Rpb1 Sumoylation in Response to UV Radiation or
Transcriptional Impairment in Yeast
Xuefeng Chen, Baojin Ding, Danielle LeJeune, Christine Ruggiero, Shisheng Li*
Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, United States of America
Covalent modifications of proteins by ubiquitin and the Small Ubiquitin-like MOdifier (SUMO) have been revealed to be
involved in a plethora of cellular processes, including transcription, DNA repair and DNA damage responses. It has been well
known that in response to DNA damage that blocks transcription elongation, Rpb1, the largest subunit of RNA polymerase II
(Pol II), is ubiquitylated and subsequently degraded in mammalian and yeast cells. However, it is still an enigma regarding
how Pol II responds to damaged DNA and conveys signal(s) for DNA damage-related cellular processes. We found that Rpb1
is also sumoylated in yeast cells upon UV radiation or impairment of transcription elongation, and this modification is
independent of DNA damage checkpoint activation. Ubc9, an E2 SUMO conjugase, and Siz1, an E3 SUMO ligase, play
important roles in Rpb1 sumoylation. K1487, which is located in the acidic linker region between the C-terminal domain and
the globular domain of Rpb1, is the major sumoylation site. Rpb1 sumoylation is not affected by its ubiquitylation, and vice
versa, indicating that the two processes do not crosstalk. Abolishment of Rpb1 sumoylation at K1487 does not affect
transcription elongation or transcription coupled repair (TCR) of UV-induced DNA damage. However, deficiency in TCR
enhances UV-induced Rpb1 sumoylation, presumably due to the persistence of transcription-blocking DNA lesions in the
transcribed strand of a gene. Remarkably, abolishment of Rpb1 sumoylation at K1487 causes enhanced and prolonged UV-
induced phosphorylation of Rad53, especially in TCR-deficient cells, suggesting that the sumoylation plays a role in
restraining the DNA damage checkpoint response caused by transcription-blocking lesions. Our results demonstrate a novel
covalent modification of Rpb1 in response to UV induced DNA damage or transcriptional impairment, and unravel an
important link between the modification and the DNA damage checkpoint response.
Citation: Chen X, Ding B, LeJeune D, Ruggiero C, Li S (2009) Rpb1 Sumoylation in Response to UV Radiation or Transcriptional Impairment in Yeast. PLoS ONE 4(4):
Editor: Michael Freitag, Oregon State University, United States of America
Received December 30, 2008; Accepted March 24, 2009; Published April 22, 2009
Copyright: ? 2009 Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by NIH grant ES012718 from the National Institute of Environmental Health Sciences, and by NSF grant MCB-0745229. The
funders played no role in the research.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
The integrity of cellular DNA is constantly challenged by both
endogenous and exogenous sources, including oxygen radicals
within cells, environmental UV light, ionizing radiation and other
genotoxic agents . Maintenance of the fidelity of genetic
material is critical for preserving normal cell function and
preventing tumorigenesis of normal cells. To survive and
generate viable progeny, cells must assess the damage and then
either repair it or trigger the apoptotic program. A major
component of the response is the DNA damage checkpoint,
which arrests the cell cycle to provide time for carrying out DNA
repair. In budding yeast, Mec1, the counterpart of mammalian
ATR (Ataxia-Telangiectasia mutated and Rad3-related), and
Tel1, the counterpart of mammalian ATM (Ataxia-Telangiecta-
sia Mutated), are the kinases that sense DNA damage . Mec1
is activated by long 39-ended single stranded DNA (ssDNA) tails
generated during resection of double strand breaks or by ssDNA
gaps arising in repair. Tel1 is activated by unresected, blunt-
ended DNA . Rad53, the counterpart of the mammalian
Chk2, is the major effector of the DNA damage checkpoint, and
its phosphorylation by Mec1 has been considered a hallmark of
checkpoint activation in yeast. Phosphorylated Rad53 targets a
number of substrate proteins, resulting in stabilization of stalled
replisomes, suppression of recombination, and prevention of cell
cycle progression .
Multiple mechanisms have evolved to repair damaged DNA,
including the versatile nucleotide excision repair (NER) which is
capable of removing a variety of bulky helix-distorting lesions,
such as UV-induced cyclobutane pyrimidine dimers (CPDs) and 6-
4 photoproducts . NER has been grouped into two pathways,
i.e., global genomic repair (GGR) and transcription coupled repair
(TCR). GGR is operative throughout the genome, and is
dependent on XPC (xeroderma pigmentosum complemention
group C)  in mammals or Rad7 and Rad16 in S. cerevisiae .
TCR, which is believed to be triggered by the stalling of RNA
polymerase II (Pol II), is dedicated to rapid repair of the
transcribed strand of actively transcribed genes . In human
cells, Cockayne’s syndrome (CS) complementation group A and B
(CSA and CSB) proteins are specifically required for TCR but
dispensable for GGR [6,7,8]. In S. cerevisiae, Rad26, the homolog
of human CSB , and Rpb9 [10,11], a nonessential subunit of
Pol II, have been shown to be specifically involved in TCR.
