Báo cáo khoa học: DNA polymerase e associates with the elongating form of RNA polymerase II and nascent transcripts pot

DNA polymerase e associates with the elongating form of
RNA polymerase II and nascent transcripts
Anna K. Rytko
¨
nen
1,2,
*, Tomi Hillukkala
1,
*, Markku Vaara
2
, Miiko Sokka
2
, Maarit Jokela
1,†
,
Raija Sormunen
3
, Heinz-Peter Nasheuer
4
, Tamar Nethanel
5
, Gabriel Kaufmann
5
, Helmut Pospiech
1
and Juhani E. Syva
¨
oja
2
1 Biocenter Oulu and Department of Biochemistry, University of Oulu, Finland
2 Department of Biology, University of Joensuu, Finland

3 Biocenter Oulu and Department of Pathology, University of Oulu, Finland
4 National University of Ireland, Department of Biochemistry, Cell Cycle Control Laboratory, Galway, Ireland
5 Department of Biochemistry, Tel Aviv University, Ramat Aviv, Israel
RNA polymerase II (RNA pol II) transcribes protein-
encoding genes in eukaryotes. It can be purified as a
‘core’ enzyme containing 10–12 subunits with a
molecular mass of  500 kDa. However, larger RNA
pol II-containing complexes, capable of transcribing
from model promoters in vitro with minimal addition
of general transcription factors, have also been purified
[1]. These ‘holoenzyme’ complexes contain general
transcription factors, other transcriptional mediators,
as well as various sets of accessory proteins, such as
chromatin remodelling factors [2]. The carboxy-ter-
minal domain (CTD) of RNA pol II has been implica-
ted in mediating interactions with other factors
involved in transcription and mRNA processing, and
appears to be a major target of regulation. The
CTD comprises tandem heptapeptide repeats of the
Keywords
DNA polymerase e; DNA replication;
immunoelectron microscopy; nucleotide
excision repair; RNA polymerase II
Correspondence
H. Pospiech, Department of Biochemistry,
PO Box 3000, FIN-90014 Oulu, Finland
Fax: +358 8 5531141
Tel: +358 8 5531155
E-mail: Helmut.Pospiech@oulu.fi
†Present address

Department of Internal Medicine, University
of Oulu, Finland
*These authors contributed equally to this
work
(Received 15 September 2006, revised 16
October 2006, accepted 18 October 2006)
doi:10.1111/j.1742-4658.2006.05544.x
DNA polymerase e co-operates with polymerases a and d in the replicative
DNA synthesis of eukaryotic cells. We describe here a specific physical
interaction between DNA polymerase e and RNA polymerase II, evidenced
by reciprocal immunoprecipitation experiments. The interacting RNA
polymerase II was the hyperphosphorylated IIO form implicated in tran-
scriptional elongation, as inferred from (a) its reduced electrophoretic
mobility that was lost upon phosphatase treatment, (b) correlation of the
interaction with phosphorylation of Ser5 of the C-terminal domain hepta-
peptide repeat, and (c) the ability of C-terminal domain kinase inhibitors
to abolish it. Polymerase e was also shown to UV crosslink specifically
a-amanitin-sensitive transcripts, unlike DNA polymerase a that crosslinked
only to RNA-primed nascent DNA. Immunofluorescence microscopy
revealed partial colocalization of RNA polymerase IIO and DNA poly-
merase e, and immunoelectron microscopy revealed RNA polymerase IIO
and DNA polymerase e in defined nuclear clusters at various cell cycle sta-
ges. The RNA polymerase IIO–DNA polymerase e complex did not relo-
calize to specific sites of DNA damage after focal UV damage. Their
interaction was also independent of active DNA synthesis or defined cell
cycle stage.
Abbreviations
BrdU, bromodeoxyuridine; CTD, carboxyterminal domain; DRB, 5,6-dichloro-1-beta-
D-ribobenzimidazole; Pol, DNA polymerase; RNA pol II,
RNA polymerase II; TFIIH, transcription factor II H.

FEBS Journal 273 (2006) 5535–5549 ª 2006 The Authors Journal compilation ª 2006 FEBS 5535
consensus sequence Y-S-P-T-S-P-S and varies in length
among eukaryotes. During or shortly after initiation,
mainly serine 5 of the heptapeptide repeat is phosphor-
ylated in a transcription factor II H (TFIIH)-depend-
ent manner [3]. In the elongation stage of mRNA
synthesis, serine 5 is dephosphorylated and this change
is accompanied by extensive phosphorylation of serine
2. After transcription termination, the CTD is com-
pletely dephosphorylated, rendering RNA pol II com-
petent for subsequent initiation of transcription. The
differential phosphorylation of the CTD could provide
the signal for the co-ordinated sequestration of pro-
cessing factors required for mRNA capping, splicing
and 3¢ end processing [4].
The RNA pol II holoenzyme may also integrate
mRNA transcription with DNA repair, DNA repli-
cation and recombination. Maldonado and coworkers
[5] purified an RNA pol II holoenzyme complex con-
taining multiple DNA replication and repair proteins,
including DNA polymerase (Pol) e, Ku, Rad51, RPA
and RFC. This finding was confirmed and extended
by reports showing that BRCA1 [6–8], BRCA1-asso-
ciated RING domain protein BARD1 [9], MCM
proteins [10–12] and Ku [13] associate with RNA
pol II. Variations in the content of the DNA replica-
tion and repair factors in the RNA pol II holoen-
zyme complexes are described in the different reports
and may be accounted for by different purification
approaches employed [2,7,14].

Transcription factors may be involved in regulating
DNA replication in eukaryotic cells indirectly, by
modifying chromatin structure, or directly, by recruit-
ing protein complexes [15,16]. Their effects are also
reflected by a global co-ordination of cellular tran-
scription and replication. In fact, the more transcrip-
tionally active a chromosomal region, the greater the
likelihood that replication initiates early in S phase
within that domain [17]. Studies in yeast suggest that
recruitment of the RNA pol II complex activates
replication [18] and that the CTD is sufficient for
this positive regulation of DNA replication initiation
[19]. On the other hand, DNA replication and repair
factors, such as MCM5, BRCA1 and Ku, have been
implicated as positive or negative regulators of tran-
scription [8,13,20]. Transcription has also been shown
to induce homologous recombination [21].
Pol e is an essential replication protein involved in
many cellular transactions [22]. Pol e, together with
Pols a and d, is required for synthesizing the bulk of
DNA during replication in mammalian cells [23,24],
but its specific role in this process remains uncertain.
In addition, Pol e is implicated in DNA repair and cell
cycle regulation [22,25–27].
Here, we demonstrate, by co-immunoprecipitation,
immunofluorescence and immunoelectron microscopy,
that Pol e associates specifically with a transcriptional-
ly active, hyperphosphorylated form of RNA pol II.
Results
Pol e associates with RNA pol II

While studying the crosslinking of human replicative
DNA polymerases with newly synthesized nucleic acids,
we found that Pol e-crosslinked nascent RNA not rela-
ted to DNA replication. This hinted at a possible
association of Pol e with the transcription apparatus.
To investigate this possibility, we performed reciprocal
immunoprecipitations with antibodies against RNA
pol II and the three replicative Pols (a, d and e). Preci-
pitates were subjected to extensive washing at physiolo-
gical salt concentration. The immunocomplexes were
collected and analyzed by immunoblotting with anti-
bodies against the cognate proteins (see Table 2, in
Experimental procedures). A polyclonal antibody
against RNA pol II coprecipitated Pol e, but not
Pols a and d (Fig. 1). Reciprocal immunoprecipitation
with antibodies against the replicative Pols a, d and e
indicated that RNA pol II was co-immunoprecipitated
only by the monoclonal antibody against Pol e. Immu-
noprecipitations utilizing polyclonal antiserum K27,
raised against a different region of Pol e, gave identical
results (data not shown). We also excluded the possibil-
ity that the RNA pol II–Pol e interaction was mediated
by the presence of DNA, by performing the experi-
ments also in the presence of ethidium bromide at
concentrations known to disrupt protein–DNA interac-
tions [28]. It is also noteworthy that no interaction
between replicative pols could be observed under the
moderately stringent conditions employed in our
experiments.
Pol e interacts with the hyperphosphorylated

elongating isoform RNA pol IIO
RNA pol II exists in a dynamic equilibrium between
the hypophosphorylated initiation isoform IIA and
the hyperphosphorylated elongating isoform IIO that
exhibits reduced electrophoretic mobility compared
with the IIA isoform. Whereas the polyclonal anti-
RNA pol II antibody precipitated both the fast and
slowly migrating forms of RNA pol II, antibodies to
Pol e coprecipitated only the slowly migrating form of
RNA pol II (Fig. 1). This suggested that Pol e associ-
ates with the RNA pol IIO isoform. To characterize
further the phosphorylation status of the RNA pol II
DNA polymerase e associates with RNA polymerase II A. K. Rytko
¨
nen et al.
5536 FEBS Journal 273 (2006) 5535–5549 ª 2006 The Authors Journal compilation ª 2006 FEBS
complexed with Pol e, we examined the effect of
phosphatase on the precipitated RNA pol II. Immuno-
precipitates were incubated at +37 °C, in the presence
or absence of calf intestinal phosphatase, prior to
western analysis (Fig. 2A). Following phosphatase
treatment, the cellular fraction of RNA pol II that
associated with Pol e showed increased mobility, cor-
responding to that of isoform IIA. The isoform IIO
was also lost from RNA pol II precipitates after phos-
phatase treatment. Thus, Pol e could interact specific-
ally with hyperphosphorylated isoform RNA pol IIO,
possibly during transcriptional elongation. Therefore,
we studied this interaction more closely during the
transcription cycle.

