Moreover, pol d neutralizing antibodies inhibited replicative DNA synthesis most efficiently in late S-phase nuclei, whereas antibodies against pol e were most potent in early S phase.. I
Trang 1DNA replication
Anna K Rytko¨nen1,2, Markku Vaara2, Tamar Nethanel3, Gabriel Kaufmann3, Raija Sormunen4, Esa La¨a¨ra¨5, Heinz-Peter Nasheuer6, Amal Rahmeh7, Marietta Y W T Lee7, Juhani E Syva¨oja2 and Helmut Pospiech1
1 Biocenter Oulu and Department of Biochemistry, University of Oulu, Finland
2 Department of Biology, University of Joensuu, Finland
3 Department of Biochemistry, Tel Aviv University, Israel
4 Biocenter Oulu and Department of Pathology, University of Oulu, Finland
5 Department of Mathematical Sciences, University of Oulu, Finland
6 National University of Ireland, Department of Biochemistry, Cell Cycle Control Laboratory, Galway, Ireland
7 Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY, USA
DNA polymerases (pols) have a central role in DNA
replication and maintenance of chromosomal DNA
[1] At least 14 pols have been identified in the
mam-malian cell, but only three – pols a, d and e – are
needed to synthesize the bulk of DNA during nuclear
DNA replication These pols are structurally related,
belonging to the family B DNA polymerases [2]
Nonetheless, all three perform additional roles in other
DNA transactions as well as transduce signals of cell
cycle control and DNA damage response [1]
Only pol a is capable of initiating DNA synthesis
de novo owing to its associated primase activity [3] The major function of pol a⁄ primase is synthesizing a short RNA–DNA primer of 30–40 nucleotides that serves both to initiate leading strand DNA replication and to provide precursors of the 200 nucleotide-long Okazaki fragments on the lagging strand [4–6] Pol
a⁄ primase is then replaced by the elongating pols d or
e This switch from pol a to pol d is controlled by rep-lication factor C, which loads the processivity factor,
Keywords
cell cycle; DNA polymerase; DNA
replication; electron microscopy; UV
cross-linking
Correspondence
H Pospiech, Department of Biochemistry,
PO Box 3000, FIN-90014 University of Oulu,
Finland
Fax: +358 8 553 1141
Tel: +358 8 553 1155
E-mail: helmut.pospiech@oulu.fi
(Received 20 March 2006, revised 3 May
2006, accepted 5 May 2006)
doi:10.1111/j.1742-4658.2006.05310.x
The contributions of human DNA polymerases (pols) a, d and e during S-phase progression were studied in order to elaborate how these enzymes co-ordinate their functions during nuclear DNA replication Pol d was three to four times more intensely UV cross-linked to nascent DNA in late compared with early S phase, whereas the cross-linking of pols a and e remained nearly constant throughout the S phase Consistently, the chro-matin-bound fraction of pol d, unlike pols a and e, increased in the late
S phase Moreover, pol d neutralizing antibodies inhibited replicative DNA synthesis most efficiently in late S-phase nuclei, whereas antibodies against pol e were most potent in early S phase Ultrastructural localization of the pols by immuno-electron microscopy revealed pol e to localize predomin-antly to ring-shaped clusters at electron-dense regions of the nucleus, whereas pol d was mainly dispersed on fibrous structures Pol a and prolif-erating cell nuclear antigen displayed partial colocalization with pol d and
e, despite the very limited colocalization of the latter two pols These data are consistent with models where pols d and e pursue their functions at least partly independently during DNA replication
Abbreviations
BrdU, bromodeoxyuridine; CLSM, confocal laser-scanning microscopy; EM, electron microscopy; immuno-EM, immuno electron microscopy; MCM2, minichromosome maintenance 2; NP-40, Nonidet P-40; PCNA, proliferating cell nuclear antigen; pol, DNA polymerase.