The fate of the stalled Pol II remains one of the major enigmas
concerning how the cell reacts to damaged DNA . Strikingly,
in response to transcription-blocking DNA damage, Rpb1, the
largest subunit of Pol II, is ubiquitylated and subsequently
degraded [13,14,15,16]. An earlier study in human cells showed
PLoS ONE | www.plosone.org1April 2009 | Volume 4 | Issue 4 | e5267
that the TCR-specific proteins CSA and CSB are required for
Rpb1 ubiquitylation and subsequent degradation, which led to the
proposition that Pol II may need to be degraded for TCR to take
place . However, a recent report showed that the defects in
Rpb1 ubiquitylation observed in CS cells are caused by an indirect
mechanism: these cells shut down transcription in response to
DNA damage, effectively depleting the substrate for ubiquityla-
tion, namely elongating Pol II . In yeast, several proteins,
including Rsp5 , Elc1, Def1  and Rpb9 , have
been shown to be involved in Rpb1 ubiquitylation and subsequent
degradation. However, Rsp5 , Elc1  and Def1  were
shown to play no role in TCR. Interestingly, the domains of Rpb9
that are required for Rpb1 ubiquitylation are different from those
that are involved in TCR [15,21]. Together, the recent results
indicate that Pol II ubiquitylation and degradation do not play a
role in TCR.
The Small Ubiquitin-like MOdifier (SUMO) has been revealed
to be involved in a plethora of cellular processes, including
transcription, DNA repair, cell cycle progression, chromatin
organization, nuclear transport, signal transduction and protein
degradation [22,23]. Like ubiquitin, SUMO is linked to its
substrates via an amide bond between its C-terminal carboxyl
group and the e-amino group of a K residue in the substrate
[22,23]. SUMO modification in yeast is catalyzed by a three-step
enzyme reaction, involving the heterodimeric activating enzyme
(E1) Uba2/Aos1, the conjugating enzyme (E2) Ubc9, and a few
ligases (E3). Sumoylation is a reversible process, and two specific
isopeptidases, Ulp1 and Ulp2, were shown to be responsible for
removing SUMO from modified proteins .
In this study, we identified SUMO as a novel covalent
modification of Rpb1 in response to UV radiation or impairment
of transcription elongation. We further characterized the sumoyla-
tion and found that it plays no role in transcription elongation or
TCR and does not crosstalk with Rpb1 ubiquitylation. However,
Rpb1 sumoylation was found to be affected by activities of NER,
particularly TCR. Remarkably, the sumoylation appears to
function in restraining the DNA damage checkpoint response
caused by transcription-blocking lesions.
Rpb1 is sumoylated in response to UV-induced DNA
TCR is believed to be triggered by the stalling of RNA
polymerase II (Pol II) . In response to UV-induced DNA
damage, Rpb1, the largest subunit of Pol II, is ubiquitylated and
subsequently degraded in both human and yeast cells [13,14]. The
early studies proposed that Pol II ubiquitylation and subsequent
degradation may be required for TCR to take place. However, it
was later found that these events are not related to TCR in either
human  or yeast [15,16,19] cells. To explore potential Pol II-
related signal(s) for TCR and/or other DNA damage responses,
we examined other possible modifications of Rpb1 following UV
irradiation. Rpb1 was immunoprecipited from yeast cells using
antibody 8WG16 which specifically recognizes the C-terminal
heptapeptide repeats of Rpb1 . The immunoprecipitates were
subject to Western blot and probed with antibodies that were
known to recognize potential covalent modifications. Interestingly,
when the immunoprecipitated Rpb1 was probed with an anti-
SUMO antibody, several bands could be seen in the UV
irradiated samples, but not in the unirradiated ones (Fig. 1 A),
indicating that Rpb1 was sumoylated in response to UV-induced
DNA damage. To confirm this finding, a reciprocal immunopre-
cipitation was carried out. Sumoylated proteins were immuno-
precipitated from normally cultured and UV-irradiated cells using
an anti-SUMO antibody, and the immunoprecipitates were
probed with 8WG16 on a Western blot. Several bands could be
detected in the UV irradiated sample, but not in the unirradiated
one (Fig. 1B), indicating that Rpb1 is sumoylated in response to
UV-induced DNA damage. The different bands may reflect
different forms of sumoylated Rpb1 (e.g., mono-, poly- or multi-
Activation of DNA damage checkpoint is not required for
UV-induced Rpb1 sumoylation
DNA damage to a cell can activate checkpoint response, which
promotes cell-cycle arrest, DNA repair, senescence or apoptosis
[2,26,27]. To test if activation of DNA damage checkpoint is
required for UV-induced Rpb1 sumoylation, we examined the
modification in cells lacking Mec1, which plays a key role in
activation of checkpoint in response to UV DNA damage .
Mec1 is essential for cell viability even in the absence of DNA
damage . The inviability of mec1 cells is suppressed by
increasing the activity of cellular ribonucleotide reductase (RNR)
rather than by restoring DNA damage checkpoint function
[28,29], and the essential role of Mec1 during normal cell growth
appears to be in stabilizing stalled replication forks [30,31].
Simultaneously ablating Sml1, an inhibitor of the cellular RNR,
restores the viability of mec1 cells . UV-induced Rpb1
sumoylation was slightly higher in mec1 sml1 cells than in the
isogenic wild type and sml1 cells (Fig. 1C), indicating that this
covalent modification is independent of the checkpoint activation.
The slightly enhanced Rpb1 sumoylation in mec1 sml1 cells is
presumably due to the persistence (slower repair) of DNA damage
in the absence of the checkpoint activation.