RNA Pol II is sequentially phosphorylated during
the transcription cycle, mainly by cyclin-dependent
kinase activities. During transcription initiation, serine
5 residues of the CTD repeat become mainly phos-
phorylated by Cdk7, a subunit of TFIIH [29,30]. Sub-
sequent phosphorylation on serines at position 2 by
the Cdk9 ⁄ pTEFb is required for transcription elonga-
tion. We employed phospho-specific antibodies for
immunoprecipitation and western blot analysis to
study whether the Pol e interaction is specific in terms
of CTD phosphorylation of RNA pol II. Polyclonal
antibody (N20) recognizes both hyperphosphorylated
IIO and hypophosphorylated IIA forms of RNA
pol II. H14 antibody is specific for early stage RNA
pol II phosphorylated at Ser5, and 8WG16 antibody
recognizes RNA pol II that is not phosphorylated at
Ser2, but may or may not have phosphate at Ser5. H5
is specific for RNA pol II having phosphate at Ser2,
and is considered a marker for elongating RNA pol II
[31]. The form of RNA pol II coprecipitating with
Pol e was recognized by all antibodies to be IIO, but
not IIA (Fig. 2B). Conversely, all different RNA pol II
antibodies always coprecipitated Pol e. The results
indicate that the interaction does not depend on phos-
phorylation of Ser2, because Pol e is coprecipitated by
antibody 8WG16. Alternatively, antibody 8WG16
could have precipitated incompletely phosphorylated
RNA pol II. Phosphorylation of Ser5 alone is suffi-
cient for the interaction. The results do not exclude the
possibility that phosphorylation of Ser2 alone would

also be sufficient for the interaction.
In order to study the interaction with RNA pol II
and Pol e in more detail, we employed chemicals
known to inhibit transcription. Whereas a-amanitin
inhibits transcription both in the initiation and elonga-
tion phases by preventing translocation of DNA and
RNA through the enzyme [32,33], the cdk inhibitors
5,6-dichloro-1-beta-d-ribobenzimidazole (DRB) and
roscovitine inhibit phosphorylation of the CTD and
thereby prevent transition from the initiation to the
elongation complex [34,35]. RNA pol II complexes,
already engaged in elongation, are not affected. As
expected, treatment of the cells with DRB or roscovi-
tine strongly decreased the hyperphosphorylation of
RNA pol II in the whole-cell extract and in RNA
pol II immunoprecipitate samples, because mainly the
IIA form can be detected (Fig. 2C). Treatment with
DRB or roscovitine also significantly decreased the
amount of RNA pol II co-immunoprecipitating with
Pol e (Fig. 2C). In contrast, a-amanitin did not affect
the hyperphosphorylation of RNA pol II, and had no
effect on the interaction between RNA pol II and
Pol e (Fig. 2C). These results confirm that Pol e associ-
ates specifically with elongation-competent RNA
pol IIO. Moreover, this association persists, even when
transcription is stalled.
Pol e associates with nascent RNA
We employed a UV crosslinking technique to study the
association of Pol e with nascent nucleic acids. This
method is a modification of the polymerase trap tech-

nique used to link newly synthesized DNA covalently
to the synthesizing Pol or to various replication proteins
intimately interacting with the nascent DNA [23,36,37].
We labelled permeabilized HeLa cells with either radio-
active UTP or dATP in the presence of the photoreac-
tive DNA precursor bromodeoxyuridine triphosphate
(BrdUTP), and purified respective protein–nucleotide
Fig. 1. RNA polymerase (pol) II and DNA polymerase (Pol) e
co-immunoprecipitate and copurify. Pol e, but not Pol a and Pol d,
co-immunoprecipitates with RNA pol II, and RNA pol IIO co-immu-
noprecipitates with Pol e. Immunoprecipitations were performed
with antibody to RNA pol II (N20), antibody to Pol a (SJK 132–20),
antibody to Pol d (K32), antibody to Pol e (GIA) or control antibody
(mouse IgG). Immunopreciptitation (IP) and western blot analysis
(WB) were performed as described in Experimental procedures.
Where indicated, immunoprecipitations were performed in the
presence of 50 lgÆmL
)1
ethidium bromide (EtBr) to exclude interac-
tions mediated by DNA. Hyperphosphorylated RNA pol IIO is indica-
ted with a solid arrow and hypophosphorylated RNA pol IIA is
indicated with an open arrow.
A. K. Rytko
¨
nen et al. DNA polymerase e associates with RNA polymerase II
FEBS Journal 273 (2006) 5535–5549 ª 2006 The Authors Journal compilation ª 2006 FEBS 5537
complexes after UV crosslinking by immunoprecipita-
tion with antibodies against Pol a, Pol d or Pol e. All
three pols were labelled with dATP (Fig. 3A), as expec-
ted, because all three Pols are required for DNA

replication in mammalian cells [23]. Pol a and Pol e,
but not Pol d, were labelled also with UTP. The label-
ling of Pol a by UTP was expected because this enzyme
synthesizes continuously RNA–DNA hybrid primers
required for the initiation of the continuous strand and
production of Okazaki fragments. Whether Pol e could
also crosslink RNA-primed nascent DNA was not
known. To find out, we compared the labelling of Pol a
and Pol e by UTP using a panel of controls (Fig. 3B).
In one control, BrdUTP, which facilitates the crosslink-
ing to the DNA moiety of RNA-primed nascent DNA,
was omitted. In a second control, DNA replication was
inhibited by omitting dNTPs from the reaction mixture
and including in it aphidicolin, an inhibitor of the repli-
cative Pols [38]. In the last control, transcription was
inhibited with a-amanitin. As expected, the labelling of
Pol a by UTP was abolished when BrdUTP was omit-
ted or replication inhibited, whereas a-amanitin had no
effect. These results confirmed that the labelling of
Pol a by UTP was linked to DNA replication, specific-
ally, the synthesis of RNA–DNA primers. In contrast,
Pol e labelling by radioactive UTP was not affected
when the DNA crosslinker was omitted or DNA syn-
thesis inhibited, but was abolished by the transcription
inhibitor a-amanitin, which prevents incorporation of
radioactive ATP in the polymerase trap reaction. It is
noteworthy that the photolabelling of Pol e with incor-
porated radioactive dATP absolutely depends on the
presence of BrdUTP [23]. Thus, both Pols a and e could
be UV crosslinked to newly synthesized RNA, but the

RNA associated with Pol a primed DNA replication,
whereas that associated with Pol e represented nascent
RNA transcripts.
Pol e and RNA pol IIO colocalize in the nucleus
Immunofluorescence and immunoelectron microscopy
were next performed to study if the association of RNA
pol IIO and Pol e is also reflected in their cellular local-
ization. T98G cells were stained by indirect immunoflu-
orescence, with antibody H5 recognizing RNA pol IIO
and with Pol e antibody G1A. T98G cells showed quite
even RNA pol IIO staining, with some cells showing
very few distinct foci. Pol e localized to numerous foci
(data not shown). These staining patterns correspond
well to previous reports [39,40]. RNA pol II foci became
more prominent after hypotonic permeabilization of
cells and removal of the bulk DNA by restriction diges-
tion prior to fixation (Fig. 4A). Most RNA pol IIO foci,
detectable after this treatment, were found to colocalize
or overlap with Pol e foci. As Pol e foci were by far
more abundant, only few Pol e foci colocalized with
RNA pol IIO.
A
B
C
Fig. 2. DNA polymerase (Pol) e interacts with hyperphosphorylated
RNA polymerase (pol) II. (A) Pol e co-immunoprecipitates the hyper-
phosphorylated form RNA pol IIO. Immunoprecipitations were per-
formed with antibody to RNA Pol II (N20), antibody to Pol e (GIA) or
control antibody (mouse IgG). Precipitates were treated with calf
intestinal phosphatase (CIP) where indicated and RNA pol II was