Trang 2proliferating cell nuclear antigen (PCNA), onto the 3¢
end of the RNA–DNA primer [7]
Simian virus 40 (SV40) has provided the
predomin-ant mammalian model system for DNA replication in
the last three decades In SV40 replication, the host
cell provides all the replication factors, except for the
viral large T antigen, which acts as the initiator protein
and replicative helicase [8] Interestingly, only pols a
and d are required for the virus to replicate, whereas
pol e seems dispensable [9–11] In contrast, studies in
yeasts and in animal systems indicate that both pols d
and e are required for nuclear DNA replication [1,12]
Although pol e is essential for viability both in the
budding yeast, Saccharomyces cerevisiae [13,14], and
the fission yeast Schizosaccharomyces pombe [15,16], it
is the C-terminal checkpoint domain [17], rather than
the N-proximal catalytic pol domain, that executes the
essential function [18–20] Nevertheless, the catalytic
activity of pol e seems to partake in DNA replication
in a number of eukaryotic models [9,11,20–22]
Several hypotheses have been proposed to account
for the requirement of both pols d and e in nuclear
DNA replication Most models placed the two pols on
opposite arms of the replication fork [12] This view is
supported by genetic studies that demonstrate a strand
bias in replication fidelity of proofreading-deficient
pols d and e yeast mutants [23,24] A bias for
replica-tion errors on the leading and lagging strands also
appears to be established by origins of replication [25]
However, pols d and e still await specific assignment to
the leading or the lagging strand by this method
Moreover, the contributions of DNA checkpoint
con-trol and DNA repair processes on strand-specific error
bias also need to be established in more detail [26,27]
Other models have allocated a role for pol e during
specific stages of DNA replication Mostly, they
impli-cate pol e in the initiation of replication [28–31] On
the other hand, a role in late DNA replication has
been proposed for human pol e, based on confocal
laser-scanning microscopy (CLSM) [32]
In the present study we addressed the specific
contri-butions of pols a, d and e to nuclear replication by
fol-lowing their behaviour during S-phase progression,
using four different methods, namely (a) studying their
cross-linking to newly synthesized DNA, (b)
determin-ing their association with chromatin, (c) followdetermin-ing the
effect of cognate inhibitory antibodies on DNA
repli-cation in isolated nuclei and (d) localizing the pols
by immuno-electron microscopy (immuno-EM) The
results suggest that pol a is continuously involved in
replication throughout the S phase, pol e is more
act-ive in early S phase and pol d is actact-ive during the later
stages Moreover, pol e colocalized with pol a in
ring-shaped clusters within electron-dense regions of the nucleus, whereas pol d was mainly dispersed in fibrous structures Taken together, these data are con-sistent with models where pols d and e pursue their functions independently during DNA replication
Results
The cross-linking efficiencies of the three replicases to nascent DNA change during S-phase progression
To evaluate the specific contributions of pols a, d and
e to DNA replication, we studied their association with nascent DNA as a function of S-phase progression HeLa cells were synchronized with mimosine, which blocks cells at the G1⁄ S border prior to initiation of replication [33,34] Two hours after release from the block, cells have entered S phase, and after 14 h they were found to have entered the G2⁄ M phase of the cell cycle (Fig 1A) We utilized the DNA polymerase trap technique to tag the pols with their DNA products [11,35] Nascent nuclear DNA was briefly pulse-labelled with bromodeoxyuridine (BrdU) UTP and [32P]dATP[aP] in a monolayer of nuclei isolated from synchronized cells Subsequent digestion with DNase left the pols photolabelled with a residual radioactive DNA adduct The photolabelled proteins were then separated from the bulk DNA and the pols were immunoprecipitated with an excess of specific anti-bodies Analysis of precipitated protein and superna-tant indicated that the immunoprecipitation efficiency remained constant at different time points (data not shown) After resolution on SDS⁄ PAGE and transfer
to poly(vinylidene difluoride) membrane, the specific photolabelled products could be related to the corres-ponding immunoblotting signals (Fig 1B) Although this method did not reveal the absolute level of the pols engaged in DNA synthesis, it allowed evaluation
of the relative changes in their level as a function of S-phase progression
If pols a, d and e function co-ordinately in a com-mon replication fork, one would expect similar chan-ges in their photolabelling intensity during S-phase progression However, as indicated by the results shown in Fig 1B,C, pols a, d and e consistently dem-onstrated different behaviours during the S phase The photolabelling intensity of pol a (Fig 1C, upper panel) increased only slightly during the later stages of the