Impairment of Pol II transcriptional elongation also
induces Rpb1 sumoylation
UV-induced DNA lesions in the transcribed strand of a gene
block Pol II transcription elongation . We wondered whether
Rpb1 sumoylation occurs specifically in response to UV-induced
DNA damage or is due to blockage of Pol II transcription
elongation. Several chemicals, such as mycophenolic acid (MPA),
thiolutin and 1, 10-phenanthroline, have been used to inhibit
transcription in yeast . MPA inhibits transcription elongation
by depleting cellular GTP pool, and sensitivity to this drug has
been widely used as a landmark of transcription elongation
deficiency . Thiolutin inhibits transcription by all three RNA
polymerases, mainly at the stage of transcription initiation . 1,
10-phenanthroline is a metal chelator that most likely inhibits
transcription by sequestering divalent metal ions . Thiolutin
and 1, 10-phenanthroline did not induce detectable Rpb1
sumoylation (Fig. 1D). However, MPA induced Rpb1 sumoylation
to a certain level, which is lower than that induced by UV
(Fig. 1D). These results suggest that Rpb1 sumoylation can be
induced by impairment of transcription elongation. The reason
that MPA induces a lower level of Rpb1 sumoylation than UV
may reflect the fact that UV induced DNA damage may cause a
more severe blockage of elongating Pol II.
K1487 of Rpb1 is a major sumoylation site
We attempted to identify the site(s) of sumoylation on Rpb1.
Sumoylation usually occurs on a lysine (K) residue located in the
consensus motif YKxE/D (where Y is a hydrophobic residue and
x is any residue) . Rpb1 is a high molecular-weight protein
(192 kD) with a total of 93 K residues. A sequence search
indicated that K1487 is located in the sumoylation motif (VKDE).
Sumoylation of Rpb1
PLoS ONE | www.plosone.org2 April 2009 | Volume 4 | Issue 4 | e5267
We created centromeric LEU2 plasmids encoding the wild type
and a mutant Rpb1 with an R replacing the K at site 1487
(K1487R). The LEU2 plasmids were shuffled into yeast cells whose
genomic RPB1 gene was deleted. Yeast cells expressing the mutant
Rpb1 grew normally under all conditions examined (not shown).
Wild type and the mutant Rpb1 were immunoprecipitated from
the respective cells following UV irradiation and probed with an
anti-SUMO antibody on a Western blot. The K1487R mutation
caused disappearance of a major and a minor band reflecting
different forms of sumoylated Rpb1 (Fig. 1E). This indicates that
K1487 is the major sumoylation site in response to UV-induced
Several Ks of Rpb1, namely K431 (WKVE), K1221 (FKND),
K1286 (MKYD), K1720 (PKQD) and K1725 (QKHN) are
located in sequences that are similar to the sumoylation motif. To
test if these Ks are the minor sumoylation sites, we created
plasmids encoding mutant Rpb1 with Rs replacing K1487 and
each of these Ks. The additional K to R mutations did not change
the pattern of UV induced Rpb1 sumoylation (Fig. 1F), indicating
that these Ks (other than K1487) are not the minor sumoylation
Ubc9 and Siz1 play important roles in Rpb1 sumoylation
In S. cerevisiae, a single essential gene, SMT3, encodes the
SUMO (Smt3) protein . The SUMO is activated by an E1
activating enzyme and then passed to an E2 conjugase. An E3
ligase acts as an adapter to interact with both E2 and substrates
and promote the transfer of SUMO from E2 to specific substrates
Ubc9, which is essential for cell viability, is the only SUMO E2
conjugase identified so far in yeast . To examine if Ubc9 is
involved in Rpb1 sumoylation, we inserted the degron-myc
sequences [38,39] in-frame at the 59 end of the coding region of
the genomic UBC9 gene, thereby expressing the Ubc9 protein with
Figure 1. Western blots showing Rpb1 sumoylation in response to UV radiation or impairment of transcription elongation. (A) Rpb1
was immunoprecipitated from the unirradiated and UV irradiated cells using antibody 8WG16 (anti-Rpb1) and probed with anti-SUMO and 8WG16
antibodies. (B) Sumoylated proteins were immunoprecipitated from the unirradiated and UV irradiated cells and probed with 8WG16 and anti-SUMO
antibodies. (C) UV-induced Rpb1 sumoylation in wild type (JKM179), sml1 (YFD756) and sml1 mec1 (YAA25) cells. (D) Sumoylation of Rpb1 in response
to UV or treatments of transcription inhibitors. (E) UV-induced Rpb1 sumoylation in cells expressing wild type (CX84) or K1487R mutant (CX79) Rpb1.
Bars on the left of the blot indicate distinct bands formed by wild type Rpb1. Arrow heads on the right of the blot mark bands abolished by the
K1487R mutation. (F) UV-induced Rpb1 sumoylation in cells expressing wild type (CX84) or K to R mutant (CX79, CX105, CX106, CX108, CX110 and
CX110) Rpb1. Bars on the left of the blot indicate distinct bands formed by wild type Rpb1. Arrow heads on the right of the blot mark bands not
shown by the mutant Rpb1. WT, wild type.
Sumoylation of Rpb1
PLoS ONE | www.plosone.org3 April 2009 | Volume 4 | Issue 4 | e5267
a degron-myc tag at the N-terminus. Degron tagging has been
successfully used to conditionally degrade an essential protein in
yeast [38,39]. A degron tagged protein can function normally in
cells at permissive temperature (24uC), but is rapidly degraded
through a ubiquitin-mediated pathway at non-permissive temper-
ature (37uC) . The myc tag following the degron tag is used for
detection of a tagged protein with a generic anti-myc antibody
[38,39]. The tagged Ubc9 was degraded to an undetectable level
,2.5 hours after the cells were shifted to 37uC (Fig. 2A). In cells
expressing the degron-myc tagged Ubc9, UV induced Rpb1
sumoylation occurred normally at the permissive temperature but
was undetectable at the non-permissive temperature (Fig. 2B). On
the other hand, Rpb1 sumoylation occurred normally in cells
expressing the native Ubc9 at the nonpermissive temperature.
