Volume 5 Number 3 March 1978
N u c l e i c A c i d s Research
DNA replication in Physarum polycephalum: bidirectional replication of DNA within replicons
Steinar Funderud, Rolf Andreassen and Finn Haugli
Institute of Medical Biology, University of Tromsd, 9000 Troms6, Norway
Received 5 December 1977
ABSTRACT
The direction of replication of DNA within replicons of
Physarum polycephalum was studied by pulse.-labelling with
5-bromouracil-deoxyriboside (BrdUrd) and 3H-adenosine deoxyriboside (dAdo), followed by ultraviolet- (UV) - photolysis
and analysis of molecular weights of single strand DNA fragments on alkaline sucrose gradients. Newly made DNA within
replicons at all stages of completion is split in two equal
halves upon UV irradiation when BrdUrd was given at the time
of initiation of DNA synthesis. This shows that replication
within replicons of Physarum polycephalum starts at an origin
located in the center of each unit, proceeding bidirectionally
from this origin.
INTRODUCTION
The bidirectional mode of DNA replication within replicating units of eukaryotic chromosomes was first demonstrated
by Huberman and Riggs in Chinese hamster cells, using DNA
fiber autoradiography. Bidirectional replication has since
been demonstrated in a number of other eukaryotic cells using
2-1)
5
autoradiography
, and electron microscopy . Biochemical
evidence in support of a bidirectional mode of DNA synthesis
within replicons was obtained by Weintraub (1972) , using UVirradiation to specifically cut newly made DNA from chick
erythroblasts at points preselected by pulse labelling with
BrdUrd. A similar biochemical approach was employed by
7
McKenna and Masters (1972) to show that DNA replication in
the E . coli replicon is also bidirectional from the origin of
replication. Recently Planck and Mueller (1977) used UV
induced photolysis of BrdUrd labelled DNA to show that repli© Information Retrieval Limited 1 Falconberg Court London W1V5FG England
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Nucleic Acids Research
eating units of HeLa cells are synthesized bidirectionally.
Thus bidirectional DNA synthesis within replicons is very widespread, but not universal, since unidirectional replication
n
occurs in some bacteriophages and in metazoan mitochondrial
DNA
. DNA replication in Physarum proceeds with natural,
perfect synchrony at the molecular level
and this organism is
therefore well suited for biochemical analysis of the mechanisms
of replication. The question of bidirectional vs. unidirectional replication in Physarum was never analyzed before, although
suggested
Brewer (1972)
and Brewer, Evans and Evans (1971!)
that synthesis might be unidirectional. We have previously
presented evidence that DNA replication in Physarum proceeds
with the "discontinuous" synthesis of Okazaki fragments on both
strands of progeny DNA molecules ' . Recently we have also
obtained biochemical evidence that replicons exist in Physarum,
that their single strand molecular weight is 107 - 3x10 7
daltons and that replicons occur in temporal clusters where
little or no joining of adjacent replicons takes place during
-1 o
the first hour after initiation
In the present investigation we take advantage of the
precise natural synchrony, and the previous finding that the
first temporal sets of replicons are not joined measurably
until 40-60 minutes after initiation. Under these conditions
extremely short pulses of BrdUrd allows precision photolysis
of nascent DNA in order to determine the topological relationship within replicons during DNA synthesis.
METHODS OF ANALYSIS
Strain and culture techniques
Strain TU291 is used in all experiments.
tions have been described
Culture condi-
Labelling; procedures
'.-.'hole, synchronous surface plasmodia were pulse-labelled
with 5-bromodeoxyuridine (BrdUrd) at 50 yg/ml on regular semidefined medium without any pre-treatment. The pulse was for
30 seconds immediately after completion of anaphase, at which
time DNA replication is initiated. Excess BrdUrd was removed
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Nucleic Acids Research
by blotting on a pad of filter paper before the plasmodium was
transferred to medium containing cold thymidine (dThd) at
20 pg/ml.
After a 60 second chase of the BrdUrd on this
medium, discs of plasmodia were pulse-labelled with
H-deoxy-
adenosine ( H-dAdo) for 2 minutes at radioactive concentration
250 yCi/ml of semidofined medium.
dAdo was chosen for radio-
active labelling to have the advantage of non-competing pools
for the two pulse labelling agents.
The radioactive pulse was
followed by chase on regular medium for the desired time
periods, as given in the Results section.