S phase Statistical modelling indicated that the photo-labelling of pol a could be fitted well into a linear model The increase of relative photolabelling for pol a, expressed as a linear trend coefficient of the log
Trang 3over the whole time range, was 0.025Æh)1(SE¼ 0.018),
implying a 1.3-fold increase in relative photolabelling
over the time range
Pol e behaved similarly to pol a, the estimated slope
being 0.046Æh)1 (SE¼ 0.018) However, the increase
was not monotonic; a plateau was observed at mid S
phase 8 h after release from mimosine block (Fig 1C,
bottom panel) In contrast, pol d showed a continuous
rise in relative photolabelling throughout the S phase
(Fig 1B,C) The estimated slope was 0.113Æh)1 (SE¼
0.018) This corresponds to a three- to fourfold
increase from immediate early to late S phase and was clearly higher than the fluctuation of 1.5-fold observed for pols a and e (P¼ 0.0036 for this compar-ison, see supplementary Doc S1 for a more detailed description of the statistical analysis)
Chromatin association of the replicases during
S phase The increase in relative photolabelling of pol d could
be attributed to an increase in cross-linking to nascent
B
Fig 1 Photolabelling of DNA polymerases (pols) a, d and e during the S phase The activity of the pols during the S phase was studied by using a UV cross-linking technique HeLa cells were synchronized with mimosine, which blocks cells at the G1 ⁄ S border, then released from the block for 2 h (very early S phase), 5 h (early S phase), 8 h (middle S phase) or 12 h (late S phase) and photolabelled Pols a, d and e, and their photolabelled derivatives, were monitored as described in the Experimental procedures (A) Cell synchronization Flow cytometric analy-sis indicates the DNA content of HeLa cells throughout the time course of a typical mimosine synchronization (B) Autoradiogram and west-ern blot analysis of a representative experiment (C) Photolabelling efficiency (autoradiography) and immunoreactive protein (westwest-ern blot analysis) were densitometrically quantified and the ratios of these values were normalized against the average of the respective experiment The results of five independent experiments on pols a, d and e, respectively, are presented with different marks The average for each pol is shown as a bold line.
Trang 4DNA in late S phase or to a decrease in the protein
level associated with chromatin, because the calculated
cross-linking intensities represent the ratio of these
qualities Inspection of our DNA polymerase trap
experiments indicated an increase of the
immunoreac-tive pol d protein level during S phase (Fig 1B, lower
panel) We therefore determined the association of pols
a, d and e directly with chromatin We utilized a
sim-ple high-salt extraction scheme that permitted
compar-ison with the results from the concurrent polymerase
trap experiments
HeLa cells were synchronized with mimosine at the
G1⁄ S boundary After release from the block, cells at
defined stages of the S phase were lysed in hypotonic
buffer in the presence of Nonidet P-40 (NP-40)
deter-gent to release deterdeter-gent-soluble protein, including
nucleosolic proteins (soluble fraction) The second
fraction contained proteins released by high-salt extraction from the remaining monolayer of open nuc-lei and included the chromatin-associated proteins (‘bound’) The remaining material was solubilized in SDS (rest fraction) The quality of the fractionation was monitored by western blot analysis of marker pro-teins (Fig 2A) from asynchronous cells Markers for the soluble fraction included the Golgi marker GM130, the endoplasmic reticulum-specific marker protein disulfide isomerase (PDI) and b-tubulin These proteins were found exclusively in the soluble fraction, indicating that the high-salt and rest fractions are largely free of soluble contaminants The chromatin marker, minichromosome maintenance deficient-2 (MCM2), was distributed between the soluble and the bound fraction, as expected [36] A similar distribution was found for PCNA (Fig 2A) Lamins A⁄ C were
Fig 2 Association of DNA polymerases (pols) to chromatin during the S phase Proteins were synchronized with mimosine and fractionated
to result in a Nonidet P-40 soluble fraction, a high-salt (bound) fraction, and a remaining matrix fraction (rest), as outlined in the Experimental procedures (A) Western blot analysis of marker proteins in a cell fractionation from asynchronous cells Extracts representing an equal num-ber of cells were loaded from each fraction The pan-histone antibody recognized multiple bands, corresponding to core and linker histones,
as indicated by dots (B) Levels of bound pols a, d and e during the S phase, as determined by western blot analysis SYPRO orange staining was used to monitor and normalize loading of the gel Lane A is an asynchronous control (C) Densitometric quantification of the bound pro-tein levels The results represent the average of two independent cell fractionations Repetitions of the western blot analysis were averaged for each fractionation and pol.