These results indicate that the SUMO E2 conjugase Ubc9 plays
an important role in Rpb1 sumoylation.
In yeast, four SUMO E3 ligases have been identified: Siz1, Siz2
(Nfi1) [40,41], Mms21/Nse2 , and Zip3 . To identify the
E3 ligase(s) that is/are involved in Rpb1 sumoylation, we started
with testing Siz1 and Siz2, because they have been shown to be the
major SUMO E3 ligases . The prominent bands of
sumoylated Rpb1 shown in wild type cells were essentially
abolished in siz1 cells (Fig. 2C), indicating that Siz1 plays an
important role in Rpb1 sumoylation. However, some high
molecular weight bands/smear of sumoylated Rpb1 appear to
be somewhat enhanced in siz1 cells (Fig. 2C, upper part of the
blot), presumably reflecting a higher induction of highly
sumoylated Rpb1 in the mutant cells.
The pattern of bands reflecting different forms of sumoylated
Rpb1 was altered in siz2 cells: the prominent slower migrating
bands were fainter whereas the intensity of the fastest migrating
band (at the bottom of the blot) was slightly increased (Fig. 2C,
compare the siz2 and WT lanes). This suggests that Siz2 may, to a
certain extent, facilitate induction of poly-sumoylation of Rpb1.
Sumoylation of Rpb1 does not affect its UV-induced
Sumoylation takes place on lysine residues, which can also be
modified by ubiquitylation. A growing body of evidence shows the
existence of cross-talk between the two processes. Sumoylation of a
substrate could stabilize the protein by antagonizing ubiquitylation
[for a recent review see ]. Recently, it was found that
sumoylation can also promote the degradation of the modified
protein by facilitating its ubiquitylation . As UV induces both
sumoylation and ubiquitylation of Rpb1, we wondered if there is
crosstalk between the two processes.
First, we examined if sumoylation of Rpb1 affects UV-induced
Rpb1 degradation, which has been shown to be dependent on a
prior ubiquitylation event [13,15,16]. UV induced Rpb1 degra-
dation was not compromised in siz1 cells (Fig. 3A), where Rpb1
sumoylation was virtually abolished (see above, Fig. 2C). More-
over, a K to R mutation at residue 1487, the major sumoylation
site of Rpb1 (see above, Fig. 1E and F), does not cause any
noticeable alteration in the UV induced Rpb1 degradation
(Fig. 3B). These results indicate that UV-induced Rpb1 degrada-
tion, which is dependent on prior ubiquitylaiton, is not dependent
on Rpb1 sumoylation.
Hex3 (Slx5) and Slx8 are yeast proteins with important
functions in DNA damage control and maintenance of genomic
stability [45,46,47,48]. Several recent studies showed that the
Hex3/Slx8 complex is an E3 ligase that specifically ubiquitylates
sumoylated proteins in yeast [48,49,50]. Deficiency in the Hex3/
Slx8 ubiquitin ligase causes the accumulation of sumoylated
proteins . To examine if the UV induced Rpb1 sumoylation is
the substrate of the Hex3/Slx8 E3 ubiquitin ligase, we examined
UV induced Rpb1 degradation in hex3 slx8 cells and found that the
degradation rate was similar to that in wild type cells (Fig 3C).
Also, UV induced sumoylated Rpb1 did not accumulate in hex3
Figure 2. Western blots showing the roles of Ubc9 and Siz1 in
UV-induced Rpb1 sumoylation. (A) Degradation of degron-myc
tagged Ubc9 upon shifting to nonpermissive temperature (37uC) in
galactose containing medium (to induce the expression of plasmid
pKL142 encoded Ubr1, a ubiquitin E3 ligase). Tubulin serves as an
internal loading control. (B) Abolishment of UV-induced Rpb1
sumoylation when Ubc9 was depleted. Rpb1 was immunoprecipitated
from the cells cultured at the indicated conditions using antibody
8WG16 and probed with anti-SUMO and 8WG16 antibodies. (C) The
roles of Siz1 and Siz2 in UV-induced Rpb1 sumoylation. Rpb1 was
immunoprecipited from the UV irradiated wild type (BY4741) and
mutant (strains 4245 and 2412) cells using antibody 8WG16 and probed
with anti-SUMO and 8WG16 antibodies. The control was a sample
prepared from unirradiated wild type cells. WT, wild type.
Sumoylation of Rpb1
PLoS ONE | www.plosone.org4 April 2009 | Volume 4 | Issue 4 | e5267
slx8 cells (not shown), indicating that the sumoylated Rpb1 is not a
substrate for the Hex3/Slx8 ubiquitin ligase.
A poly-SUMO chain is required for some sumoylated proteins
to be targeted for ubiquitylation and subsequent degradation
[51,52]. Formation of poly-SUMO chains on a substrate needs
K11, K15 or K19 of the Smt3 (SUMO) protein . K to R
mutations at all 3 sites block the formation of poly-SUMO chains
but still allow mono-sumoylation on substrates . We used cells
expressing a mutant Smt3 whose K11, K15 and K19 were
replaced by R residues to determine whether blockage of poly-
sumoylation of Rpb1 affects Rpb1 degradation. Rpb1 was
degraded normally in the mutant cells (Fig. 3D), indicating that
the degradation is independent of Rpb1 poly-sumoylation. Taken
together, our results strongly suggest that sumoylation of Rpb1
does not affect UV-induced Rpb1degradation. As Rpb1 degrada-
tion is dependent on a prior ubiquitylation event [13,15,16], it is
unlikely that the sumoylation affects UV-induced Rpb1 ubiquity-
Rpb1 ubiquitylation does not affect its sumoylation
We wondered if ubiquitylation of Rpb1 play a role in its
sumoylation. Def1  and Elc1  are required for UV induced
Rpb1 ubiquitylation and degradation in yeast. The patterns of
Rpb1 sumoylation were similar between def1 or elc1 and their
respective isogenic wild type cells (Fig. 4), indicating that Rpb1
ubiquitylation does not affect its sumoylation.
