holrnes, A. H., Hesslewood, 1. P. &Johnston, I. R. (1974) Eur. J. Biochem. 43, 487-499
Kates, J. R. & Jones, R. F. (1964) J. Cell. Comp. Physiol. 63, 157-164
Loeb, L. A. (1969) J. Biol. Chent. 244, 1672-1681
McLennan, A. G. & Keir, H. M. (1975~)Biochem. J. 151, 227-238
McLennan, A. G. & Keir, H. M. (19756) Biochern. J. 151, 239-247
Schonherr, 0. Th. & Keir, H. M. (1972) Biochem. J. 129, 285-290
Wintersberger, U. & Wintersberger, E. (1970) Eur. J. Biochem. 13, 20-27
Role of Deoxyribonucleic Acid Polymerase p in Nuclear Deoxyribonucleic
Acid Synthesis
TAUSEEF R. BUTT, WILLIAM M. WOOD* and ROGER L. P. ADAMS
Department of Biochemistry, Uniwrsity of Glasgow, Glasgow GI 2 SQQ, Scotland, U.K.
Homogenization of mouse L929 cells in hypo-osmotic buffer releases most of the DNA
polymerase a into the supernatant fractions. However, nuclei isolated under these conditions still contain both DNA polymerase a and DNA polymerase B ( A d a m et al.,
1973). DNA polymerase a shows an increase in activity as cells leave a resting phase
and enter into S phase (Lindsay et al., 1970; Chang et al., 1973), but the activity of DNA
polymerase fl is independent of the state of growth of the cells. Although this points to a
possible rolc for DNA polymerase Bin repair of DNA, it does not exclude some role in
DNA replication. It has been reported ( A d a m &Wood, 1973) that removal of up to 50 %
of the total nuclear DNA polymerase has little effect on the ability of isolated nuclei to
synthesize DNA. Here we present some evidence that DNA polymerase /?alone is sufficient to catalyse this nuclear DNA synthesis in uitro.
L929 cells were grown as described by Lindsay et al. (1970), and nuclei were prepared
by homogenizing the cells in 0.25 M-sucrose containing 20m~-Tris/HCI,pH7.5, and
5m-2-mercaptoethanoI. DNA synthesis was assayed in nuclei, washed twice with the
same buffer, by incubation in the reaction mixture of Hershey et al. (1973). This contains
1OmM-MgCI,, 4Om~-Tris/HCl, pH 7.8, 100m~-NaC1,0.5 m - E D T A , 4 m~-2-mercaptoethanol, SrnM-ATP, 0.1 mM each of dATP, dCTP, dGTP and r3H]dTTP (specific
radioactivity 5OpCi/pmol and 2mCi/pmol).
To extract DNA polymerase a differentially, nuclei from S-phase cells were suspended
gently inTris/sucrose buffer containing02~~KC1,
left for 15minand sedimentedat 8OOg.
* Present address :Department of Physiology and Biophysics, University of Illinois at UrbanaChampaign, IL 61801, U.S.A.
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BIOCHEMICAL SOCIETY TRANSACTIONS
5
10
15
0
5
10
15
Fraction no.
Fig. I . Sucrose density gradient (5-20%) of extracts of S-phase nuclei
(a) Total O.~M-KCI
extract; (h) O.~M-KCIextract of nuclei previously extracted with
O.~M-KCI.
The above procedure was repeated once more, which gives nuclei completely free of
DNA polymerase u. Evidence of the absence of DNA polymerase u was obtained by
exhaustively extracting nuclei with Tris/sucrose buffer containing O . ~ M - K CThe
~ . extract
was layered on to 5-20% linear sucrose gradients containing O.~M-KCI.
The centrifugation was performed at 4°C in SW56 rotor at 50000rev./min for 12-16h in a Beckman
L2-65B ultracentrifuge. Then 15 fractions were collected by using an MSE gradient
harvester, and the fractions were assayed for DNA polymerase activity as described
previously (Adams et al., 1973), but with 'activated' DNA as template. Markers of
haemoglobin (4.1 S), immunoglobulin G (7s) and catalase ( 1 I .3S) were used.
Characterization of DNA synthesized in vitro was performed on alkaline sucrose
gradients (Krokan et al., 1975), with Simian virus 40 DNA as marker.
Nuclei isolated from S-phase cells are capable of synthesizing DNA when incubated
with the standard test mixture, and this synthesis continues for about 30min. The maximum amount of dTTP incorporated into DNA by such nuclei was 300pmol/mg of DNA.
These results are quite comparable with results obtained by Hershey et al. (1973), who
showed that incorporation represented the continuation of DNA synthesis initiated
in vivo. Calculations show that an average of 160 nucleotides could be added to each
growing chain.
1976
809
563rd MEETING, LONDON
4
3
c
6
a
a,
x
*
‘5
2
*
.d
3
.-0
4
00
d
X
N
21
0
0
0
0
0 0
00 0
0
0
I
I
-
10
20
30
Fraction number
Fig. 2. Alkaline-sucrose-density-gradient centrifugation of DNA synthesized in isolated
nuclei
Nuclei were prepared from cells pre-labelled with 5pCi of [14C]thymidine( 0 ) .They were
incubated for 5min in standard test mixture as described in the text, except that the final
concentration of [3H]dTTP was 1 2 . 5 (specific
~ ~
radioactivity 2mCi/pmol) ( 0 ) . Samples
(50pg of DNA) were prepared for centrifugation as described by Krokan et al. (1975),
except that centrifugation was performed at 4°C in a SW56 Spinco rotor for 4 h at
50 OOO rev./min.
As predicted, nuclei isolated from stationary-phase cells fail to synthesize any DNA,
demonstrating that the observed DNA synthesis reflects the state of the initial cells.
