Cell, Vol.

13, 573-580, March 1978, Copyright 0 1978 by MIT

Formation of Okazaki Fragments in Polyoma QNA

Synthesis Caused by Misincorporation of Uracil

Kerstin Brynolf, Rolf Eliasson and Peter Reichard Machida, Okazaki and Okazaki, 1977; Scherzinger
Medical Nobel Institute et al., 1977).
Biochemistry Department I It has recently been found that in E. coli, short
Karolinska Institute fragments might also arise at the replication fork
S-10401 Stockholm, Sweden by asecond mechanism (Tye et al., 1977), originally
discovered by Lindahl and his co-workers (Lindahl,
1974; Ljungquist and Lindahl, 1974; Ljungquist
Summary and Lindahl, 1977; Lindahl et al., 1977) as a repair
mechanism, which prevents the accumulation of
When dUTP”replaced dTTP during polyoma DNA mutations created by spontaneous deamination of
replication in isolated cell nuclei, radioactivity cytosine residues during the .lifetime of a DNA
from labeled deoxynucleoside triphosphates was molecule. Figure 1 shows that according to this
almost exclusively recovered in very short Oka- mechanism, uracil is removed by a specific N-
zaki fragments and incorporation ceased after a glycosidase, the diester linkage at the apyrimidinic
short time. Addition of uracil, a known inhibitor of site is then cleaved by an endonuclease and the
the enzyme uracil-DNA glycosidase (Lindahl et resulting chain break is finally repaired by an exo-
al., 1977), increased total synthesis and shifted nuclease-initiated process.
the incorporation to longer progeny strands. The Uracil is not a normal component of DNA even
presence of as little as 2.5% of dUTP in a dTTP- though dUTP, formed by reduction of ribonucleo-
containing system gave a distinct increase in tides, is a normal intermediate in dTMP formation
isotope incorporation into Okazaki pieces accom- (Bertani, Haggmark and Reichard, 1961; Green-
panied by a corresponding decrease in longer berg and Somerville, 1962; Bertani, Haggmark and
strands. This effect was reversed completely by Reichard, 1963), and can serve as substrate in
uracil. The short strands formed from dUTP could place of dTTP for DNA polymerase (Bessman et al.,
be chased efficiently into long strands. Our re- 1958). It has been proposed that in the cell, a
sults suggest that dUTP can be incorporated in specific dUTPase rapidly transforms dUTP to dUMP
place of dTTP ‘into polyoma ‘DNA, and that poly- (Bertani et al., 1963) and thereby makes it unavail-
oma-infected nuclei, similar to E. coli (Tye et al., able for the polymerase. The importance of
1977), contain an excision-repair system which by dUTPase in this connection was demonstrated by
removal of uracil causes strand breakage and Tye et al. (1977), who showed that mutants of E.
under certain circumstances may contribute to coli, deficient in dUTPase, accumulate short Oka-
the formation of Okazaki fragments. zaki fragments, and that this accumulation disap-
pears in revertants which have regained dUTPase
Introduction activity. Short fragments in the mutant are clearly
generated by a mechanism different from that orig-
The elongation of DNA strands of papova viruses inally visualized by Okazaki, and Tye et al. (1977)
(polyoma, SV40) is a discontinuous process, both ask whether and to what extent such “repair-type”
in vivo (Fareed and Salzman, 1972) and in vitro Okazaki fragments might also be produced during
(Magnusson et al., 1973; Francke and Hunter, replication of DNA in wild-type organisms.
1974; Pigiet, Eliasson and Reichard, 1974), with This paper investigates the question whether the
the intermediate formation of short fragments sequence of reactions outlined in Figure 1 occurs
about 100-200 nucleotides long. Similar short frag- in polyoma-infected nuclei. We find that addition
ments, but with a length of about 1000 nucleotides, of very small amounts of dUTP in the presence of a
were earlier detected during DNA replication in E. large excess of dTTP to an in vitro system synthe-
coli (Okazaki et al., 1968). Such “Okazaki frag- sizing polyoma DNA (Magnusson et al., 1973) leads
ments” are believed to originate at the replication to a transient accumulation of short DNA frag-
fork during the synthesis of the strand which is ments, and we present evidence that these frag-
elongated in the 3’ to 5’ direction. In polyoma ments arise by the mechanism outlined in Figure 1.
(Eliasson, Martin and Reichard, 1974; Reichard,
Eliasson and Soderman, 1974), these fragments Results
are initiated by oligoribonucleotides close to 10
nucleotides long (= initiator RNA), and evidence In a first experiment, we tested the ability of dUTP
for a similar involvement of short oligoribonucleo- to replace dTTP during polyoma DNA replication in
tides has also been obtained in other eucaryotic isolated nuclei. dUTP was added to the incubation
and procaryotic systems (Tseng and Goulian, 1975; mixture containing the other three dNTPs under
Waqar and Huberman, 1975; Kurosawa et al., 1975; standard conditions, and the incorporation of CY-
Cell
574