detected as described in Experimental procedures. Whole-cell
extract (WCE) represents 5–10% of the input. (B) Characterization
of the carboxyterminal domain phosphorylation status of RNA pol II
co-immunoprecipitated with Pol e. Immunoprecipitations and west-
ern blot analyses were performed with the indicated phosphospe-
cific RNA pol II antibodies, anti-Pol e antibody (GIA) or control
antibody (mouse IgG), as described in Experimental procedures.
The phosphorylation status of the heptapeptide repeat is indicated
on the right. (C) Effects of the kinase inhibitors 5,6-dichloro-1-beta-
D-ribobenzimidazole (DRB) and roscovitine, and of the transcription
inhibitor, a-amanitin, on RNA pol II co-immunoprecipitation with
Pol e. Cells were treated with the indicated inhibitors 1.5 h prior to
preparation of cell extract, as described in Experimental proce-
dures. Immunoprecipitations and western blot analyses were per-
formed with antibody to RNA Pol II (N20), antibody to Pol e (GIA) or
control antibody (mouse IgG), as in Fig. 1A. Whole-cell extract
(WCE) represents 5–10% of the input. In all panels, hyperphosphor-
ylated RNA pol IIO is indicated with a solid arrow and hypophos-
phorylated RNA pol IIA with an open arrow.
DNA polymerase e associates with RNA polymerase II A. K. Rytko
¨
nen et al.
5538 FEBS Journal 273 (2006) 5535–5549 ª 2006 The Authors Journal compilation ª 2006 FEBS
For immunoelectron microscopy, T98G cells were
serum deprived followed by serum stimulation, and
cells were collected at different time points, ranging
from 4 h (G
0
⁄ G
1

) to 22 h (late S ⁄ G
2
phase) after serum
stimulation, as determined by flow cytometry analysis
of cultures (see supplementary Figs S1 and S2 for syn-
chronization profiles). Cells were subjected to successive
staining for Pol e (antibody G1A) and RNA pol IIO
(antibody H5), using antibodies conjugated indirectly
to gold particles of 5 and 10 nm, respectively. Both
Pol e and RNA pol IIO localized predominantly to
distinct sites or centres, represented by irregular or
ring-shaped aggregates of gold particles of  50–
100 nm diameter (Fig. 4B). Staining with antibody
G1A is specific for Pol e, because it colocalizes exten-
sively with antibody to Pol e (H3B) in human
fibroblasts (data not shown). The two antibodies recog-
nize different epitopes of Pol e [41]. Quantitative analy-
sis of staining series derived from two independent
synchronizations indicate that, in general, 52% of Pol e
and 66% of RNA pol IIO localizes to centres contain-
ing at least three gold particles (Table 1). Pol e and
RNA pol IIO demonstrated striking colocalization
(Fig. 4B). Most of the centres identified contained
both proteins, although centres containing only RNA
pol IIO or Pol e were also present (see Fig. 4B, right).
Micrographs from 64 nuclei were scored for the pres-
ence of centres. Fifty-two per cent of all centres identi-
fied contained both proteins, 23% contained only Pol e
and 25% only RNA pol IIO. We did not detect any
apparent differences in the localization pattern of the

two proteins at the different time points investigated,
indicating that colocalization is not limited to a specific
cell cycle stage. We repeated immunoelectron microsco-
py staining using RNA pol II antibody N20, which
recognizes both the IIA and the IIO form. Although
less RNA pol II appears to be recognized by the anti-
body in this application, the focal colocalization of
RNA pol II and Pol e remained apparent (Fig. 4C).
Although only 45% of RNA pol II detected was in cen-
tres, still 37% colocalized with Pol e (Table 1, Fig. 4C),
confirming the authenticity of the observed colocaliza-
tion. The reduced focal staining of RNA pol II by anti-
body N20 compared with antibody H5 could reveal the
functional difference between total RNA pol II and iso-
form IIO, but may also simply reflect the apparently
lower affinity of antibody N20 in the application.
Taken together, the cell stainings indicate extensive,
although not complete, colocalization of Pol e with
RNA pol IIO at near-molecular level, as could be
expected from the physical interaction studies.
The RNA pol IIO–Pol e interaction is not linked
to nucleotide excision repair
TFIIH and Pol e are both components of an RNA
pol II holoenzyme, and both have been implicated in
nucleotide excision repair [25,26,42]. In addition, RNA
Pol IIO has an important function in the transcription
coupled repair pathway of nucleotide excision repair
[43]. When the transcription apparatus encounters dam-
age on the transcribed strand, nucleotide excision repair
is performed, after which transcription can be resumed.

Therefore, we studied whether the interaction between
RNA pol IIO and Pol e could be linked to nucleotide
A
B
Fig. 3. UV crosslinking of replicative DNA polymerases to nascent
DNA or RNA chains. (A) DNA polymerases (Pols) a and e, but not
Pol d, are photolabelled with radioactive UTP. HeLa cells were per-
meabilized, incubated in the presence of radioactive UTP or dATP
and the photoreactive DNA precursor BrdUTP, and subjected to UV
crosslinking. The photolabelled derivatives of Pols a, d and e were
then resolved by SDS ⁄ PAGE, as described in Experimental proce-
dures. (B) The photolabelling of Pol a or Pol e with radioactive UTP
depends on DNA replication or RNA transcription, respectively. UV
crosslinking of the Pols to nascent RNA labelled from radioactive
UTP in permeabilized HeLa cells was performed as described
above (complete), without BrdUTP (no BrdUTP), without dNTP pre-
cursors and with aphidicolin (+ aph, no dNTP) or in the presence of
a-amanitin (a-amanitin). The photolabelled derivatives of Pols a and
e were then resolved by SDS ⁄ PAGE and monitored as described in
Experimental procedures.
A. K. Rytko
¨
nen et al. DNA polymerase e associates with RNA polymerase II
FEBS Journal 273 (2006) 5535–5549 ª 2006 The Authors Journal compilation ª 2006 FEBS 5539
excision repair. Staining patterns of Pol e and RNA
pol IIO appeared unchanged after uniform UV expo-
sure of T98G cells (data not shown). In order to evalu-
ate changes in staining intensities caused by UV
exposure, cells were UV irradiated through an 8 lm
filter to cause local UV damage [44]. The sites of UV

damage inside nuclei were visualized by indirect immu-
nofluorescence staining, with antibody H3 recognizing
cyclobutane thymidine dimers. As shown previously
[45], nucleotide excision repair factor XP-B relocalized
to damaged areas, where it gave a uniform staining
(Fig. 5, upper panel). Staining with an antibody against
nucleotide excision repair factor XP-A gave a compar-
able staining pattern (data not shown). Pol e detected
with antibody G1A showed the punctuate staining typ-
ical for nondamaged cells also at the damaged areas
(Fig. 5, middle panel). We noticed a small, but very
reproducible, increase in the staining intensity of Pol e
in almost all UV irradiated areas compared to nonirra-
diated areas of the same nuclei. The staining intensity of
RNA pol IIO detected with antibody H5 in UV exposed
area remained constant or appeared decreased
Fig. 4. Pol e and RNA pol II colocalize in the nucleus. (A) Immunofluorescent staining of T98G cell nuclei with a-RNA pol II H5 (red) and
a-Pol e G1A (green). Yellow indicates colocalization in the merge image. Cells were permeabilized in hypotonic buffer in the presence of
detergent, and DNA was digested to enhance visibility of RNA pol II foci. (B,C) A significant fraction of RNA pol II and Pol e colocalize in
immunoelectron microscopy. Ultrathin cryosections of human T98G cells were subjected to immunostaining of Pol e (antibody G1A) followed
by staining of RNA pol IIO (antibody H5) (B) or total RNA pol II (antibody N20) (C), as described in Experimental procedures. The antibodies
were indirectly coupled to 5 nm and 10 nm gold particles, as indicated. Images represent serum-deprived cells 4 h (G
1
phase; panel B left),
10 h (G
1
⁄ S phase; panel C left) 12 h (early S phase; panel B middle) and 16 h (mid S phase; panel B right) after serum stimulation or syn-
chronization to the G
1
⁄ S boundary with mimosine (G

1
⁄ S phase; panel C right). No apparent differences were detected in the staining pattern
at different time points. The scale bar is 100 nm.
DNA polymerase e associates with RNA polymerase II A. K. Rytko
¨
nen et al.
5540 FEBS Journal 273 (2006) 5535–5549 ª 2006 The Authors Journal compilation ª 2006 FEBS
compared to the control area of the same nuclei (Fig. 5
lower panel). Overall, the staining intensities of RNA
pol II and Pol e do not correlate, and there is only a
very minor effect of local UV damage on the nuclear
localization of RNA pol II and Pol e.
The RNA pol II–Pol e interaction does not depend
on DNA replication
Pol e is one of the three major replicases in the eukary-
otic cell [22]. Therefore, we tested the effect of the
replicative state of the cell on the interaction between
Pol e and RNA pol II. A possible confinement of the
interaction to late G
1
and S phases (the cell cycle sta-
ges dedicated to initiation and execution of DNA
replication) would have strong implications on a func-
tional interplay between transcription and DNA repli-
cation. We therefore examined the interaction between
RNA pol II and Pol e during the cell cycle. T98G
cells were serum deprived, followed by stimulation to
proliferate, to create samples at G
0
⁄ G