There are two DNA polymerases present in nuclei isolated from S-phase cells. They
are DNA polymerase /3 (4s) and DNA polymerase a (8S), which are easily separated
on sucrose gradients (Fig. la). Extraction of these nuclei with O.~M-KCI
removes all
DNA polymerase activity and abolishes their DNA-synthesizing capability in uitro. It is
possible to remove all the assayable DNA polymerase a from these nuclei by gentle
washing with 0.2M-KCI (Fig. 16). Surprisingly, this treatment has little effect on the
DNA-synthesizing capability of these nuclei, thus excluding the role of polymerase a
in the reaction studied in these nuclei. Characterization on alkaline sucrose gradients
of DNA synthesized in uitro by nuclei containing both DNA polymerases a and B
shows that only small ‘0kazaki’-type pieces are synthesized, and the same is true for
nuclei containing only DNA polymerase B(Fig. 2). These results show the involvement of
DNA polymerase B in the synthesis of DNA observed in these nuclei.
Thanks are due to Professor R.M. S. Smellie and Professor A. R.Williamson for their interest
and for providing necessary facilities. T.R. B. is supported by a grant from M.R.C., who also
provided funds.
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BIOCHEMICAL SOCIETY TRANSACTIONS
Adams, R. L. P. & Wood, W. M. (1973) Biochem. SOC.Trans. 1, 627-629
Adams, R. L. P., Henderson, M. A. L., Wood, W. M. & Lindsay, J. G. (1973) Biochem. J. 131,
237-246
Chang, L. M. S., Brown, M. & Bollum, F. J. (1973) J. Mol. Biol. 74, 1-8
Hershey, H. V., Stieber, J. F. & Mueller, G. C. (1973) Eur. J. Biochem. 34, 383-394
Krokan, H., Birklid, E. & Prydz, H. (1975) Biochemistry 14, 42274232
Lindsay, J. G., Berryman, S. & Adams, R. L. P. (1970) Biochem. J. 119, 839-848
The Nature of ‘Activated’ Deoxyribonucleic Acid used in
Deoxyribonucleic Acid Polymerase Studies
JAMES M. MORRISON
Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ,
Scotland, U.K.
DNA polymerases catalyse the template-directed transfer of nucleotide residues to the
3’-hydroxyl group of a poly- or oligo-nucleotide chain hydrogen-bonded to that
template. The preferences of various polymerases towards different forms of DNA template have been the subject of much study, and in particular, DNA that has been
‘activated’ by the action of certain DNAases* has found wide application. ‘Activated’
DNA falls into two groups:
(i) lightly ‘nicked‘ DNA, or duplex DNA into which a low density of single-strand
breaks has been introduced by the action of nanogram amounts of pancreatic DNAase
(Aposhian & Kornberg, 1962);
(ii) extensively degraded DNA, which has been acted on by microgram amounts of
pancreatic DNAase until an appreciable amount (usually 5-20 %) has been rendered
acid-soluble or else by lower amounts of DNAase followed by limited digestion by a
3’: 5’-exonuclease acting non-processively on duplex DNA (e.g. exonuclease I11 from
Escherichia coli). This latter is sometimes termed ‘gapped’ DNA (Kornberg & Gefter,
1972).
Use of type (i) DNA has been largely restricted to work with prokaryotic polymerases
of the poll class, as these are the only enzymes so far described which are capable of the
strand displacement and/or 5’: 3’ hydrolysis necessary for extensive synthesis on this
template (Kornberg, 1974). Type (ii) DNA has been widely used in studies on polymerases from eukaryotic and bacteriophage-infected sources, as well as the prokaryotic
class I1 and 111enzymes. It should be noted that almost all methods for the preparation
of activated DNA involve a heating step intended to inactivate the DNAase used. The
importance of this heating in the activation process does not appear to have been
evaluated to date.
In the course of studies on the DNA polymerase and DNA exonuclease induced by
herpes simplex virus, DNA of both types has been extensively used. Several useful properties of the purified, virus-induced enzymes have been exploited in order to gain a
better understanding of the nature of ‘activated’ DNA.
In order to decrease the number of experimental variables, the DNA was rendered
small and relatively homogeneous by ultrasonic vibration before DNAase treatment.
This had the added advantage of allowing comparability with the very useful physical
studies of Hays & Zimm (1970). On the basis of hydrodynamic studies, these authors
concluded that the main effect of single-strand breakage on duplex DNA occurred when
two neighbouring breaks were sufficiently close to permit either (i) release of short oligonucleotides, when the breaks were on the same strand, or (ii) breakage of the duplex into
two shorter lengths with single-stranded tails, when the breaks were on opposite strands.
Both of these structures are, of course, ideal substrates for polymerase activity.
Herpes-virus-induced DNA polymerase has proved a useful reagent for these studies
because of its low apparent K,,, for both deoxyribonucleoside triphosphates and DNA
* Abbreviation: DNAase, deoxyribonuclease.
1976