We also directly examined if blockage of Rpb1 ubiquitylation
sites affects Rpb1 sumoylation. On Rpb1, K330 and K695 are the
major and minor ubiquitylation sites, respectively . While a
K695R mutant Rpb1 was almost normally degraded upon UV
irradiation , a K330R mutant Rpb1 was essentially not
degraded following UV irradiation (not shown), in agreement with
a previous report . However, neither the K330R nor the
K695R mutation affects the pattern of UV-induced Rpb1
sumoylation (Fig. 4). This indicates that the multiple Rpb1 bands
detected by anti-SUMO antibody on the Western blots are not
caused by concomitant ubiquityaltion, but may reflect different
forms (mono-, poly- or multi-) of Rpb1 sumoylation. Taken
together, our results suggest that Rpb1 ubiquitylation does not
affect its sumoylation and vice versa: there is no apparent cross-talk
between the two processes.
Deficiency in TCR or entire NER enhances UV-induced
Next, we examined if Rpb1 sumoylation interplays with NER,
especially TCR. The UV-induced Rpb1 sumoylation was
Figure 3. Sumoylation of Rpb1 does not affect its degradation
in response to UV radiation. Whole cell extracts were prepared from
the cells that had been incubated for different times following UV
irradiation. Rpb1 in the whole cell extracts were probed with antibody
8WG16 on the Western blots. Tubulin serves as an internal loading
control. (A) Levels of Rpb1 in isogenic wild type (BY4741) and siz1 (strain
4245) cells. (B) Levels of wild type and K1487R mutant Rpb1 expressed
in isogenic cells (CX84 and CX79). (C) Levels of Rpb1 in wild type (Y452)
and hex3 slx8 (MHY3861) cells. (D) Levels of Rpb1 in isogenic cells
expressing wild type (JD74-13c) or K11,15,19R mutant Smt3 (YKU116).
WT, wild type.
Figure 4. Ubiquitylation of Rpb1 does not affect its sumoyla-
tion. Rpb1 was immunoprecipitated from the unirradiated (control,
BJ5465) and UV irradiated isogenic wild type [BJ5465 (lane 2) and Y452
(lane 4)], elc1 (CR105) and def1 (SL128) cells and cells expressing wild
type [CX84 (lane 6)], K330R (CR191) or K695R (CR192) mutant Rpb1
using antibody 8WG16 and probed with anti-SUMO and 8WG16
Sumoylation of Rpb1
PLoS ONE | www.plosone.org5 April 2009 | Volume 4 | Issue 4 | e5267
measured in wild type, rad7 (GGR-deficient) , rad26 rpb9 (TCR-
deficient) , rad7 rpb9 rad26 (TCR- and GGR-deficient) 
and rad14 (TCR- and GGR-deficient)  cells. UV-induced
sumoylation of Rpb1 occurred in all of these strains (Fig. 5A),
indicating that neither GGR nor TCR activity is essential for
Rpb1 sumoylation. However, the levels of sumoylated Rpb1 were
significantly higher in rad26 rpb9, rad7 rpb9 rad26 and rad14 cells
than in wild type and rad7 cells (Fig. 5A). These results indicate
that deficiency in NER, particularly TCR, enhances the induction
of Rpb1 sumoylation.
To confirm that the deficiency in TCR, rather than a specific
repair factor, enhances the induction of Rpb1 sumoylation, we
compared UV-induced Rpb1 sumoylations in rad7 rpb9, rad7 rpb9
rad26 and rad7 rpb9 rad26 spt4 cells. Deletion of the SPT4 gene,
which encodes a transcription elongation factor, partially restores
TCR in rad7 rad26  and rad7 rad26 rpb9  cells.
Interestingly, the level of UV-induced Rpb1 sumoylation in rad7
rpb9 rad26 spt4 was similar to that in rad7 rpb9 cells but significantly
lower than that in rad7 rpb9 rad26 cells (Fig. 5B), suggesting that the
deficiency in TCR is the cause for the enhanced UV-induced
Abolishment of Rpb1 sumoylation at K1487 does not
The observation that deficiency in TCR enhances UV-induced
Rpb1 sumoylation can be explained by the persistence (or slower
removal) of transcription-blocking lesions in the transcribed strand
of the genes, as the mere impairment of transcription elongation
by MPA treatment also induces Rpb1 sumoylation (Fig. 1D).
Alternatively, Rpb1 sumoylation may serve as a TCR signal,
which may be removed (Rpb1 de-sumoylated) during or after the
TCR process in TCR-proficient cells. To test the second
possibility, we examined the effect of K1487R mutation of Rpb1
on repair of CPDs in the constitutively transcribed RPB2 gene in
rad16 cells where GGR is abolished [5,57] and TCR can be
unambiguously analyzed. Yeast cells were cultured to late log
phase, UV irradiated, and incubated in a repair medium for
various lengths of time. Total DNA was isolated, digested with a
restriction enzyme to excise the fragment of interest, and incised at
the UV-induced CPDs with an excess amount of T4 endonuclease
V . The incised fragments were strand-specifically end-labeled,
resolved on a DNA sequencing gel, and exposed against a
Phosphoimager screen. The band intensities in the gel lane of ‘‘0’’
time repair indicate the yields of CPDs at different sites. A
decrease in band intensities with time at respective sites indicates
CPD repair at these sites. In rad16 cells expressing the wild type
Rpb1, fast repair can be seen in the transcribed strand of the RPB2
gene, initiating at ,40 nucleotides upstream of the transcription
start site (Fig. 6A), in agreement with our previous results .