1
(4 h), late G
1
(10 h), early S (14 h), and late S ⁄ G
2
(24 h) phases of
the cell cycle, and T98G cells were blocked at the
G
1
⁄ S boundary by mimosine (see supplementary
Fig. S1 for flow cytometric cell cycle analysis). RNA
Pol IIO and Pol e co-immunoprecipitated with com-
parable efficiencies throughout the cell cycle (Fig. 6A).
Although minor differences in the interaction may not
be revealed by changes in immunoprecipitation, these
results demonstrated that the interaction is not con-
fined to a specific stage of the cell cycle. This is consis-
tent with immunoelectron microscopy, revealing that
RNA Pol II and Pol e colocalize throughout the cell
cycle. What is more, the interaction prevailed in the
presence of aphidicolin (Fig. 6B). Although our data
do not address potential effects of the observed inter-
action during DNA replication, they suggest that the
interaction is not dependent on DNA replication.
Discussion
We describe here an extensive interaction of RNA
pol II with Pol e. Our observation is based on detailed
analysis of co-immunoprecipitation using multiple anti-
bodies against the cognate proteins. This interaction
appears to be specific for Pol e, because Pols a and d

were absent from RNA pol II immunoprecipitates con-
taining Pol e, RNA pol II was not precipitated with
Pols a or d. What is more, the interaction is limited to
the hyperphosphorylated RNA pol IIO, based on elec-
trophoretic shift, treatment of immunoprecipitates with
phosphatase and use of antibodies specific for differen-
tially phosphorylated forms of the CTD.
Maldonado et al. [5] reported the purification of an
RNA pol II holoenzyme complex that also comprised
several factors involved in DNA repair and recombina-
tion, including Pol e. The latter complex contained
mainly hypophosphorylated RNA pol IIA, included
several general transcription factors and several sub-
units of human Mediator and was capable of tran-
scribing from a model promoter in vitro with minimal
addition of general transcription factors, consistent
with a transcription initiation complex [30]. The pres-
ence of DNA repair or recombination factors in puri-
fied RNA pol II has been found to depend strongly on
the purification strategy employed. We see a striking
difference in our results compared with those of
Maldonado et al. [5], because the interaction described
here correlated strictly with phosphorylation of the
CTD. In support of this view, chemical kinase inhibi-
tors that prevent phosphorylation-dependent transition
from the initiation to the elongation of transcription
strongly reduced the interaction observed by us. How-
ever, the initiation and elongation inhibitor a-amanitin
had no effect on this interaction, suggesting that it per-
sists also when transcription stalls. What is more, we

observed that part of the hyperphosphorylated RNA
Table 1. Comparison of colocalization and focal staining of Pol e and RNA pol II by immunoelectron microscopy. The number of gold parti-
cles representing Pol e (5 nm) and RNA pol II (10 nm), and the fraction of particles colocalizing and in centres, were determined in double
stainings. Clusters of three or more gold particles were considered as centres. The table represents the summary of the analyses of sam-
ples from G
0
until late S phase (0–22 h after serum stimulation) derived from serum-deprived T98G cells stimulated to proliferate. Typically,
20 separate images, representing seven to nine nuclei, were scored for each sample. Eight samples from two independent synchronizations
were stained for Pol e (antibody G1A) and transcriptionally active RNA pol II phosphorylated at Ser2 (antibody H5), and four samples derived
from one synchronization were analysed for staining of Pol e (antibody G1A) and all forms of RNA pol II (antibody N20). No difference in focal
staining and colocalization was detected at different time points.
Staining Particles counted Particles in centres Particles colocalizing Nuclei counted Images counted
RNA Pol II Ser2-P Pol e 2519 1312 (52%) 976 (39%) 64 158
RNA Pol II 1610 1058 (66%) 656 (41%)
All RNA Pol II Pol e 1133 680 (60%) 192 (17%) 33 80
RNA Pol II 177 80 (45%) 66 (37%)
A. K. Rytko
¨
nen et al. DNA polymerase e associates with RNA polymerase II
FEBS Journal 273 (2006) 5535–5549 ª 2006 The Authors Journal compilation ª 2006 FEBS 5541
pol IIO form copurified during the three conventional
column steps of the standard Pol e purification [46]
(data not shown). Finally, Pol e could be UV cross-
linked to newly synthesized a-amanitin-sensitive RNA,
indicating close association of Pol e with nascent RNA
pol II transcripts. All these results are consistent with
the view that Pol e interacts with elongating RNA
pol IIO.
The CTD of RNA pol II has a central role in the
co-ordination of the transcription cycle because it is

capable of recruiting several factors involved in tran-
scription and mRNA processing to the transcription
apparatus [4]. The differential phosphorylation of the
CTD provides means of discrimination during the
transcription cycle. As we found that hyperphosphory-
lation of the CTD correlates with the interaction with
Pol e, it is tempting to speculate that the interaction
may be mediated by the CTD, in particular when
phosphorylated at the repeated serine 5 residues,
although such an interaction could be mediated by
another protein, or other regions of RNA pol II.
Bulky DNA lesions block the passage of elonga-
ting RNA pol II, thereby stalling the transcription
apparatus [43]. To avoid consequent detrimental
effects, nucleotide excision repair is preferentially
directed to the impaired DNA template by the
stalled transcription apparatus in a process called
transcription-coupled repair. As Pol e, together with
Pol d, are implicated in the DNA synthesis step of
nucleotide excision repair [25,26], one could antici-
pate that the interaction between Pol e and RNA
pol IIO would facilitate transcription-coupled repair.
Fig. 5. Pol e and RNA pol II do not relocalize to a nuclear site of UV exposure. T98G cells were UV irradiated at 60 JÆm
)2
through an 8 lm
pore polycarbonate filter. Cells were fixed 1 h after UV exposure and subjected to immunofluorescence staining. DNA staining (blue) shows
positions of nuclei. The UV damage marker XP-B (S-19) localizes, as expected, to sites of UV damage visualized by an antibody specific to
cyclobutane pyrimidine dimers (CPD) (upper panel), as opposed to Pol e (visualized by antibody G1A; middle panel) and RNA pol IIO (antibody
H5; lower panel).
DNA polymerase e associates with RNA polymerase II A. K. Rytko

¨
nen et al.
5542 FEBS Journal 273 (2006) 5535–5549 ª 2006 The Authors Journal compilation ª 2006 FEBS
Notwithstanding this expectation, the observed RNA
pol IIO–Pol e interaction did not depend on, or was
augmented in response to, DNA damage. This was
most clearly demonstrated by the failure of the pro-
tein to relocalize to a defined UV-irradiated nuclear
area (Fig. 5). Although Sarker et al. [47] described
recently an interaction between hyperphosphorylated
RNA pol II and XP-G in undamaged cells, we were
not able to detect XP-G, XP-A or XP-B in our
Pol e or RNA pol II immunoprecipitates (data not
shown), supporting the view of a sequential assembly
of nucleotide excision repair factor at the sites of
DNA damage [45]. Although we cannot exclude
beneficial effects of the observed interaction on tran-
scription-coupled repair, there is no indication that
such repair represents its predominant function.
In theory, an interaction between RNA pol IIO
and Pol e could be important for the nondisruptive
bypass of a transcription bubble by a replication
fork, as has been reported previously, for example,
in bacteria [48,49]. Nevertheless, the interaction
between Pol e and RNA pol II is not limited to
S phase, indicating that it does not depend on DNA
replication. This is also reflected by the fact that the
replication inhibitor, aphidicolin, does not affect the
interaction. What is more, the extent of interaction
and ultrastructural colocalization observed between

the two proteins is difficult to reconcile with such a
specific function as the passing of colliding replica-
tion and transcription apparatus.
There are several direct links between transcription
and DNA replication. Adolph et al. [50] showed that
a-amanitin blocks cells specifically in the G
1
phase of
the cell cycle. This effect could be attributed to inhi-
bition of transcription of cell cycle-regulated genes at
this phase of the cell cycle [51]. Nevertheless, it was
shown that cell cycle block by a-amanitin at the ori-
gin decision point in G
1
phase does not depend on
protein synthesis of gene products [52]. It has been
long known that actively transcribed genes are repli-
cated early, and it has been proposed that the posit-
ive regulation of DNA replication is a direct effect of
the transcription apparatus, or is caused by remodel-
ling of the chromatin mediated by transcription
factors [16,19,53–55]. It is also worth considering that
the RNA pol IIO–Pol e interaction may help in load-
ing Pol e at replication origins within transcribed
domains. Links between transcription and DNA repli-
cation have also been supported by ultrastructural
studies. Sites of replication and transcription colocal-
ized in human cells when studied by confocal micros-
copy [56]. Using high-resolution electron microscopy,
centres of DNA replication were found to contain

