The CTD comprises tandem heptapeptide repeats of the Keywords DNA polymerase e; DNA replication; immunoelectron microscopy; nucleotide excision repair; RNA polymerase II Correspondence H
Trang 1RNA polymerase II and nascent transcripts
Anna K Rytko¨nen1,2,*, Tomi Hillukkala1,*, Markku Vaara2, Miiko Sokka2, Maarit Jokela1,†,
Raija Sormunen3, Heinz-Peter Nasheuer4, Tamar Nethanel5, Gabriel Kaufmann5, Helmut Pospiech1 and Juhani E Syva¨oja2
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 polypoly-merase 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.
Trang 2consensus 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
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] Trantran-scription 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 hinrela-ted 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
Trang 3complexed with Pol e, we examined the effect of
phosphatase on the precipitated RNA pol II
Immuno-precipitates were incubated at +37C, 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 translocaelonga-tion 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)1ethidium bromide (EtBr) to exclude
interac-tions mediated by DNA Hyperphosphorylated RNA pol IIO is
indica-ted with a solid arrow and hypophosphorylaindica-ted RNA pol IIA is
indicated with an open arrow.
Trang 4complexes 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 inhibiomit-ted, 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 fixadiges-tion (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
hyphosphorylated 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.
Trang 5For immunoelectron microscopy, T98G cells were
serum deprived followed by serum stimulation, and
cells were collected at different time points, ranging
from 4 h (G0⁄ G1) to 22 h (late S⁄ G2phase) 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 antianti-body 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.
Trang 6excision 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 (G1phase; panel B left),
10 h (G1⁄ 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.
Trang 7compared 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 G1 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 G0⁄ G1 (4 h), late G1
(10 h), early S (14 h), and late S⁄ G2 (24 h) phases of
the cell cycle, and T98G cells were blocked at the
G1⁄ 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 partiparti-cles 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 G0until 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
Trang 8pol 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 trantran-scription
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)2through 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).
Trang 9Notwithstanding 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 transcripreplica-tion apparatus
There are several direct links between transcription and DNA replication Adolph et al [50] showed that a-amanitin blocks cells specifically in the G1 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 G1 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 microsmicros-copy, 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 G1⁄ 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 antianti-body (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.
Trang 10cell 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 37C 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.
Pol a catalytic
subunit
[68]
Pol d catalytic
subunit
Pol e catalytic
subunit
Biotechnologies
Products
Biotechnologies
Biotechnologies Cyclobutane pyrimidine
dimers
a Chen S, Kremmer E, Weisshart K, Hubscher U & Nasheuer H-P, unpublished results.