dATP, dCTP, dGTP, dUTP, dTTP

1A
1000 -

ACGUTAC E
500 -

-I
DNA glycosidase
( inhibited by uracil ) Fraction number from bottom
Figure 2. Length of Progeny Strands when dUTP Replaces dTTP

A C GCHOT A C Nuclei were incubated under standard condrtions for 1.5 min (A)
or 15 min (6) with either 50 PM dTTP (O-O) or 50 PM dUTP
(0- - -0) using ws2P-dGTP as the radioactive substrate. Por-
JPJPJPJPJPJPJP + uraci’ tions of Hirt supernatant fluid were centrifuged through an alka-
line sucrose gradient.

A C G
1 CHO
endonuclease

T A C
there is no apparent transfer of radioactivity
heavy peak, and the increase in total radioactivity
is exclusively located to the Okazaki peak region.
to the

Furthermore, this material sediments slower than

4S, both at 1.5 and 15 min, and is thus shorter than
PJPJPJOH + P.JPiPJPJP the Okazaki pieces formed from dTTP.
dUTP thus substitutes for dTTP, but only very
exonuclease short DNA fragments are formed. This could mean
that with dUTP, chain elongation is much slower
than with dTTP, possibly caused by a decreased
A C G CHO T A C,
ability of the system to elongate strands once uracil
is incorporated. As an alternative, elongation of
uracil-containing strands might continue equally
Figure 1. Fragmentation of DNA Strands after incorporation of
well, but the newly formed strands are cleaved
dUTP according to the reactions outlined in Figure 1. It
The scheme shows the incorporation of uracil from dUTP, but seemed possible to distinguish between these two
uracil in DNA may also arise by spontaneous deamination of alternatives. Uracil is an inhibitor of the DNA gly-
cytosine. cosidase (Lindahl et al., 1977), and according to
the second alternative, addition of the pyrimidine
32P-dGTP was used to monitor polyoma DNA syn- should inhibit chain breakage.
thesis. The incorporation of 32P into polyoma DNA We therefore investigated the effect of uracil on
after 1.5 and 15 min was 14 and 25 pmole/mg DNA, DNA synthesis and the size of progeny strands. In
respectively, as compared to values of 24 and 80 a control containing dTTP under standard condi-
with dTTP in place of dUTP. Thus dUTP substituted tions, 0.94 pmole of dGMP were incorporated into
quite well for dTTP early during incubation but did DNA during 5 min, and this value did not change
not support a sustained replication of DNA. on addition of 6 mM uracil. When dUTP was substi-
Figure 2 shows results from the same experiment tuted for dTTP, the value dropped to 0.30 pmole
in which ,the length of the daughter strands was and increased to 0.70 pmole in the presence of 6
measured by’alkaline sucrose centrifugation. The mM uracil. As shown in the alkaline sucrose pat-
dTTP experiment gives at 1.5 min a bimodal pat- terns of Figure 3, addition of uracil in the dUTP
tern, where the peak at the top of the gradient experiment resulted not only in an increase of the
corresponds to the 4s Okazaki pieces formed by total radioactivity incorporated into DNA, but also
discontinuous synthesis, while the heavier material in a shift of radioactivity from the 4S region to
represents the growing daughter strands. The ra- heavier material. Whereas in the control 38% of the
dioactivity is about equally distributed between the total radioactivity sedimented with the Okazaki
two peaks. After 15 min, most radioactivity is re- fragments (fractions 19-26 in Figure 3), the corre-
covered in the heavier peak. The situation changes sponding value was 88% in the dUTP experiment.
dramatically when dUTP is substituted for dTTP. With uracil this value decreased to 63% and at the
Now 85% of the label is recovered in the Okazaki same time, the rota/ amount of radioactivity in the
peak at 1.5 min and this value increases to 92% heavy peak increased 4.5 fold. Thus addition of
after a 15 min incubation. Between 1.5 and 15 min, uracil to a dUTP-utilizing system considerably in-
Okazaki Fragments from Uracil Incorporation
575