Isolation of nuclei and alkaline sucrose gradient analysis
Isolation of nuclei was carried out according to the
19
as previously
method of Mohberg and Rusch (1969)
described
' '.
0.5 - 1.0x10
nuclei suspended in 0.25 ml of
0.15 M NaCl 10 mM EDTA, pH 9.0, were gently mixed with an
equal volume of 4 % sarkosyl 0.2 M NaOH, 10 mM EDTA, and left
at 50 C for 20 minutes for lysis and denaturation of DNA.
Centrifugation analysis was on a 4-20 % alkaline sucrose
gradient
in a SW40 rotor in Spinco L65B centrifuge at 22°C
and 30,000 rpm for 5 or 6 hours.
Ultraviolet irradiation
0.5 - 1.0x10
nuclei, suspended in 125 pi 0.15 M NaCl,
10 mM EDTA, pH 9.0 were placed in a plastic Petri dish 20 cm
below a Westinghouse PS20 Sunlamp and irradiated at 2°C for
10 minutes without any shield.
Marker DNA and determination of S-values and molecular weights
Phage X
14
17
C-DNA was prepared as previously described
and included in all samples to allow calculation of sedimentation constants and molecular weights.
S..
for alkaline
tU j W
single
strand X
is
40 and molecular
weights1965)
are 2 °.
givenWeby
0 H
the relation:
S o DNA
= 0.0528'M
• °° (Studier,
n
assume that a 4-20 % sucrose gradient in the 11.5 ml tubes of
the SW40 rotor of Spinco L65B provides an approximation of
isokinetic conditions, although the use of the X marker to
estimate the molecular weight of the Okazaki fragments in
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Nucleic Acids Research
Physarum leads to an overestimation of S-value and molecular
weight . However, the deviations for DNA molecules much
larger than the Okazaki fragments and closer to the X DNA in
molecular weight, is much smaller and do not influence the
basic conclusions of the present work.
RESULTS
Control experiments
To obtain results which
with a maximum of clarity, we
investigation to analyze only
very first sets of temporally
reflect the ongoing processes
have chosen in the present
the processes occurring in the
isochronous replicons. Prom
-i r_
-i o
previous studies on DNA replication
we conclude that no
replicons are terminated, nor joined to neighboring replicons
within the first 40-60 minutes of the S-period. Thus, during
this time interval only molecules at various stages of completion towards the full replicon size occurs, allowing the
desired simpliciti for analysis.
We attempted to maintain the precision for analysis
which follows from the naturally synchronous DNA replication
of Physarum by giving extremely short pulses of the photolyses
agent, BrdUrd, as well as of the radioactive marker. Control
experiments showed that the labelling regimen given in the
Methods section allowed scission of photo sensitized DNA, when
isolated nuclei were irradiated with the Westinghouse PS20
Sunlamp from a distance of 20 cm, without shield. The temperature was 2 C and 10 minutes of irradiation proved sufficient
to break essentially 100 % of the labelled molecules. DNA
which had not incorporated any BrdUrd remained unchanged even
after 25 minutes of irradiation.
Analysis of the size of nascent DNA with and without photolysis
If replication proceeds unidirectionally from the origin,
a pulse labelling regime where BrdUrd is given at initiation,
immediately followed by radioactive labelling, should yield
fragments of single stranded DNA which at all stages of growth
within the replicon should not change its molecular weight
after UV irradiation. However, if replication proceeds
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Nucleic Acids Research
bidirectionally from the origin, and joining of the discontinously made Okazaki fragments occurs rapidly, then UV irra.diation should break the nascent DNA in two fragments. These
photolysis products will be of equal length if both replication forks proceed at the same rate, but will be of different size if the two rates are different. These possibilities
as well as the expected patterns on alkaline sucrose gradients are displayed in Pig. 1
The results of experiments where DNA was pulse labelled
at initiation and chased for various times are shown in Fig.2.
When DNA, pulsed with H-dAdo at initiation of DNA synthesis,
but with BrdUrd only at the very end of a 35 minute chase, was
irradiated and analyzed in the same manner, no change in
A
B
C
o
o
o
Pig. 1:
Models displaying possible modes of replicon growth
and the expected results on alkaline sucrose
gradients.
A: unidirectional synthesis from origin, 0
B: bidirectional synthesis with different growth rates
at the two forks.
C: bidirectional synthesis with similar growth rates
at the forks.
(/VAX): BrdUrd pulse. (...):^H-deoxyadenosine.