Trang 5detected in the high-salt and rest fractions, but not in
the soluble fractions Histones were identified mainly
in the high-salt fraction In asynchronous cells, pols a,
d and e were distributed, to various extents, between
the soluble and the high-salt fractions (Fig 2A) These
results indicate that the NP-40-resistant high-salt
frac-tion represents a good approximafrac-tion for the
chroma-tin-bound pols
We then followed the NP-40-resistant high-salt
frac-tion of the pols as a funcfrac-tion of cell cycle progression
Results from representative western blot analyses of
their high-salt fractions are presented in Fig 2B All
three pols were detected in these fractions Notably,
pol e appeared as a double band after long runs in
low-percentage gels These bands could be accounted
for by post-translational modification, proteolysis,
alternative splicing or alternative promoter usage [37]
The relative abundance of the two forms did not vary
during the S phase
As can be seen from the densitometric quantification
of the experiments presented in Fig 2C and their
statistical modelling, NP-40-resistant levels of pol e
appeared to be largely constant or slightly decreasing
(slope)0.014Æh)1; SE¼ 0.016, corresponding to a
1.3-fold decrease during 17 h) In contrast, NP-40-resistant
pol a seems to have an increasing trend (slope
0.024Æh)1, SE¼ 0.012, corresponding to a 1.5-fold
increase) The pol d levels increased even more rapidly,
approximately twofold during the S phase (slope
0.043Æh)1, SE¼ 0.013) The changes detected in the
levels of the pols in the high-salt fraction are only
moderate during the S phase; however, the difference
between the replicative pols becomes apparent by
pairwise comparison of the time trend of chromatin
association The difference between the slopes of
pol d and pol a was 0.019Æh)1 (SE¼ 0.018, P ¼ 0.3),
between pol d and pol e was 0.057Æh)1 (SE¼ 0.020,
P¼ 0.005), and for pol e vs pol a it was )0.038Æh)1
(SE¼ 0.020, P ¼ 0.05) The time trend deviated
from a linear pattern for pol e, but allowing for
curvature had no effect on the contrasts of its average
slope vs those of pol a and d, respectively Therefore,
the change in NP-40-resistant, high-salt-extractable
pol e appears to be different from those of pols a
and d
Inhibitory effects of antibodies against the
replicases at different S-phase stages
We further evaluated the temporal differences between
the contributions of pols d and e to DNA replication
by studying the effects of cognate neutralizing
antibod-ies on the pol activitantibod-ies in nuclei isolated at different
stages of the S phase [38–40] We have previously shown that polyclonal antibody K18 against pol e inhibits replication in isolated nuclei from asynchro-nous HeLa cells to a level similar to that of the well-characterized, neutralizing antibody, SJK-132-20, against pol a [9,41] We extended this study by inclu-ding antibody 78F5, which neutralizes specifically the pol d activity [42] and by following the effect of the antibodies against the three pols as a function of S-phase progression For this purpose, synchronized HeLa monolayer cells were released from the mimosine block, and the resultant G1⁄ S, early, middle and late S-phase cells were studied in the DNA replication assay
Antibody SJK-132-20 against pol a inhibited consis-tently 50% of the replicative DNA synthesis, irres-pective of the S-phase stage (Fig 3A, estimated slope 0.3%Æh)1, SE¼ 0.51%) In contrast, the inhibition of replicative DNA synthesis by antibody 78F5 against pol d increased almost threefold, from 17 to 48%, as cells progressed from the G1⁄ S boundary to the late
S phase (Fig 3A) (slope 2.6%Æh)1, SE¼ 0.70%) At the same time, inhibition of DNA replication by anti-body K18 against pol e dropped from 45 to 24%, reaching a minimum 8 h after release from mimosine block (slope )2.7%Æh)1, SE¼ 0.81% for the first 8 h) The difference between pols d and e was striking The difference between the slopes of pol d and pol e was 3.7%Æh)1(SE¼ 1.0%, P ¼ 0.0003), using a model with separate linear and quadratic terms to allow for the nonlinear behaviour of pol e in late S phase