also RNA processing components [57]. In our study,
RNA pol IIO and Pol e displayed a partly dispersed
staining with numerous foci in conventional immuno-
fluorescence microscopy, but colocalization of the
proteins became more prominent after hypotonic
extraction and removal of part of the DNA. In con-
trast, using cryo-electron microscopy without such
manipulations, we found both proteins to colocalize
extensively and to cluster into centres in the vicinity
of small electron-dense domains of the nucleus. The
RNA pol II staining corresponds well to previous ul-
trastructural studies, where RNA pol II was concen-
trated into clusters that overlapped with the sites of
RNA synthesis [58,59].
Another example for prominent replication factors
that interact with RNA pol II are the MCM proteins
[10]. MCM proteins can be detected in RNA pol II
holoenzyme affinity-purified against elongation factor
TFIIS, but not in holoenzyme purified by anti-cdk7
[10,14]. Furthermore, the TFIIS holoenzyme form
appears to be more abundant in the G
1
⁄ S phase of the
A
B
Fig. 6. The interaction between RNA pol II and Pol e does not
depend on DNA replication. (A) Pol e and RNA pol II co-immunopre-
cipitate throughout the cell cycle. T98G cells were synchronized by
serum deprivation and released, or blocked with mimosine, to
obtain samples from different stages of the cell cycle. Immunoprec-

ipitations were performed with antibody to RNA pol II (N20), anti-
body to Pol e (GIA) or control antibody (mouse IgG), and analysed
by western blot, as described in Experimental procedures. Whole-
cell extract (WCE) represents 5–10% of the input. (B) Inhibition of
DNA replication by aphidicolin does not affect the co-immunoprecip-
itation of Pol e and RNA pol II. Cells were treated with aphidicolin
for 1.5 h before preparation of the extract. Immunoprecipitations
were performed and analysed as above. Hyperphosphorylated RNA
Pol IIO is indicated with a solid arrow and hypophosphorylated RNA
Pol IIA with an open arrow.
A. K. Rytko
¨
nen et al. DNA polymerase e associates with RNA polymerase II
FEBS Journal 273 (2006) 5535–5549 ª 2006 The Authors Journal compilation ª 2006 FEBS 5543
cell cycle. Antibodies against Mcm2 inhibited tran-
scription, and, conversely, the yeast CTD mutants
interacted genetically with mcm5 mutants and showed
adverse effects on minichromosome maintenance and
DNA replication [19].
Whereas other DNA repair and replication factors
interacting with RNA pol II appear to regulate tran-
scription positively [6,10,13], yeast Pol e has been
implicated in transcriptional silencing of ribosomal
DNA, the mating type locus and subtelomeric regions
[60–62]. The B subunit of mouse Pol e interacts with
SAP18, which is known to associate with the transcrip-
tional corepressor, Sin3 [63]. Sin3 is a component of a
protein complex possessing histone deacetylase activity,
which interacts with several transcriptional corepres-
sors and functions in transcriptional silencing [64].

Thus, a possible role of Pol e in transcription attenu-
ation or silencing remains to be investigated.
The physical and structural association between
RNA pol II and Pol e presented here provide a direct
link between central components of the transcription
and DNA replication apparati. Further functional
characterization of this interaction may provide valu-
able insight into the crosstalk between transcription
and DNA replication.
Experimental procedures
Antibodies
Primary antibodies used are listed in Table 2. Rabbit poly-
clonal antibody K32, against the human Pol d catalytic
subunit, was raised against a peptide corresponding to
amino acids 297–542 (swissprot entry P28340) and
expressed as glutathione S-transferase fusion protein, as
described previously [24]. Purified mouse IgG was pur-
chased from Pierce (Rockford, IL, USA), rabbit anti-mouse
IgG (Zymed, San Francisco, CA, USA) and rabbit anti-rat
IgG (Jackson Immunoresearch, West Grove, PA, USA)
were used as secondary antibodies in immunoelectron
microscopy, and horseradish peroxidase-conjugated anti-
bodies (Jackson Immunoresearch or Chemicon, Chandlers
Ford, UK) were used as secondary antibodies in western
blotting.
Cell culture, synchronization and UV irradiation
HeLa CCL2 monolayer cells [American Type Culture
Collection (ATCC), Manassas, VA, USA] were cultured
DMEM (Invitrogen, Paisley, UK) containing 10% fetal
bovine serum and antibiotics, at 37 °C in a 5% carbon

dioxide atmosphere. T89G cells (ATCC) were grown in
Table 2. Primary antibodies used in this study. IEM, immunoelectron microscopy; IF, immunofluorescence microscopy; IP, immunoprecipita-
tion; Pol, DNA polymerase; pol, polymerase; WB, western blot; X-LINK, UV crosslinking.
Target Clone Species ⁄ type Application Reference
Pol a catalytic
subunit
1Ct102, 2Ct25 Mouse monoclonal WB [67]
SJK-132–20 Mouse monoclonal IP ATCC CRL-1640
[68]
p140 Rabbit polyclonal X-LINK [69]
Pol d catalytic
subunit
PDG-1E8 Rat monoclonal WB This study
a
K30 Rabbit polyclonal X-LINK, WB [70]
K32 Rabbit polyclonal IP This study
Pol e catalytic
subunit
G1A Mouse monoclonal IP, X-LINK, WB, IEM [41]
H3B Mouse monoclonal IF, WB [41]
E24C Mouse monoclonal WB [41]
K27 Rabbit polyclonal IP [71]
RNA pol II N20 Rabbit polyclonal IP, WB, IF, IEM Santa Cruz
Biotechnologies
H5 Mouse monoclonal, IgM IP, WB, IF, IEM Covance Research
Products
H14 Mouse monoclonal IgM IP, WB Nordic Biosite
8WG16 Mouse monoclonal IgG2a IP, WB Nordic Biosite
XP-A 12F5 Mouse monoclonal IP NeoMarkers
XP-B S-19 Rabbit polyclonal IF Santa Cruz

Biotechnologies
XP-G 8H7 Mouse monoclonal IP Santa Cruz
Biotechnologies
Cyclobutane pyrimidine
dimers
H3 mouse monoclonal IF Affitech
a
Chen S, Kremmer E, Weisshart K, Hubscher U & Nasheuer H-P, unpublished results.
DNA polymerase e associates with RNA polymerase II A. K. Rytko
¨
nen et al.
5544 FEBS Journal 273 (2006) 5535–5549 ª 2006 The Authors Journal compilation ª 2006 FEBS
Eagle’s minimum essential medium (Sigma-Aldrich Fin-
land, Helsinki, Finland) supplemented with 10% fetal
bovine serum, glutamax, nonessential amino acids,
sodium pyruvate and antibiotics, at 37 °Cin5%CO
2
.
T98G cells were synchronized by arresting them at the
G
0
phase for 3–6 days using starvation medium (growth
medium containing 0.5% serum). Cells were stimulated to
grow by addition of equal volumes of complete and bal-
anced growth medium. The efficacy of the synchroniza-
tion was demonstrated by fluorescence cytometry analysis
(Becton Dickinson, Helsinki, Finland) of propidium iod-
ide-stained cells [65] in parallel cell cultures. Where indi-
cated, cells were cultured in the presence of the indicated
concentrations of one of the following inhibitors:

20 lgÆmL
)1
a-amanitin, 40 lm roscovitine, 50 lm DRB or
5 lgÆmL
)1
aphidicolin (all Sigma-Aldrich Finland). These
inhibitors were added for 1.5 h before preparing the cell
extracts.
Immunoprecipitation
HeLa cells were washed with NaCl ⁄ P
i
and scraped into
lysis buffer [100 mm Tris ⁄ HCl, pH 7.5, 80 mm NaCl, 10%
glycerol, 0.1% Nonidet P-40, Complete
TM
protease inhibi-
tors (Roche, Espo, Finland), 10 mm Na
3
VO
4
and 10 m m
NaF]. The cell extract from synchronized T98G cells used
for analyzing protein–protein interactions during the cell
cycle was prepared in lysis buffer II (50 mm Tris ⁄ HCl
pH 7.5, 100 mm KCl, 5% glycerol, 0.1% Nonidet P-40,
Complete
TM
protease inhibitor, 1 mm EDTA, 10 mm NaF,
2mm Na
3