(
): cold DNA made in progressively longer chase.
Panels: (
) unirradiated DNA, and (
) irradiated
DNA on alkaline sucrose gradients.
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Nucleic Acids Research
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Pig. 2:
Photolysis of growing DNA molecules in replicons.
A plasmodium was labelled with BrdUrd and 3H-deoxyadenosine as described in Methods. The chase was on
regular semidefined medium for 153 20, 25j 305 35 and
40 min, as shown. All nuclear samples were divided
in two equal halves, one of which was irradiated for
10 minutes as described in Methods. Irradiated and
unirradiated DNA were analyzed separately with X DNA
as marker, on 4-20 % alkaline sucrose gradients, SW40,
30 K, 22°C. The 15' sample was centrifuged for 6
hours, the others for 5 hours. Results for each
chaseset are superimposed on each other by alignment
of the X-marker position (arrow). Direction of sedimentation is from right to left. o
o unirradiated.
o
o irradiated.
molecular weight could be detected (data not shown). The
molecular weights or unirradiated and irradiated DNA, calculated from the positions of peaks and marker DNA in Fig. ?,
is displayed in Fig. 3We conclude that DNA in replicons at all stages of completion
grows in a manner compatible v:ith the model outlined in Fig.lC.
That is, initiation of replication occurs in the center of the
replicon and proceeds bidirectionally at equal rates for both
forks. The cumulative, average rate at these two forks is
3-2 • 10 daltons per minute as calculated from the data in
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Nucleic Acids Research
c
o
4-f
(D
T>
O
ight
X
13121110876-
<u
3
Lecu
l(0
4321-
ri
15
!
20
-I!II
25
Minutes
30
40
35
chased.
Pig.
3:
Chain growth rate and reduction in molecular weight
by irradiation of BrdUrd substituted nascent DNA.
The molecular weights of the DNA species from Pig.2
have been plotted in this histogram, as a function
of the chase period.
|
| irradiated
ynna non-irradiated
Pig.
3.
Thus, we suggest a growth rate per strand per fork
of approximately 1.6x10
daltons per minute.
DISCUSSION
-1
We have obtained evidence elsewhere
Q
that replicons of
size corresponding to a single strand molecular weight of
7
7
10 - 3x10 daltons exist in Physarum. These are synthesized
on both templates via the rapid joining of discontinuously made
Okazaki fragments.
In the present investigation we have
analyzed the mechanisms of DNA synthesis in replicons in more
detail by means of BrdUrd photolysis of the growing chains at
the site of initiation of replicon DNA synthesis.
Considering
the models presented in Pig. 1, and the results given in Pig. 2
and Fig. 3 the following conclusions can be reached:
1.
Replicons grow with an overall rate of about 3.2 • 10
daltons per strand per minute, as calculated from the
non-irradiated samples in Pig. 2 or Pig. 3.
Calculations
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Nucleic Acids Research
2.
3-
based on shorter intervals give results which are higher
or lower (eg. 7 • 10 daltons per minute at 15-20 minutes
and 1 • 10 daltons per minute at 30-35 minutes).
This scattering is likely to be due to timing errors,
but the possibility of rate change with time can not be
excluded.
UV-irradiacion of DNA pulse-labelled at initiation with
BrdUrd and H-dAdo and then chased for various time
periods causes a reduction in molecular weight to approximately one half of the original, as can be seen from
Fig. 2 or Pig. 3.
Rates of chain growth for the photolysis fragments and
for the whole molecules respectively, are 8.0x10 and
1.67x10 daltons per minute after 15 minutes chase
following the pulse label with BrdUrd and H-dAdo;
1.55xl05 and 3-OxlO5 after 20 minutes of chase; 1.84xl05
and 3-6xl05 after 25 minutes; 1.67xlO5 and 3-5x10^ after
30 minutes; 1.71x10 and 3.14x10 after 35 minutes; and
1.8x10 and 3-25x10 daltons per minute after 40 minutes
of chase. It can be concluded that replicon chain growth
occurs bidirectionally with approximately equal rates at
both forks.
Since this work only includes the analysis of those
replicons that initiate at the time of master initiation of
DNA synthesis, immediately following anaphase, the conclusions
reached here are strictly valid only for this class. We do
suggest, however, that the mechanisms are likely to be similar
throughout the S-phase, although it is possible that rates of
chain growth and replicon size could change at later stages,
21
as has been found in other systems
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34.
56.
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