Mimosine, which has been utilized for cell synchron-ization in this study, has been found to induce DNA damage [43] Therefore, we considered that the detec-ted differences between pols a, d and e could be influ-enced by checkpoint response, or may reflect DNA repair, rather than differences in the contribution to DNA replication We therefore stimulated T98G cells
to proliferate after prolonged serum deprivation and tested the effect of neutralizing antibodies on replica-tion in nuclei from these cells Nuclei were from T98G cells, 12 h (early S phase) and 20 h (late S phase) after serum stimulation (see Fig S1 for flow cytrometric analysis of a typical serum stimulation) Comparable
to replication in mimosine-synchronized HeLa nuclei, DNA synthesis was found to be reduced by 60% by the anti-pol a Ig, both in early and late S phases (Fig 3B) Inhibition by pol e antibodies dropped, in general, from 60% in early, to 18% in late, S phase
At the same time, inhibition by antibodies against pol d showed a general increase, from 8 to 69% These results are comparable to the results obtained with mimosine-synchronized HeLa cells The contrast
Trang 6between pols d and e are even more pronounced in
nuclei undergoing an unperturbed S phase than in
nuc-lei synchronized by mimosine The differences observed
between pols d and e are therefore not provoked by
possible DNA damage caused by mimosine
synchron-ization
The results presented above indicate that the
requirement of pol a activity remains constant
throughout the S phase As the other replicative pols
depend on primer synthesis by pol a-primase, the
effect of the anti-pol a Ig could well represent the
cumulative inhibition of pol a and the subsequent
elongating enzyme Pol e activity contributes to DNA
replication more at the G1⁄ S transition, and its relative
importance diminishes as the S phase progresses On
the other hand, the requirement of pol d activity is
lowest in the early S phase and increases as the
S phase proceeds
Pols d and e localize differently during the
S phase Next, we studied the nuclear localization of pols a, d and e as a function of S-phase progression Human IMR-90 primary fibroblasts were synchronized with mimosine, after splitting from confluency, to achieve a sharp entry into S phase (Fig S2) Cells were then col-lected at different time points to study the localization pattern of the three pols and PCNA during the indica-ted cell cycle stages by immuno-EM We chose EM, because this technique permits studying localization at near molecular resolution, and localizations can be related to nuclear structures after standard contrasting Moreover, there is no requirement for treatment with detergent or other manipulations that remove part of the protein from the nucleus Ultrathin cryo-sectioning was performed directly from extensively fixed cells
Fig 3 Effect of inhibitory antibodies on
replicative DNA synthesis in isolated,
permeabilized nuclei during the S phase.
Replicative DNA synthesis using isolated
nuclei in the presence of excess
cytoplas-mic extract was measured as incorporation
of radioactive dCMP into newly synthesized
DNA Levels of inhibition by specific
anti-bodies from independent replication
reac-tions are plotted for each DNA polymerase
(pol) The line represents the average for
each pol (A) Inhibition of replication in
isolated HeLa cell nuclei synchronized with
mimosine (B) Inhibition of replication in
serum-stimulated T98G cells Results of
ind-ividual experiments for pols a, d and e are
plotted as triangles, filled circles and
dia-monds, respectively Lines indicate the
aver-age inhibition by antibodies against the
cognate pols.