1000

8 500

.,
10 20
Fraction number
Figure 3. Effect of Uracii on Size of Progeny Strands Synthesized
from dUTP
Incubation of nuclei was carried out under standard conditions
for 5 min with 50 PM dTTP (O-O), 50 PM dUTP (0- -- 0) or
50 PM dUTP + 6 mM uracil (A-A) using ar-32P-dGTP as the
radioactive substrate. The arrow shows the position of a marker
(about 400 nucleotides long) in the alkaline sucrose gradient.

creased the capacity of nuclei to incorporate dGTP

into long strands. It seems probable that uracil
acted by inhibiting the first reaction of Figure 1, 0
1 I 1
and that the inability of the system to form long c ’
strands from dUTP is indeed caused by this exci- 10 20
sion-repair mechanism.
Fraction number
We next investigated the effects of different con- Figure 4. Effect of Uracil on Size of Progeny Strands Synthesized
centrations of uracil (O-6 mM) in experiments con- from a Mixture of dTTP (50 wM) and dUTP (1.25 PM)
taining a small amount of dUTP (initial concentra- Nuclei were incubated under standard conditions for 5 min with
tion 1.25 PM) in the presence of a large excess of 50 KM dTTP (O-O); 50 pm dTTP + 1.25 PM dUTP ( 0- - - 0); or
dTTP (50 PM). The total amount of isotope incor- 50 PM dTTP, 1.25 PM dUTP + 2 mM uracil (A-A). a-32P-dGTP
poration from a-32P-dGTP during 5 min was iden- was the radioactivesubstrate. Analysis was carried out by alkaline
sucrose gradient centrifugation. The data are plotted as the
tical in all experiments and corresponded to that of percentage of total radioactivity in each fraction.
a control containing no dUTP. As shown in Figure
4, however, the distribution of isotope between
strands of different chain lengths differed signifi- Thus uracil at this low dUTP concentration com-
cantly. The data are plotted as the percentage of pletely counteracted the effect of dUTP on chain
3ZP present in each fraction, and are given for the length.
control and the two experiments containing dUTP So far our experiments in which synthesis was
with 0 and 2 niM uracil, respectively. The results at measured by incorporation of labeled dGMP pro-
higher uracil concentrations were indistinguisha- vide only indirect evidence for the utilization of
ble from those obtained with 2 mM uracil. dUTP for DNA synthesis. In the next experiment,
The presence of dUTP resulted in a clear in- dUMP incorporation was demonstrated directly.
crease in the amount of isotope present in the 4s Nuclei were incubated for 5 min with 3H-dTTP and
peak, accompanied by a corresponding decrease with increasing concentrations of &‘P-dUTP, and
in the longer chains. It is interesting to note that the incorporation of the two isotopes into polyoma
the decrease occurred in strands of all size classes DNA was measured (results given in Table 1). The
above fraction 18 in the gradient, corresponding to effect of uracil on DNA synthesis from the two
chain lengths above approximately 500 nucleo- nucleotides was also determined. The results es-
tides. Inclusion of uracil together with dtJTP re- tablish that dUMP is incorporated into polyoma
verted the distribution back to that of the control. DNA. At equal concentrations, 3H-dTTP and (Y-~~P-
 Cell
576

Table 1. Incorporation of 3H-dTTP and @*P-dUTP into Polyoma 3(

DNA

Initial Substrate Nucleotide Incorporation into DNA

Concentration (mole)

&*p-
dUTP dTMP +
(PM) Uracil (mM) dTMP dUMP dUMP 2(
2.6 2.8
6 2.7 2.7

0.5 2.6 0.008 2.6

0.5 6 2.8 0.03 2.8

10.0 1 .o 0.5 1.5 IO

10.0 6 1.4 1.6 3.0

All incubations were for 5 min with 10 PM 3H-dTTP.