The TCR rate in rad16 cells expressing K1487R Rpb1 was similar
to those expressing the wild-type Rpb1 (Fig. 6A). In agreement
with previous results [9,10,11,57], deletion of RAD26 dramatically
diminishes TCR in the RPB2 gene (Fig. 6B). The TCR rate in
rad16 rad26 cells expressing K1487R Rpb1 was also similar to
Figure 5. UV-induced sumoylation in wild type and NER-deficient cells. Log phase cells were irradiated with UV and incubated in a rich
medium at 30uC. Rpb1 was immunoprecipitated from the cells at different times of the post-UV incubation using antibody 8WG16 and probed with
anti-SUMO and 8WG16 antibodies. (A) UV-induced Rpb1 sumoylation in wild type (BJ5465), rad7 (GGR-deficient) (SL212), rad26 rpb9 (TCR-deficient)
(SL81), rad7 rad26 rpb9 (GGR- and TCR-deficient) (SL244) and rad14 (GGR- and TCR-deficient) (CR14) cells. As Rpb1 was gradually degraded during the
post-UV incubation in RPB9+(WT, rad7 and rad14) cells , the loadings of samples from these cells at the different time points were adjusted to
approximately the same level of Rpb1 remaining. (B) UV-induced Rpb1 sumoylation in rad7 rpb9 (SL221), rad7 rad26 rpb9 (SL244) and rad7 rad26 rpb9
spt4 (SL243) cells.
Sumoylation of Rpb1
PLoS ONE | www.plosone.org6 April 2009 | Volume 4 | Issue 4 | e5267
those expressing the wild-type Rpb1 (Fig. 6B). These results
indicate that the K1487R mutation affects neither the overall
TCR nor the Rad26-independent TCR.
In agreement with our TCR analysis results, mutating K1487 of
Rpb1 to an arginine does not affect UV sensitivities of otherwise
wild type, rad16 and rad16 rad26 cells (not shown). Also, yeast cells
expressing K1487R mutant Rpb1 are not sensitive to the
nucleotide depletion drug MPA (not shown), suggesting that the
mutation does not significantly affect transcription elongation.
Abolishment of Rpb1 sumoylation at K1487 enhances
UV-induced Rad53 phosphorylation, especially in TCR-
In human cell lines with defective TCR, stalled Pol II causes
an increase in p53 levels and eventual induction of apoptosis
. Stalling of Pol II, caused by DNA damage, a DNA
intercalating agent (actinomycin D) or microinjection of anti-Pol
II antibodies in the nuclei, leads to p53 induction in a manner
that depends on ATR and the single stranded DNA binding
protein RPA , indicating that Pol II may function as a
damage sensor for the DNA damage checkpoint response
[60,61]. Although S. cerevisiae lacks p53 and a long checkpoint
arrest in G1 phase or a robust apoptotic pathway, the organism
has a DNA damage checkpoint system that is similar to that in
mammals . In S. cerevisiae, phosphorylation of Rad53 (the
human Chk2 homologue), an effector of DNA damage
checkpoint, is an essential step for the cellular response to
DNA damage and has been widely used as a marker for DNA
damage checkpoint activation [2,62,63].
To examine if Rpb1 sumoylation plays a role in DNA damage
checkpoint response, we analyzed UV-induced Rad53 phosphor-
ylation in log phase yeast cells. In wild type cells, UV irradiation
caused rapid phosphorylation of Rad53, which is reflected by the
slower migrating Rad53 bands on a Western blot (Fig. 7A, see
Rad53 bands marked with ‘p’). The level of Rad53 phosphory-
lation, as indicated by the ratio of the phosphorylated to
unphophorylated Rad53, peaked ,30 minutes after UV irradia-
tion and gradually decreased afterwards (Fig. 7A and C). UV-
induced Rad53 phosphorylation was somewhat weakened in rad16
cells, and dramatically impaired and delayed in rad16 rad26 cells
(Fig. 7, compare panels A, D and G, and panels C, F and I). These
results agree well with previous reports showing that both GGR
mediated by Rad16 and TCR mediated by Rad26 contribute to
DNA damage checkpoint response . In fact, all yeast mutants
deficient in incision during NER have been shown to be deficient
in the rapid phosphorylation of Rad53 in response to UV
K1487R mutation of Rpb1 caused slightly enhanced and
prolonged phosphorylation of Rad53 in otherwise wild type (for
NER genes) cells in response to UV radiation (Fig. 7A, B and C).