Trang 7After selection of suitable antibodies and optimization
of the conditions, double staining was carried out with
two successive antibodies and protein A conjugated
with 5 and 10 nm gold particles, respectively
As can be seen from the double stainings of pols a
and e, mouse mAb CL22-2-42B, against the catalytic
subunit of pol a, is well suited for immuno-gold
label-ling (Fig 4, left panel) [44] The same antibody has
been previously employed successfully for immuno-EM
of resin-embedded cells [45,46] Similarly to the
previ-ous studies, pol a could be detected mainly at
electron-dense regions of the nucleus Pol a labelling appears,
in part, as ring-shaped, focal structures alone, or it
colocalizes with pol e (Fig 4, left panel, asterisks), or
as more disperse staining of discrete nuclear regions,
which is particularly visible at later stages of the S
phase (Fig 4D,E) In earlier studies, the ring-shaped
foci of pol a were shown to coincide with sites of
DNA synthesis [45,46] They were also shown to
repre-sent replication factories that appear as ovoid bodies
attached to the nucleoskeleton in thick sections [46–
48] Although the foci are largest and most abundant
during G1⁄ S transition and early S phase, pol a
appears to be rather evenly distributed between
ring-shaped and dispersed structures When enumerating
the gold particles from 17 pol a⁄ e double staining
ser-ies from two independent synchronizations, we found
that 41% of pol a localized in foci (Table 1) The
rel-ative level of pol a in foci was rather constant until
late S phase⁄ G2 transition, where the levels appeared
to decrease (data not shown) This is consistent with
the work of Lattanzi and coworkers [46], who reported
the ring-shaped pol a foci to disappear in the G2⁄ M
phase
As evident from Fig 4 (left panel), pol a colocalizes
at a near-molecular level with pol e stained with mAb
H3B [49] mAb G1A [49], against pol e, gave a similar
localization pattern as mAb H3B, and both mAbs
colocalized in double stainings (data not shown),
indi-cating the specificity of the staining The colocalization
of pol a and e is largely confined to the ring-shaped
foci In fact, more than half of all detectable foci
con-tained both pol a and e (data not shown) This is not
surprising, because pol e staining is concentrated in
foci, 75% of pol e being focal in pol a⁄ e double
stai-nings (Table 1) Similarly to pol a, pol e in foci
appears to be most pronounced from G1 up to early
S phase (Fig 4A–C, asterisks), but pol e levels in the
staining decreased relative to pol a as the S phase
pro-gressed (Fig 5)
For detection of pol d, we utilized rat mAb
PDK-7B4 against p50, the B subunit of human pol d [50]
(Fig 4, right panel, 5 nm gold particles) p50 has
previously been shown, by immunofluorescence micro-scopy, to colocalize with the catalytic subunit [51] From double stainings of pol d and e, it became apparent that pol d mainly localizes outside the ring-shaped foci, which are typical of pols a and e (Fig 4, right panel) In 17 pol d⁄ e double staining series from different S-phase stages, only 30% of pol d-directed gold particles were found in the foci of three or more particles, whereas 74% of pol e was focal in the same series (Table 1) This difference persisted throughout the cell cycle period studied from G1 until late S phase Although some pol d-directed gold parti-cles could be detected in foci, the abundance of pol d
in the foci was small compared with Pol e Pol d stain-ing was instead dispersed, but restricted to distinct ter-ritories of the nucleus It is notable that whereas some areas of the nucleus showed strong staining, neigh-bouring regions remained largely free of pol d (Fig 4I,K) Pol d directed gold particles located in the vicinity of fibrous structures and often adopted a
‘beads-on-a-string’ structure (Fig 4I,K, arrowheads) The overall staining intensity of pol d relative to pol e increased as the S phase progressed, and peaked in mid⁄ late S phase 8–12 h after release from mimosine block This was accompanied by a sharp drop, of 32%, in the fraction of the pol e-directed gold particles from 0 to 8 h (Fig 5), consistent with an augmented role of pol d in later S phase
We next repeated pol d⁄ e double labelling in T98G cells at different time points after cells were stimulated
to proliferate by serum addition Major features of the pol d and e staining appeared to be conserved between mimosine-synchronized fibroblasts and serum-stimula-ted T98G cells Analysis of a series of pol d⁄ e double-stained cells from the G1⁄ S boundary until the late
S phase (22 h) revealed a similar pattern of mainly focal staining for pol e (65%) and predominantly dis-persed staining for pol d (30%) (Fig 6 and Table 1) Pol e staining was strongest in the early S phase, where large foci prevailed In several foci, residual pol d staining could also be detected As S phase proceeds, relative pol e staining and abundance of foci decreased, as well as the size Foci contained, on aver-age, nine gold particles in early S phase, but only about five gold particles per focus in the mid and late
S phase (Fig 6) Late S-phase samples showed more heterogeneity, probably as a result of cells that failed
to proliferate (Fig S1) For pol d, the dispersed stain-ing detected in fibroblasts prevailed also in the T98G cells throughout S phase, with a minor part of pol d colocalizing to large pol e foci, or forming, less fre-quently, small own foci (typically three gold particles), that may well have arisen from a single pol d molecule
Trang 8owing to the amplification process using secondary
antibodies and protein A
PCNA colocalizes partly with pol d and partly
with pol e
Attempts to detect sites of ongoing DNA replication by
means of BrdU incorporation failed because various
methods of DNA denaturation, required for