>I
dUTP are used equally well when uracil is present .Z
>
during incubation, and the sum of the two isotopes P
equals the amount of 3H-dTMP incorporated in the g 10
control (no dUTP added). In the absence of uracil, .-
however, the sum of the isotopes,is only about 50% BL
of the control, and only a third of the total incor-
poration arises from dUMP. The stimulation of s
dUMP incorporation by uracil is also evident at the
low dUTP concentration.
Alkaline sucrose gradients of polyoma DNA from
the experiment containing 50% dUTP are shown in 5
Figure 5A. There is a complete overlap for the
incorporation of 3H-dTMP and 32P-dUMP into
strands of different chain lengths both in the ab-
sence and presence of uracil. Addition of uracil
increased the relative amount of isotope recovered
in longer strands, but did not completely change
J /,
the pattern to that of the control. The latter is
I I I I
shown in Figure 5B, together with the results
obtained at the low dUTP concentration. Only the 10 20
results for 3H-dTMP incorporation are given, since
too little 32P was recovered to permit a meaningful
Fraction number from bottom
Figure 5. Simultaneous Incorporation of 3H-dTTP and a-32P-
analysis. It can be seen that the addition of dUTP dUTP into Progeny Strands
resulted in a shift of isotope from longer to shorter In all experiments, nuclei were incubated with 10 PM 3H-dTTP for
strands, and that the further addition of uracil 5 min, and the size of progeny strands was analyzed by alkaline
switched the pattern back to that of the control. sucrose gradient centrifugation. (A) shows the results from exper-
iments with 10 &M aG2P-dUTP [(A) 3H; (0) anP] or 10 PM L+~P-
At this point it became important to determine to dUTP + 6 mM uracil [(A) 3H; (0) 32P]; (B) shows the results from
what extent dUTP was degraded during incubation experiments without dUTP (O-O), 0.5 PM dUTP ( 0- - - 0) or
by the powerful dUTPase present in isolated nuclei. 0.5 yM dUTP + 6 mM uracil (A-A). In all cases the data are
By paper chromatography (Krebs and Hems, 1953), plotted as the percentage of total radioactivity.
we found that after a 5 min incubation at 0.5 PM
dUTP, all 32P was recovered as dUMP, while at IO DNA. Figure 6 shows data from a determination of
PM dUTP, only 30% of dUTP had been transformed the K, for dUTPase carried out with nuclei under
to dUMP. Uracil had no effect on dUTP degrada- the conditions used for DNA synthesis. From the
tion. In all experiments, >95% of 3H-dTTP re- Lineweaver-Burk plot, a value of about 0.1 PM is
mained undegraded. In separate experiments not derived. From these data we can calculate that in
detailed here, we found that isolated nuclei at 25°C the experiment of Table 1, dUTP, present at an
quite reproducibly transformed between 0.5-l .O initial concentration of 0.5 FM, has disappeared
nmole of dUTP to dUMP per minute and mg of completely after a l-2 min incubation.
Okazaki Fragments from Uracil Incorporation
577

1
v
50

-10 0 10 20 30 40

( PM-' 1
Figure 6. Lineweaver-Burk Plot for dUTPase
Nuclei were diluted 20 (O-O) or 100 fold (O---+) with isotonic HEPES (Winnacker et al., 1972) and Incubated for 10 min at 25°C with
different concentration& of u-Y-dUTP.