VO
4
). Cells were broken by sonication (2 · 15 s)
and the extract was further incubated on ice for 15 min
and centrifuged in an Eppendorf 5415 table centrifuge
(Eppendorf AG, Hamburg, Germany; 10 min, 20 000 g,
4 °C). For immunoprecipitations, 0.2–3 mg of cell extract
and 2–4 lg of antibody was used per sample. After pre-
clearing, the antibody–protein complex was allowed to
form overnight at 4 °C. Rabbit anti-mouse IgM linkers
were added in H14 and H5 immunoprecipitations to facili-
tate collection of mouse IgM on protein G–Sepharose. Ten
microlitres of GammaBind protein G–Sepharose (Amer-
sham Biosciences, Helsinki, Finland) was used to collect the
immunocomplex. Immunoprecipitates were washed five
times with modified lysis buffer (without inhibitors, 100 mm
NaCl). Samples were eluted with SDS loading buffer, separ-
ated through 6% SDS ⁄ PAGE and transferred to poly(viny-
lidene difluoride) membrane (Millipore, Espo, Finland).
Proteins were detected using chemiluminescence reagents
(Pierce). Antibodies GIA, E24C and H3B, or N20 were
used for the detection of Pol e and RNA pol II, respect-
ively.
For dephosphorylation, immunoprecipitates were first
washed as usual and then treated with 15 U calf intestinal
phosphatase (Amersham Biosciences) for 30 min at +37 °C
prior to elution to SDS ⁄ PAGE loading buffer.
UV crosslinking
UV crosslinking of proteins to nascent DNA ⁄ RNA in a
monolayer of isolated nuclei was performed, as described

previously [23], with minor modifications. Cells were
washed with buffer KM (10 mm Mops-NaOH, pH 7.0,
10 mm NaCl, 1 mm MgCl
2
,2mm dithiothreitol, 1 · com-
plete
TM
protease inhibitor) and lysed by incubation for
30 min on ice with buffer KM containing 0.5% Nonidet
P-40, resulting in a monolayer of isolated nuclei. The
nuclear monolayer was then washed twice with buffer
KAc (30 mm Hepes-KOH, pH 7.5, 5 mm K-acetate,
0.5 mm MgCl
2
,2mm dithiothreitol, 1 · complete
TM
pro-
tease inhibitor). The reaction mixture for UTP labelling
contained 30 mm Hepes-KOH, pH 7.8, 50 mm K-acetate,
5mm MgCl
2
,2mm dithiothreitol, 0.05% Nonidet P-40,
2 lm each of dATP, dGTP and dCTP, 20 lm BrdUTP,
1 lm [
32
P]UTP[aP] (specific activity 410 CiÆ mmol
)1
), 2 mm
ATP and 10 lm CTP and GTP. a-amanitin and aphidic-
olin were used at 50 lgÆmL

)1
. Each 6 cm plate received
400 lL of reaction mixture and the reactions proceeded
for 2.5 min at 30 °C. The mixture was used for a total
of three successive plates. After labelling, the nuclei were
washed with buffer KAc and UV irradiated with a stand-
ard UV transilluminator for 6 min. The irradiated nuclei
were treated with HindIII and PstI (100 UÆmL
)1
) for
30 min at 37 °C to remove bulk DNA. Protein–nucleic
acid complexes were extracted with phenol, precipitated
with acetone, collected at 0 °C and washed three times.
The dried protein–nucleic acid pellet was resuspended by
boiling in denaturation buffer (50 mm Tris ⁄ HCl, pH 7.5,
0.5% SDS, 70 mm b-mercaptoethanol) and renaturated
(50 mm Tris ⁄ HCl, pH 7.5, 150 mm NaCl, 0.5% Nonidet
P-40, 1 · Complete
TM
protease inhibitors). Proteins were
then ready for immunoprecipitation. For immunoprecipi-
tations, 7 mg of protein A– or G–Sepharose and 3 lLof
p140 serum, 4 lL of K30 serum and 8 lg of purified
GIA antibody were used to precipitate, in parallel, Pols
a, d and e, respectively. Immunoprecipitates were washed
three times with washing buffer (50 mm Tris ⁄ HCl,
pH 7.5, 150 mm NaCl, 0.5% Nonidet P-40). Samples
were eluted with SDS loading buffer and separated
through 6% SDS ⁄ PAGE, transferred to poly(vinylidene
difluoride) membrane and exposed to BioMax MS film

(Kodak, Vantaa, Finland). After autoradiography, pro-
teins were detected using enhanced chemiluminescence
reagents (Pierce). Antibodies 1Ct102 and 2Ct25, PDG-
1E8, and a combination of G1A, H3B and E24C were
used for detection of Pols a, d and e, respectively.
Indirect immunofluorescence
T98G cells were grown on glass slides to logarithmic phase
and washed twice with hypotonic KM buffer (see above)
on ice followed by permeabilization for 30 min in the same
A. K. Rytko
¨
nen et al. DNA polymerase e associates with RNA polymerase II
FEBS Journal 273 (2006) 5535–5549 ª 2006 The Authors Journal compilation ª 2006 FEBS 5545
buffer containing 0.5% Nonidet P-40 and complete prote-
ase inhibitors. The majority of DNA-bound proteins were
removed by digesting DNA with 100 UÆmL
)1
HindIII and
PstI for 30 min at 37 °C. Cells were fixed with 3% parafor-
maldehyde in NaCl ⁄ P
i
for 10 min, followed by quenching
with 50 mm NH
4
Cl in NaCl ⁄ P
i
.
For UV irradiation, T98G cells were grown to logarith-
mic phase on glass slides, washed once with NaCl ⁄ P
i

and
partially exposed to 60 JÆm
)2
UV light using an 8 lm
micropore filter (Millipore). Cells were allowed to recover
from the UV pulse for 1 h in balanced growth medium and
were then permeabilized with 0Æ2% Triton X-100, fixed with
3% paraformaldehyde and quenched with 50 mm NH
4
Cl
(all solutions prepared in NaCl ⁄ P
i
), and applied to the cells
for 10 min at room temperature. Samples to be stained with
H3 antibody at a later stage were treated with 2 m HCl for
7 min at room temperature to denature the DNA.
Prior to antibody treatment, cells were treated with 0.2%
coldwater fish skin gelatine (Sigma) in NaCl ⁄ P
i
for 1 h. Pri-
mary antibodies were diluted to 5 lgÆmL
)1
and secondary
antibodies to 4 lgÆmL
)1
with 0.2% gelatine in NaCl ⁄ P
i
.
Cells were sequentially stained for 30 min at 37 °C. DNA
was stained with 1 lgÆmL

)1
bisbenzimide (Hoechst 33258;
Sigma) for 5 min at room temperature, followed by mount-
ing to objective glass with Shandon Immu-Mount (Thermo
Electron Cooperation, Vantaa, Finland). Images were taken
with a charge-coupled device camera and Olympus BX-61
microscope (Olympus Finland, Vantaa, Finland) with a
·100 objective, followed by image processing using adobe
photoshop (Adobe, San Jose, CA, USA).
Immunoelectron microscopy
T98G cells stimulated to proliferate were fixed for 30 min
with 4% paraformaldehyde in 0.1 m phosphate buffer,
pH 7.5, containing 2.5% sucrose, detached and centrifuged
in an Eppendorf 5415 table centrifuge (2000 g for 3 min) to
a tight pellet. The pellet was mixed to a small volume of
2% NuSieve agarose (FMC BioProducts, Philadelphia, PA,
USA) in NaCl ⁄ P
i
at 37 °C, cooled and immersed in
NaCl ⁄ P
i
containing 2.3 m sucrose. The specimens were fro-
zen in liquid nitrogen and thin cryosections were cut with a
Leica Ultracut UCT microtome (Leica Microsystems, Wetz-
lar, Germany). The sections were first incubated in NaCl ⁄ P
i
containing 5% BSA and 0.1% gelatine. Antibodies and
gold conjugates were diluted in NaCl ⁄ P
i
containing 0.1%

BSA-C (Aurion, Wageningen, the Netherlands). All wash-
ing steps were performed in 0.1% BSA-C in NaCl⁄ P
i
. For
the double-labelling experiments, after blocking, as des-
cribed above, sections were exposed to the first primary
antibody for 60 min followed by incubation with rabbit
anti-mouse IgG at 1.9 lgÆmL
)1
for 30 min for mouse pri-
mary antibodies, and protein A–gold complex, size 5 nm
[66] for 30 min. After washing, 1% glutaraldehyde in 0.1 m
phosphate buffer was used to block free binding sites on
protein A. The sections were then incubated with the sec-
ond antibody for 60 min followed by antimouse for 30 min
for mouse primary antibodies, and protein A–gold complex
(size 10 nm) for 30 min, as described above. Antibodies
G1A, N20 and H5 were used at 5 lgÆmL
)1
for detection of
Pol e and RNA pol II, respectively. The controls were
prepared by carrying out the labelling procedure without
primary antibody. The efficiency of blocking was controlled
by performing the labelling procedure in the absence of the
second primary antibody. The sections were embedded in
methylcellulose and examined in a Philips CM100 transmis-
sion electron microscope (FEI company, Hillsboro, OR,
USA). Images were captured by charge-coupled device cam-
era equipped with TCL-EM-Menu version 3 from Tietz
Video and Image Processing Systems GmbH (Gaunting,

Germany).
Acknowledgements
This work was supported by grants from the Academy
of Finland to JES and HP and a grant by the German-
Isareli Foundation for Scientific Research and Develop-
ment to GK. We are grateful to Tomi Ma
¨
kela
¨
for
generous gifts of antibodies and a construct for recom-
binant expression of the CTD. We would like to thank
Sirpa Kellokumpu for excellent technical assistance.
References
1 Lee TI & Young RA (2000) Transcription of eukaryotic
protein-coding genes. Annu Rev Genet 34, 77–137.
2 Parvin JD & Young RA (1998) Regulatory targets in
the RNA polymerase II holoenzyme. Curr Opin Genet
Dev 8, 565–570.
3 Shilatifard A, Conaway RC & Conaway JW (2003) The
RNA polymerase II elongation complex. Annu Rev Bio-
chem 72, 693–715.
4 Howe KJ (2002) RNA polymerase II conducts a sym-
phony of pre-mRNA processing activities. Biochim
Biophys Acta 1577, 308–324.
5 Maldonado E, Shiekhattar R, Sheldon M, Cho H,
Drapkin R, Rickert P, Lees E, Anderson CW, Linn S &
Reinberg D (1996) A human RNA polymerase II com-
plex associated with SRB and DNA-repair proteins.
Nature 381, 86–89.