immunode-tection of BrdU, destroyed the fine structure of the
cryo-sections In order to obtain further insight into the function of pols d and e, we determined the locations of these proteins, relative to PCNA, by immuno-EM dou-ble staining PCNA is a processivity cofactor of both pols d and e Therefore, it is considered an important marker for active replication [52–55] Still, not necessar-ily all PCNA participate in DNA replication, as PCNA
is more abundant inside the cell than DNA replication forks at a given time, and also partakes in other DNA transactions [54] As can be seen from a comparison
G B
I D
Fig 4 Replicative DNA polymerases (pols)
show distinctive localization patterns in
human IMR-90 fibroblasts synchronized with
mimosine The cells were synchronized to
cell cycle stages, as indicated on the left,
after release from mimosine block Ultrathin
cryosections were then subjected to
immu-nostaining of pol a followed by staining of
pol e (images A–E), or immunostaining of
pol d followed by staining of pol e (images
F–K) Immunolabelling was visualized under
the electron microscope by linking the
pri-mary antibody to protein A coupled to 5 nm
(small: pol a and d) or 10 nm (large: pol e)
gold particles Ring-like focal staining of at
least four particles is marked by asterisks,
and examples of beads-on-a-string like
stain-ing of pol d is shown by arrowheads The
scale bar is 100 nm.
Trang 9between Fig 7 with Fig 4, PCNA behaved similarly to
pol a, yielding a staining pattern that is partly focal
(asterisks) and partly disperse or ‘beads-on-a-string’-like
(arrowheads) Similar patterns of PCNA staining,
coin-ciding at least partly with sites of DNA synthesis, have
been reported in previous EM studies of mammalian
and plant cells [56–60] Focal staining is most apparent
from G1 to early S phase, and foci contain often also
pol e (Fig 7F,G, asterisks), and less frequently pol d
(Fig 7A–C, asterisks) It is noteworthy that although
foci containing only PCNA were rarely detected, pol e
foci free of PCNA were common, in particular in the G1
and early S phases (Fig 7F,G,I, open circles) This
indi-cates that pol e is present in preformed structures As
S phase progresses, pol e staining decreases relative to
PCNA, although the decrease is weaker compared with
the pol d⁄ e double staining (Fig 5) In contrast, the
levels of pol d-directed gold particles remain constant,
or show a slight increase, relative to PCNA during the
S phase (Fig 5)
In double staining of pol d and PCNA, pairs of
small and large gold particles were visualized They
indicate intimate colocalization of the two proteins
(Fig 7D,E, arrows) As immunolabelling is obviously
incomplete, such double-labelling probably detects
only part of potential pol d–PCNA complexes
Taken together, the immuno-EM studies indicate
that pol e adopts mainly a ring-shaped focal staining
that dominates during the early S phase, whereas pol d
is detected mostly as disperse or beads-on-a-string-like
staining that prevails in late S phase As for pol a and
PCNA, they show staining patterns that combine focal
and dispersed features
Discussion
An outstanding question in eukaryotic DNA
replica-tion is how the elongating replicases pols d and e
co-operate to achieve efficient and faithful duplication
of the nuclear DNA We addressed this question in the present study by combining biochemical and cell biolo-gical approaches aiming to determine the spatial and temporal co-ordination of the two pols and additional replication proteins throughout the S phase The main conclusion emerging from this study is that pols d and
e pursue their functions during DNA replication with-out being physically connected, although they may per-form complementary functions at the same replication forks We infer it from the following observations First, the relative contribution of pol d to replicative DNA synthesis increases steadily with progression of the S phase at the expense of pol e This is judged from the different behaviour which the two pols exhib-ited in cross-linking nascent DNA (Fig 1) and binding chromatin (Fig 2), as well as the degree of inhibition
of replicative DNA synthesis attained with cognate inactivating antibodies (Fig 3) Second, immuno-EM visualization revealed that pols d and e localize to mainly different nuclear sites and structures through-out the S phase (Figs 4 and 6)
The more pronounced contribution of pol e in early
S phase agrees with the proposed role in replication initiation Namely, in the budding yeast, chromatin immunoprecipitation has demonstrated that pols a and
e load concurrently onto origins of replication [28–30] Subsequently, these pols transferred from origin to nonorigin DNA concomitantly with Cdc45 and MCM2-7, possibly reflecting their retention at the rep-lication fork junction as the replicated ori DNA moves away [28] Similarly, pol e loads onto chromatin prior
to initiation in Xenopus egg extracts [31] The inde-pendent behaviour of pols d and e observed in this study could further reflect distinct roles of the two enzymes during elongation, possibly participation in the lagging and leading strand DNA synthesis, respect-ively [61,62]
In a recent chromosome-wide scan in the budding yeast, Hiraga et al [61] were able to demonstrate that
Table 1 Distribution of DNA polymerases (pols) between ring-shaped, focal structures and dispersed staining in immuno-electron
microsco-py The number of 5- and 10-nm gold particles were quantified from pol a ⁄ e and pol d ⁄ e double stainings Clusters of three or more gold par-ticles were considered as foci Four to 29 separate images, representing typically eight to nine nuclei, were counted for each of 17 series derived from two independent synchronizations (IMR-90 cells) or seven series derived from one synchronization (T98G cells).