The data described so far indicate that the pres- 1972; Magnusson et al., 1973). Isolated nuclei can
ence of a small amount of dUTP together with a therefore be used to study the mechanisms in-
large excess of dTTP suffices to give rise to short volved in the synthesis of these fragments. In this
fragments of the approximate size of Okazaki paper, we demonstrate that complete replacement
pieces. It was important to establish whether these of dTTP by dUTP in the incubation mixture gave
fragments-similar to Okazaki pieces-can be rise to only very short strands and that synthesis
chased into longer strands. To investigate this apparently stopped after a short time. When both
question, nuclei were first incubated for 2 min with nucleotides were present together, a parallel incor-
10 PM 3H-dTTP with and without 1 PM dUTP. At poration into strands of different lengths was ob-
this point, all dUTP in the incubation mixture had served. A decrease in the length of strands was
been degraded by dUTPase as determined by paper found with as little as 2.5% dUTP.
chromatography. One group of samples was ana- We believe that our results are in all probability
lyzed at this point, whereas nonlabeled dTTP (120 explained by the fact that incorporation of uracil
PM) was added to a second group which was into DNA triggers the repair mechanism outlined in
further incubated for 30 min. Figure 7 which leads to strand breakage. Evidence
Both groups were analyzed by alkaline sucrose for such a mechanism is provided in particular by
gradient centrifugation as shown in Figure 7. The the effects of adding uracil to the system. Uracil is
presence of dUTP during the first 2 min of incuba- known to inhibit the DNA glycosidase (Lindahl et
tion increased the amount of labeled short strands al., 1977). As demonstrated by the results in Table
in the Okazaki region from 41-49%. During the 1 and Figures 3 and 4, addition of uracil increased
subsequent chase, all Okazaki pieces in both cases both the total incorporation of labeled nucleotides
were transferred to longer strands, demonstrating into DNA and the size of the strands. Taken to-
that the short fragments arising from the incorpo- gether, these data suggest that both an endonucle-
ration of dUTP could also be chased efficiently. ase and an exonuclease are involved in the gener-
ation of short fragments in our system. The partic-
Discussion ipation of an exonuclease is suggested by our
finding (Table 1) that addition of dUTP results in an
Earlier work showed that in isolated nuclei, labeled apparent decreased incorporation of dUMP +
deoxynucleoside triphosphates are incorporated dTMP into DNA (= 1.5 pmole) as compared to the
intact into polyoma DNA, and that such incorpora- control (= 2.8 pmole), and that further addition of
tion represents semiconservative synthesis and oc- uracil brings this value back to the control value.
curs via the intermediate formation of short frag- With high concentrations of dUTP relative to
ments (Winnacker, Magnusson and Reichard, dTTP, short fragments are poorly transformed into
Cell
578

longer strands: Only the first class, however,

should contain initiator RNA, and this might serve
as a distinction between the two classes.
Under what circumstances may Okazaki frag-
ments of the second class arise? During in vitro
conditions, dUTP could be added inadvertantly
together with other deoxynucleotides or formed
enzymatically during incubation. Figure 8 shows
that in vivo dUTP is a normal intermediate in
thymidine biosynthesis and is formed via reduction
of either UDP or CDP. In this connection, we would
like to point out some differences between mam-