However, the K1487R mutation caused significant increase of
UV-induced Rad53 phosphorylation in rad16 (Fig. 7E and F) and
rad16 rad26 cells (Fig. 7H and I). Intriguingly, the K1487R
mutation largely restored the rapid phosphorylation of Rad53 in
rad16 rad26 cells in response to UV radiation (Fig. 7H and I). We
also examined the effects of the K1487R mutation on UV-induced
Rad53 in cells synchronized at G1 (with a factor) and G2/M (with
nocodazole) phases, and in stationary phase cultures. The general
trends were similar to those obtained with the unsynchronized log
phase cells (not shown). These results indicate that abolishment of
Rpb1sumoylation at K1487 enhanced UV-induced Rad53
phosphorylation, especially in TCR deficient cells. In other words,
sumoylation of Rpb1 at K1487 may play a role in restraining
DNA damage checkpoint response caused by transcription-
We also observed that treatment of wild type, rad16 and rad16
rad26 cells expressing wild type or the K1487R mutant Rpb1 with
the nucleotide depleting drug MPA did not trigger phosphoryla-
tion of Rad53 (not shown), although the treatment triggers Rpb1
sumoylation (Fig. 1D). This indicates that mere impairment of
transcription elongation may not be sufficient for inducing DNA
damage checkpoint response in yeast.
Figure 6. Abolishment of Rpb1 sumoylation at K1487 does not
affect overall TCR or Rad26-independent TCR. (A) DNA sequenc-
ing gels showing repair of UV-induced cyclobutane pyrimidine dimers
(CPDs) in the transcribed strand of the RPB2 gene in rad16 cells
expressing wild type (CX85) or K1487R mutant (CX87) Rpb1. (B) DNA
sequencing gels showing repair of CPDs in the transcribed strand of the
RPB2 gene in rad16 rad26 cells expressing wild type (CX112) or K1487R
mutant (CX113) Rpb1. Lanes U are unirradiated controls. Other lanes are
samples from cells incubated for different times (min) following UV
irradiation. The arrow on the left of the gels indicates the transcription
start site of RPB2.
Sumoylation of Rpb1
PLoS ONE | www.plosone.org7 April 2009 | Volume 4 | Issue 4 | e5267
In this study, we identified SUMO as a novel covalent
modification of Rpb1 in response to UV DNA damage and
impairment of transcription elongation, and unraveled an
interesting connection between the modification and the DNA
damage checkpoint response in yeast. Like ubiquitylation ,
sumoylation of Rpb1 can be induced by either UV radiation or
nucleotide depletion drugs (e.g., MPA), indicating that inductions
of these modifications are not limited to DNA damage but are
triggered by impairment of transcription elongation. However,
Rpb1 ubiquitylation and sumoylation do not appear to have any
crosstalk, as the K sites for the two modifications do not overlap
and the events of UV-induced Rpb1 degradation (which is
dependent on prior ubiquitylation) and sumoylation are mutually
independent. Furthermore, Rpb1 sumoylation appears to be
independent of DNA damage checkpoint activation, as the
modification is not compromised in cells lacking Mec1 (Fig. 1C).
In many cases, without an E3 ligase, the E2 conjugase Ubc9 can
directly attach SUMO protein to a substrate [23,65]. The catalytic
cleft of Ubc9 directly interacts with many substrates via their SUMO
consensus motif (yKxE/D), but this interaction is not sufficient for
efficient SUMO transfer to the target K residue. Target modification
therefore often depends on a third class of enzymes, the E3 ligases,
which enhance SUMO transfer from the E2 to the substrate.
Interestingly, our results indicate that while E3 ligase Siz1 is critical
for Rpb1 sumoylation, the E3 ligase Siz2 may, to a certain extent,
facilitate poly-sumoylation of Rpb1. These two E3 ligases may
function competitively to achieve optimal sumoylation of Rpb1. In
the absence of Siz1, more Rpb1 molecules may be available for Siz2
mediated sumoylation, which may result in the somewhat enhanced
induction of high molecular weight forms of sumoylated Rpb1 in siz1
cells (Fig. 2C, compare WTand siz1 lanes).On the other hand, in the
absence of Siz2, more Rpb1 may be available for Siz1, which may
mainly mediate mono- or oligo-sumoylation of Rpb1.
Figure 7. Effects of Rpb1 sumoylation at K1487 on UV-induced Rad53 phosphorylation. (A–C) UV-induced Rad53 phosphorylation in log
phase wild type (for NER genes) cells expressing wild type (CX84) or K1487R mutant (CX79) Rpb1. (D–F) UV-induced Rad53 phosphorylation in log
phase rad16 cells expressing wild type (CX85) or K1487R mutant (CX87) Rpb1. (G–I) UV-induced Rad53 phosphorylation in log phase rad16 rad26 cells
expressing wild type (CX112) or K1487R mutant (CX113) Rpb1. The cells were irradiated with UV and incubated in a rich medium at 30uC. Whole cell
extracts were prepared from the cells at different times of the post-UV incubation. Rad53 in the whole cell extracts was probed with an anti-Rad53
antibody on Western blots. p and u on the left of the blots indicate bands of phosphorylated and unphosphorylated Rad53, respectively. Plots C, F
and I show ratios of phosphorylated Rad53 (Rad53p) to unphosphorylated Rad53 (Rad53u) in the wild type, rad16 and rad16 rad26 cells, respectively.
Error bars represents standard deviations.
Sumoylation of Rpb1
PLoS ONE | www.plosone.org8 April 2009 | Volume 4 | Issue 4 | e5267
TCR is generally believed to be initiated by Pol II stalled at a
lesion in the transcribed strand of a gene . However, the exact
signal for TCR is still a mystery. Here, we present evidence that
sumoylation at the major sumoylation site of Rpb1 (K1487) is not
involved in TCR, as a K to R mutation at this site does not affect
either the overall TCR or the Rad26-independent TCR (Fig. 6).
Therefore, it is less likely that sumoylation of Rpb1 serves as a
TCR signal. At present, however, we cannot rule out the
possibility that sumoylation at some as-yet-unidentified minor
site(s) of Rpb1 plays a role in TCR.