Staining
Particles counted
Particles
in foci
% particles
in foci
Nuclei counted
Images counted
Trang 10all three replicative pols a, d and e are associated with
early firing origins in cells arrested early in S phase
These data suggest that all three replicases participate
in the synthesis at each active origin What is more, the authors recognized a delayed association of pol d with origin ARS305 compared with pols a and e This
is consistent with our data presented here
The functional differences between pols d and e were underscored by their ultrastructural visualization using EM This revealed that the level of immuno-reactive pol e decreases more than threefold relative to pol d during S phase and that each enzyme exhibits a strikingly different localization pattern Whereas pol e stained mainly as ring-shaped foci, pol d adopted a more dispersed staining of discrete nuclear territories with little focal clustering
Both pol a and PCNA show staining patterns more similar to pol e in early S phase and to pol d in late S phase Where studied, pol a and PCNA partially colo-calize with pol d as well as pol e However, colocaliza-tion of pols d and e was very limited in the double staining Hence, pol a and PCNA are present in struc-tures that contain either pol d or e, but rarely, if at all, both Although pol a and PCNA are both well-estab-lished markers for the sites of DNA synthesis using immuno-EM and resin-embedded samples [45–47,52– 60,63], it is still uncertain if all the sites of their colo-calization with pols d and e are actually DNA replica-tion sites In other words, we cannot absolutely exclude the possibility that only a minority of the detected protein is actively engaged in DNA replica-tion, while most observed structures have other func-tions (e.g storage sites for the replication factors) Fuss & Linn [32] studied the localization of pol e
in proliferating primary fibroblasts by CLSM The authors found that pol e formed foci throughout the cell cycle These foci colocalized with PCNA and sites
of DNA synthesis only in late S phase, but were adja-cent to PCNA foci in early S phase, suggesting a role
of pol e in DNA replication late in S phase It is diffi-cult to relate these results to the data presented here The small foci detected by CLSM in early S phase were 300–400 nm across with an optical plane of
600 nm [32] This is considerably larger than the ring-shaped foci observed in immuno-EM (Figs 4 and 6) The latter are, in most cases, between 50 and
100 nm across, using ultrathin sections of 70–80 nm thickness Therefore, the ring-shape foci described here are probably below the detection limit of fluorescence microscopical techniques In contrast, the larger foci described by Fuss & Linn [32] in late S phase corres-pond well in size to the nuclear regions of dispersed staining of PCNA and pol d that are predominant in late S phase These regions span several hundreds of
nm, and contain both dispersed PCNA and focal pol e (Fig 7I,K) Nonetheless, direct colocalization is not
Fig 5 Quantitative analysis of DNA polymerases (pols) a, d and e,
and proliferating cell nuclear antigen (PCNA) in immuno-electron
microscopy The number of nuclear gold particles representing
the indicated proteins were quantified from images taken from the
respective double stainings from synchronized IMR-90 cells The
graphs represent the abundance of a given gold particle relative to
the total number of all gold particles in a given staining series Each
curve represents an independent experiment Typically, 17–20
ima-ges from seven to nine nuclei were analysed per time point in each
experiment.