malian and microbial systems. In microorganisms,
dUTP is formed by a direct deamination of dCTP
(Neuhard, 1968), whereas in mammalian cells,
dCMP is deaminated (Maley and Maley, 1959).
Furthermore, compartmentation of DNA synthesis
to the cell nucleus may also be of importance in
mammalian cells. It would be desirable to establish
with certainty the cellular localization of the en-
zyme reactions outlined in Figure 8.
Clearly the chances of Okazaki fragments arising
by a mechanism involving uracil incorporation will
depend upon the balance between the reactions
leading to the synthesis of dUTP and the degrada-
10 20 tion of the nucleotide by dUTPase. In dut mutants
Fraction number from bottom of E. coli (Tye et al., 1977), a moderate decrease in
Figure 7. Chase of Short Fragments Formed from dUTP in the dUTPase activity (2-8 fold) already led to a de-
Presence of dTTP crease in the size of pulse-labeled DNA strands.
Nuclei were incubated under standard conditions with IO PM 3H- Pure dUTPase from E. coli has a K, of 12 PM for
dTTP (O+) or 10 PM 3H-dTTP + 1 +M dUTP ( 0- - - 0). In (A)
dUTP (Shlomai and Kornberg, 1978). This relatively
incubation was stopped after 2 min, and the size of progeny
high value and the cellular abundance of the en-
strands was determined. In (6) unlabeled dTTP (120 FM) was
added and incubation was continued for 30 min before alkaline zyme have led to the suggestion that incorporation
sucrose centrifugation. of uracil, followed by the excision-repair mecha-
nism, may be a normal or even necessary event
longer strands. In the extreme case of the complete during DNA replication in E. coli (Shlomai and
replacement of dTTP by dUTP, no long strands are Kornberg, 1978). As demonstrated in this paper,
formed. The system probably reaches a dynamic the K, value for dUTP in our system is about two
equilibrium where synthesis is balanced by degra- orders of magnitude lower. There is as yet no direct
dation. On the other hand, when only small evidence for the involvement of the excision-repair
amounts of dUTP were added together with sub- mechanism during polyoma DNA replication. Re-
strate amounts of dTTP, and the added dUTP cent results concerning the strandedness of Oka-
during the first part of the experiment was de- zaki pieces formed in vitro (Hunter, Francke and
graded by the dUTPase present in nuclei, the short Bacheler, 1977; Flory, 1977) or in vivo (Perlman
fragments were efficiently chased into longer and Huberman, 1977) however, could possibly be
strands. Thus fragments arising from uracil incor- explained by such a mechanism. Short fragments
poration in this respect behave like “normal” Oka- were apparently found on both sides of the repli-
zaki pieces. cation fork, but the amount of fragments originat-
Thus one can visualize that during polyoma DNA ing from the strand growing in the 3’ to 5’ direction
replication, two classes of Okazaki fragments was 2-5 fold larger than from the strand with the
might originate at the replication fork-one class opposite polarity. An attractive hypothesis would
arising directly during retrograde synthesis of the be that the excision-repair mechanism was respon-
strand synthesized in the 3’ + 5’ direction, and a sible for fragments arising from both strands, while
second class arising by the excision-repair mecha- only fragments from the 3’ to 5’ strand arose via an
nism depicted in Figure 1 from synthesis of both initiating mechanism and should contain initiator
strands. Both classes of Okazaki fragments would RNA at their 5’ end.
only exist transiently and be normally ligated to A second instance in which an excision-repair-
Okazaki Fragments from Uracil lncorporahon
579