6 Scully R, Anderson SF, Chao DM, Wei W, Ye W,
Young RA, Livingston DM & Parvin JD (1997)
BRCA1 is a component of the RNA polymerase II
holoenzyme. Proc Natl Acad Sci USA 94, 5605–
5610.
7 Neish AS, Anderson SF, Schlegel BP, Wei W & Parvin
JD (1998) Factors associated with the mammalian RNA
polymerase II holoenzyme. Nucleic Acids Res 26, 847–
853.
DNA polymerase e associates with RNA polymerase II A. K. Rytko
¨
nen et al.
5546 FEBS Journal 273 (2006) 5535–5549 ª 2006 The Authors Journal compilation ª 2006 FEBS
8 Krum SA, Miranda GA, Lin C & Lane TF (2003)
BRCA1 associates with processive RNA polymerase II.
J Biol Chem 278, 52012–52020.
9 Chiba N & Parvin JD (2002) The BRCA1 and BARD1
association with the RNA polymerase II holoenzyme.
Cancer Res 62, 4222–4228.
10 Yankulov K, Todorov I, Romanowski P, Ligatalosi D,
Cilli K, McCracken S, Laskey R & Bentley DL (1999)
MCM proteins are associated with RNA polymerase II
holoenzyme. Mol Cell Biol 19, 6154–6163.
11 Holland L, Downey M, Song X, Gauthier L, Bell-
Rogers P & Yankulov K (2002) Distinct parts of mini-
chromosome maintenance protein 2 associate with his-
tone H3 ⁄ H4 and RNA polymerase holoenzyme. Eur J
Biochem 269, 5192–5202.
12 Dziak R, Leishman D, Radovic M, Tye BK & Yanku-
lov K (2003) Evidence for a role of MCM (mini-chro-

mosome maintenance) 5 in transcriptional repression of
sub-telomeric and Ty-proximal genes in Saccharomyces
cerevisiae. J Biol Chem 278, 27372–27381.
13 Mo X & Dynan WS (2002) Subcellular localization of
Ku protein: functional association with RNA polymer-
ase II elongation sites. Mol Cell Biol 22, 8088–8099.
14 Holland L & Yankulov K (2003) Two forms of RNA
polymerase II holoenzyme display different abundance
during the cell cycle. Biochem Biophys Res Commun 302,
484–488.
15 Murakami Y & Ito Y (1999) Transcription factors in
DNA replication. Front Biosci 4, D824–D833.
16 Kohzaki H & Murakami Y (2005) Transcription factors
and DNA replication origin selection. Bioessays 27,
1107–1116.
17 MacAlpine DM, Rodriguez HK & Bell SP (2004) Coor-
dination of replication and transcription along a Droso-
phila chromosome. Genes Dev 18, 3094–3105.
18 Stagljar I, Hu
¨
bscher U & Barberis A (1999) Activation
of DNA replication in yeast by recruitment of the RNA
polymerase II transcription complex. Biol Chem 380,
525–530.
19 Gauthier L, Dziak R, Kramer DJ, Leishman D, Song
X, Ho J, Radovic M, Bentley D & Yankulov K
(2002) The role of the carboxyterminal domain of
RNA polymerase II in regulating origins of DNA
replication in Saccharomyces cerevisiae. Genetics 162,
1117–1129.

20 Haile DT & Parvin JD (1999) Activation of transcrip-
tion in vitro by the BRCA1 carboxyl-terminal domain.
J Biol Chem 274, 2113–2117.
21 Aguilera A (2002) The connection between transcription
and genomic instability. EMBO J 21, 195–201.
22 Pospiech H & Syva
¨
oja JE (2003) DNA polymerase epsi-
lon – more than a polymerase. ScientificWorldJournal 3,
87–104.
23 Zlotkin T, Kaufmann G, Jiang Y, Lee MYWT, Uitto
L, Syva
¨
oja J, Dornreiter I, Fanning E & Nethanel T
(1996) DNA polymerase e may be dispensable for
SV40- but not cellular-DNA replication. EMBO J 15
,
2298–2305.
24 Pospiech H, Kursula I, Abdel-Aziz W, Malkas L, Uitto
L, Kastelli M, Vihinen-Ranta M, Eskelinen S & Syva
¨
oja
JE (1999) A neutralizing antibody against human DNA
polymerase e inhibits cellular but not SV40 DNA repli-
cation. Nucleic Acids Res 27, 3799–3804.
25 Aboussekhra A, Biggerstaff M, Shivji MK, Vilpo JA,
Moncollin V, Podust VN, Protic M, Hu
¨
bscher U, Egly
JM & Wood RD (1995) Mammalian DNA nucleotide

excision repair reconstituted with purified protein com-
ponents. Cell 80, 859–868.
26 Araujo SJ, Tirode F, Coin F, Pospiech H, Syva
¨
oja JE,
Stucki M, Hu
¨
bscher U, Egly JM & Wood RD (2000)
Nucleotide excision repair of DNA with recombinant
human proteins: definition of the minimal set of factors,
active forms of TFIIH, and modulation by CAK. Genes
Dev 14, 349–359.
27 Navas TA, Zhou Z & Elledge SJ (1995) DNA polymer-
ase epsilon links the DNA replication machinery to the
S phase checkpoint. Cell 80, 29–39.
28 Lai JS & Herr W (1992) Ethidium bromide provides a
simple tool for identifying genuine DNA–independent
protein associations. Proc Natl Acad Sci USA 89, 6958–
6962.
29 Palancade B & Bensaude O (2003) Investigating RNA
polymerase II carboxyl-terminal domain (CTD) phos-
phorylation. Eur J Biochem 270, 3859–3870.
30 Svejstrup JQ (2004) The RNA polymerase II transcrip-
tion cycle: cycling through chromatin. Biochim Biophys
Acta 1677, 64–73.
31 Patturajan M, Schulte RJ, Bartholomew M, Sefton BM,
Ronald Berezney R, Michel Vincent M, Olivier Bensa-
ude O, Stephen L, Warren SL, Jeffry L et al. (1998)
Growth-related changes in phosphorylation of yeast
RNA polymerase II. J Biol Chem 273, 4689–4694.

32 Bushnell DA, Cramer P & Kornberg RD (2002) Struc-
tural basis of transcription: a-Amanitin-RNA poly-
merase II cocrystal at 2.8 A
˚
resolution. Proc Natl Acad
Sci USA 99, 1218–1222.
33 Gong XQ, Nedialkov YA & Burton ZF (2004) a-Aman-
itin blocks translocation by human RNA polymerase II.
J Biol Chem 279, 27422–27427.
34 Kim DK, Yamaguchi Y, Wada T & Handa H (2001)
The regulation of elongation by eukaryotic RNA poly-
merase II: a recent view. Mol Cell 11, 267–274.
35 Schang LM (2004) Effects of pharmacological cyclin-
dependent kinase inhibitors on viral transcription and
replication. Biochim Biophys Acta 1697, 197–209.
36 Insdorf NF & Bogenhagen DF (1989) DNA polymer-
ase c from Xenopus laevis. J Biol Chem 264
, 21498–
21503.
37 Mass G, Nethanel T & Kaufmann G (1998) The middle
subunit of replication protein A contacts growing
A. K. Rytko
¨
nen et al. DNA polymerase e associates with RNA polymerase II
FEBS Journal 273 (2006) 5535–5549 ª 2006 The Authors Journal compilation ª 2006 FEBS 5547
RNA–DNA primers in replicating simian virus 40 chro-
mosomes. Mol Cell Biol 18, 6399–6407.
38 Mizushina Y, Kamisuki S, Mizuno T, Takemura M,
Asahara H, Linn S, Yamaguchi T, Matsukage A, Han-
aoka F, Yoshida S et al. (2000) Dehydroaltenusin, a

mammalian DNA polymerase a inhibitor. J Biol Chem
275, 33957–33961.
39 Bregman DBL, van der Zee S & Warren SL (1995)
Transcription-dependent redistribution of the large
subunit of RNA polymerase II to discrete nuclear
domains. J Cell Biol 129, 287–298.
40 Fuss G & Linn S (2002) Human DNA polymerase
e colocalizes with PCNA and DNA replication late,
but not early in S phase. J Biol Chem 277, 8658–
8666.
41 Uitto L, Halleen J, Hentunen T, Ho
¨
yhtya
¨
M & Syva
¨
oja
JE (1995) Structural relationship between DNA poly-
merases e and e* and their occurrence in eukaryotic
cells. Nucleic Acids Res 23, 244–247.
42 de Laat WL, Jaspers NG & Hoeijmakers JH (1999)
Molecular mechanism of nucleotide excision repair.
Genes Dev 13, 768–785.
43 Sveistrup JQ (2002) Mechanisms of transcription-cou-
pled DNA repair. Nat Rev Mol Cell Biol 3, 21–29.
44 Mone MJ, Volker M, Nikaido O, Mullenders LH, van
Zeeland AA, Verschure PJ, Manders EM & van Driel R
(2001) Local UV-induced DNA damage in cell nuclei
results in local transcription inhibition. EMBO Report 2,
1013–1017.