CDP

dCTP -F dCDP
I reductase

B dCMP

deaminase deaminase
( E. cob ) UDP (mammals )

reductase

dUTP _ dUDP W

Figure 8. Enzyme Reactions Involved in the Brosynthesis of Thymrdine Nucleotides

type mechanism could be involved concerns the and neutralization to pH 7.6 with triethylamrne, 40 nmole of non-
inhibition of polyoma DNA replication by hydroxy- labeled dUTP were added, and the solution was chromatographed
on a 5 ml column of DEAE-Sephadex with a triethylamine gradrent
urea (Magnusson, 1973). The drug acts by inhibit- [200 ml each of 0.05 and 0.5 M solutions (pH 7.6)]. dUTP was
ing ribonucleotide reductase, and its effect on DNA eluted after about 170 ml, well separated from remaining dCTP.
synthesis depends upon a depletion of the dGTP After evaporation to dryness in a vacuum, a@‘P-dUTP was
pool (Skoog and Nordenskjold, 1971). The size of dissolved in 0.1 ml and had a specific activity of 16,000 cpm/
the dUTP pool is not known. Addition of hydroxy- pmole.

urea to polyoma-infected cells leads to a rapid de-

Incubation of Nuclei
crease of the incorporation of labeled thymidine Polyoma virus-infected nuclei were prepared as descrrbed (Otto
into polyoma DNA. The residual incorporation oc- and Reichard, 1975). Incubation of nuclei (corresponding to
curs almost exclusively in short Okazaki fragments about 50 Fg of DNA) under standard conditions (compare Win-
(Magnusson, 1973). This situation could arise if, nacker et al., 1972) was as follows: 2 mM ATP, 5 mM MgCl2, 5
mM EGTA, 40 mM NaCl, 0.05 mM of each nonlabeled deoxynu-
after hydroxyurea addition, an excision-repair cleoside triphosphate, 0.01 mM of labeled deoxynucleoside tri-
process proceeded unabated, while chain elonga- phosphate, 5 mM phosphoenol pyruvate, pyruvate kinase (15 pg/
tion was inhibited by lack of dGTP. ml), 200 mM sucrose, 30 mM HEPES buffer (pH 8.0), 0.4 mM
The significance of our results lies in the dem- CaCll and 0.8 mM dithiothreitol. Incubation was at 25°C in a final
volume of 0.05 ml. The reaction was stopped as described
onstration that an excision-repair mechanism in a
(Winnacker et al., 1972), and polyoma DNA was selectively ex-
mammalian system can give rise to Okazaki pieces. tracted (Hirt, 1967). Total DNA synthesis was determined by
In several cases, this raises questions concerning measuring acid-preopitable radioactivity. The size of progeny
the origin of such fragments. Answers to these strands was determined by alkaline sucrose gradient centrifuga-
questions must await further experimentation. In tion (5-20% sucrose in 0.3 M NaOH and 0.7 M NaCI, using either
a Spinco rotor SW 56 for 4.5 hr at 50,000 rpm, or an SW41 for 18
this connection, we could also like to point out that
hr at 40,000 rpm). In some gradients, a mixture of fragments 5 +
other excision-repair mechanisms, not necessarily 6 (about 400 nucleotides in length) obtained by cleavage of 3H-
involving uracil incorporation, may occur and con- polyoma DNA with Hpa II (Griffin, Fried and Cowie, 1974) was
tribute to the generation of Okazaki fragments. included as an internal marker.

Experimental Procedures Activity and K, Value of dUTPase

dUTPase activity was measured by incubating drfferent concen-
Labeled Nucleotides trations of u-32P-dUTP with nuclei for 10 min under the condi-
%H-dTTP (11,000 cpm/pmole), &2P-dGTP (5-10,000 cpm/pmole) tions used for polyoma DNA synthesis. The reactron was stopped
and ol-32P-dCTP (original spec. act. 250 Ci/mmole) were obtained by immersion of the tubes in a boiling water bath for 1 min. After
from Amersham. centrifugation, 3 ~1 of the supernatant solution were applied to
cu-32P-dUTP was prepared by deamination from dCTP in the polyethylene imine plates together with carrier dUMP, dUDP and
following way: 0.5 mC of 32P-dCTP were evaporated to dryness, dUTP. The chromatograms were developed first with water 2 cm
dissolved in 0.10 ml of 3 M NaNO%, dissolved in M acetic acid and beyond the start line, and then with 1 M LiCl to the top of the
kept at room temperature for 4 hr. After dilution with 5 ml of water plate. The radioactivrty present in the dUTP. dUDP and dUMP
 Cell
580

spots was determined, and the amount of dUMP produced could Machida, Y., Okazaki. T. and Okazaki, R. (1977). Discontinuous
be calculated from the known specific activity of the triphosphate. replication of replicative form DNA from bacteriophage +X 174.
Proc. Nat. Acad. Sci. USA 74, 2776-2779.
Acknowledgments Magnusson, G. (1973). Hydroxyurea-induced accumulation of
short fragments during polyoma DNA replication. I. Characteriza-
This work was supported by grants from the Swedish Cancer tion of fragments. J. Virol. 72, 600-608.
Society and Magnus Bergvalls stiftelse.
Magnusson, G., Pigiet, V:, Winnacker, E. L., Abrams, R. and
The costs of publication of this article were defrayed in part by
Reichard, P. (1973). RNA-linked short DNA fragments during
the payment of page charges. This article must therefore be
polyoma replication. Proc. Nat. Acad. Sci. USA 70, 412-415.
hereby marked “advertisement” in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact. Maley, G. F. and Maley, F. (1959). Nucleotide interconversions.
IV. Activities of deoxycytidylate deaminase and thymidylate syn-
Received October 17,1977; revised December 14.1977 thetase in normal livers and hepatomas. J. Bial. Chem. 234, 2975-
2980.
References Neuhard, J. (1968). Pyrimidine nucleotide metabolism and path-
ways of thymidine triphosphate synthesis in Salmonella typhimu-
Bertani, L. E., Haggmark, A. and Reichard, P. (1961). Synthesis rium. J. Bacterial. 96, 1519-1527.
of pyrimidine deoxyribonucleoside diphosphates with enzymes Okazaki, R., Okazaki, T., Sakabe, K., Sugimoto, K., Kainuma, R.,
from Escherichia coli. J. Biol. Chem. 236, PC67-PC68. Sugino, A. and Iwatsuki. N. (1968). In vivo mechanism of DNA
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