45 Volker M, Mone MJ, Karmakar P, van Hoffen A,
Schul W, Vermeulen W, Hoeijmakers JH, van Driel R,
van Zeeland AA & Mullenders LH (2001) Sequential
assembly of the nucleotide excision repair factors in
vivo. Mol Cell 8, 213–224.
46 Syva
¨
oja J & Linn S (1989) Characterization of a large
form of DNA polymerase d from HeLa cells that is
insensitive to proliferating cell nuclear antigen. J Biol
Chem 264, 2489–2497.
47 Sarker AH, Tsutakawa SE, Kostek S, Ng C, Shin DS,
Peris M, Campeau E, Tainer JA, Nogales E & Cooper
PK (2005) Recognition of RNA polymerase II and tran-
scription bubbles by XPG, CSB, and TFIIH: insights
for transcription-coupled repair and Cockayne Syn-
drome. Mol Cell 20, 187–198.
48 Liu B, Wong ML, Tinker RL, Geiduschek EP &
Alberts BM (1993) The DNA replication fork can pass
RNA polymerase without displacing the nascent tran-
script. Nature 366, 33–39.
49 Liu B & Alberts BM (1995) Head-on collision between
a DNA replication apparatus and RNA polymerase
transcription complex. Science 267, 1131–1137.
50 Adolph S, Brusselbach S & Mu
¨
ller R (1993) Inhibition
of transcription blocks cell cycle progression of
NIH3T3 fibroblasts specifically in G1. J Cell Sci
105,

113–122.
51 Ohtani K (1999) Implication of transcription factor E2F
in regulation of DNA replication. Front Biosci 4, D793–
D804.
52 Keezer SM & Gilbert DM (2002) Sensitivity of the ori-
gin decision point to specific inhibitors of cellular signal-
ing and metabolism. Exp Cell Res 273, 54–64.
53 Hatton KS, Dhar V, Brown EH, Iqbal MA, Stuart S,
Didamo VT & Schildkraut CL (1988) Replication pro-
gram of active and inactive multigene families in mam-
malian cells. Mol Cell Biol 8, 2149–2158.
54 Hassan AB & Cook PR (1994) Does transcription by
RNA polymerase play a direct role in the initiation of
replication? J Cell Sci 107, 1381–1387.
55 Bodmer-Glavas M, Edler K & Barberis A (2001) RNA
polymerase II and III transcription factors can stimulate
DNA replication by modifying origin chromatin struc-
tures. Nucleic Acids Res 29, 4570–4580.
56 Hassan AB, Errington RJ, White NS, Jackson DA &
Cook PR (1994) Replication and transcription sites are
colocalized in human cells. J Cell Sci 107, 425–434.
57 Philimonenko AA, Hodny Z, Jackson DA & Hozak P
(2006) The microarchitecture of DNA replication
domains. Histochem Cell Biol 125, 103–117.
58 Iborra FJ, Pombo A, Jackson DA & Cook PR (1996)
Active RNA polymerases are localized within discrete
transcription ‘factories’ in human nuclei. J Cell Sci 109,
1427–1436.
59 Cmarko D, Verschure PJ, Martin TE, Dahmus ME,
Krause S, Fu XD, van Driel R & Fakan S (1999) Ultra-

structural analysis of transcription and splicing in the
cell nucleus after bromo-UTP microinjection. Mol Biol
Cell 10, 211–223.
60 Ehrenhofer-Murray AE, Kamakaka RT & Rine J
(1999) A role for the replication proteins PCNA, RF-C,
polymerase e and Cdc45 in transcriptional silencing in
Saccharomyces cerevisiae. Genetics 153, 1171–1182.
61 Smith JS, Caputo E & Boeke JD (1999) A genetic
screen for ribosomal DNA silencing defects identifies
multiple DNA replication and chromatin-modulating
factors. Mol Cell Biol 19, 3184–3197.
62 Iida T & Araki H (2004) Noncompetitive counterac-
tions of DNA polymerase e and ISW2 ⁄ yCHRAC
for epigenetic inheritance of telomere position effect
in Saccharomyces cerevisiae. Mol Cell Biol 24, 217–
227.
63 Wada M, Miyazawa H, Wang R-S, Mizuno T, Sato A,
Asashima M & Hanaoka F (2002) The second largest
subunit of mouse DNA polymerase e, DPE2, interacts
with SAP18 and recruits the Sin3 Co-repressor protein
to DNA. J Biochem (Tokyo) 131, 307–311.
64 Ng HH & Bird A (2000) Histone deacetylases: silencers
for hire. Trends Biochem Sci 25, 121–126.
65 Vindelo
¨
v LL, Christensen IJ & Nissen NI (1983) Stan-
dardization of high-resolution flow cytometric DNA
analysis by the simultaneous use of chicken and trout
DNA polymerase e associates with RNA polymerase II A. K. Rytko
¨

nen et al.
5548 FEBS Journal 273 (2006) 5535–5549 ª 2006 The Authors Journal compilation ª 2006 FEBS
red blood cells as internal reference standards. Cyto-
metry 3, 323–327.
66 Slot JW & Geuze HJ (1985) A novel method to make
gold probes for multiple labeling cytochemistry. Eur J
Cell Biol 38, 87–93.
67 Dornreiter I (1991) Generation of monoclonal antibodies
against DNA polymerase a-primase and their use for
investigating protein–protein interactions during viral
DNA replication. PhD Thesis. Munich University,
Germany.
68 Tanaka S, Hu SZ, Wang TS-F & Korn D (1982) Pre-
paration and preliminary characterization of monoclo-
nal antibodies against human DNA polymerase a.
J Biol Chem 257, 8386–8390.
69 Stadlbauer F, Brueckner A, Rehfuess C, Eckerskorn
C, Lottspeich F, Forster V, Tseng BY & Nasheuer
HP (1994) DNA replication in vitro by recombinant
DNA polymerase-a-primase. Eur J Biochem 222,
781–793.
70 Rytko
¨
nen AK, Vaara M, Nethanel T, Kaufmann G,
Sormunen R, La
¨
a
¨
ra
¨

E, Nasheuer HP, Rahmeh A, Lee
MYWT, Syva
¨
oja JE et al. (2006) Distinctive activities of
DNA polymerases during human DNA replication.
FEBS J 273, 2984–3001.
71 Makiniemi M, Hillukkala T, Tuusa J, Reini K, Vaara M,
Huang D, Pospiech H, Majuri I, Westerling T, Ma
¨
kela
¨
TP
et al. (2001) BRCT domain-containing protein TopBP1
functions in DNA replication and damage response.
J Biol Chem 276, 30399–30406.
Supplementary material
The following supplementary material is available
online:
Fig. S1. Cell cycle progression of human T98G glio-
blastoma cells after serum stimulation. Human T98G
cells were starved for 6 days in medium containing
0.25% serum and then stimulated to proliferate in
complete medium, or were cultured in the presence of
1mm mimosine in conditioned complete medium. The
cells are from parallel cultures of cells cultured for
immunoelectron microscopy and immunoprecipitation
studies. The figure presents the flow cytometric analy-
sis of cell cycle progression based on the DNA content
of the cells at the indicated times.
Fig. S2. Cell cycle progression of human T98G glio-

blastoma cells after serum stimulation. Human T98G
cells were starved for 6 days in medium containing
0.25% serum and then stimulated to proliferate in con-
ditioned complete medium. The cells are from parallel
cultures of cells cultured for immunoelectron microsco-
py. The figure presents the flow cytometric analysis of
cell cycle progression based on the DNA content of
the cells at the indicated times.
This material is available as part of the online article
from
A. K. Rytko
¨
nen et al. DNA polymerase e associates with RNA polymerase II
FEBS Journal 273 (2006) 5535–5549 ª 2006 The Authors Journal compilation ª 2006 FEBS 5549