Our data show that sumoylation of Rpb1 is not dependent on
TCR or the entire NER process, as the modification occurs in wild
type and TCR- or NER-deficient cells (Fig. 5). However, Rpb1
sumoylation is enhanced in cells with deficiency in TCR or entire
NER (Fig. 5). Our previous studies showed that Rpb1 ubiquityla-
tion and subsequent degradation are also enhanced in TCR- or
NER-deficient cells . These enhancements are mostly likely
due to the persistence (or slower removal) of Pol II-stalling lesions
in the transcribed strand of a gene in these cells. This notion is
supported by the findings that impairment of elongating Pol II by
nucleotide depletion drugs also induces Rpb1 sumoylation
(Fig. 1D) and ubiquitylation .
Single stranded DNA (ssDNA) is a useful common DNA
damage checkpoint signal as it is formed during nucleotide and
base excision repair, during resection at the ends of double
stranded DNA breaks and at stalled replication forks . It is
generally accepted that the DNA damage checkpoint response can
be triggered by interaction of ATR (or Mec1 in yeast) with RPA-
coated ssDNA. A recent study showed that stalling of Pol II in
mammalian cells, either by DNA damage or by non-DNA-
damaging agents results in the phosphorylation of the serine 15 site
of p53 in a RPA and ATR-dependent manner . It was
hypothesized that a region of ssDNA is formed after blockage of
transcription elongation that attracts RPA, leading to the
recruitment of ATR and the activation of p53 by phosphorylation
of the serine 15 site. Consistent with this hypothesis is a study
showing that RPA and ATR preferentially accumulate on
transcribed DNA sequences after UV irradiation, presumably at
sites of blocked RNA polymerases . In this paper we show that
abolishment of sumoylation of Rpb1 at K1487 enhanced UV-
induced Rad53 phosphorylation, especially in TCR-deficient cells,
establishing a link between Rpb1 sumoylation and checkpoint
control. The molecular mechanism underlying the enhancement
of Rad53 phosphorylation in cells expressing K1487R mutant
Rpb1 remains to be elucidated. It is less likely that the
enhancement is achieved by perturbation of transcription
elongation or TCR, as the K1487R mutation does not affect
either the sensitivity to the nucleotide depletion drug MPA (not
shown) or TCR (Fig. 6). One possibility is that the abolishment of
Rpb1 sumoylation at K1487 may alter the conformation of Pol II
stalled at lesions, leading to generation or exposure of ssDNA
regions that can trigger and/or sustain checkpoint response.
Alternatively, sumoylated Rpb1 may recruit additional factors,
which may in turn prevent the loading of a damage sensor to the
ssDNA generated by damage-stalled Pol II.
What is the physiological function for Rpb1 sumoylation? It is
believed that the major biological mission of the DNA damage
checkpoint is to coordinate cellular processes allowing time to
repair the damage so that the checkpoint-arrested cells can
eventually resume cell cycle progression and continue their
physiological program. Based on our results, one potential role
for Rpb1 sumoylation may be to prevent spurious activation of
checkpoint response by stalled Pol II. Interestingly, the K1487R
mutation of Rpb1 does not appear to affect UV sensitivity of any
cells tested, indicating that the role of Rpb1 sumoylation in the
checkpoint response is not linked to cell survival.
Besides K1487, some other minor sumoylation sites on Rpb1
remain to be identified, and the role(s) of sumoylation at the other
sites await to be determined. We observed that a mere impairment
of transcription by MPA treatment did not cause Rad53
phosphorylation, although the treatment caused induction of
Rpb1 sumoylation. In contrast, stalling of Pol II by some non-
DNA-damaging agents induces DNA damage checkpoint activa-
tion in mammalian cells . Therefore, it would be very
interesting to test if Rpb1 sumoylation and the role of the
modification are conserved between yeast and mammalian cells.
Materials and Methods
The yeast strains used in the present work are listed in Table 1.
To create gene deletion mutants, cells were transformed with
linearized plasmids or PCR products bearing a selection marker
(URA3, LEU2 or KanMX genes) flanked by sequences comple-
mentary to the genes to be deleted. Strains expressing degron-myc
tagged Ubc9 under the control of the copper-inducible promoter
were created by transforming cells with PCR products generated
using plasmid pKL187 as template . Plasmid pKL142, which
encodes Ubr1 (a ubquitin E3 ligase) under the control of the GAL1
promoter, was transformed into the cells expressing the degron-
myc tagged Ubc9 to promote rapid and conditional depletion of
the degron tagged Ubc9 upon shifting to the nonpermissive
temperature (37uC) in galactose containing media .
Plasmid construction and shuffling
Plasmid pJS670, which bears the full length wild type RPB1
gene on the centromeric LEU2 vector pRS415 , was kindly
provided by Dr. Jeff Strathern (the National Cancer Institute,
NIH, Frederick, Maryland). Plasmids encoding Rpb1 with a K to
R mutation at specific sites were created using plasmid pJS670 as
template through site-directed mutagenesis (QuickChange II
mutagenesis kit, Stratagene). The LEU2 plasmids encoding the
wild type or mutated Rpb1 were transformed into yeast strains
whose genomic RPB1 gene is deleted and complemented by a
centromeric URA3 plasmid encoding the wild type Rpb1. The
URA3 plasmid encoding the wild type Rpb1 was then evicted from
the cells by selecting the transformed cells on plates containing 5-
fluoroorotic acid (5-FOA), which will kill the cells with a functional
URA3 gene .
Cell culture and UV irradiation
Unless otherwise indicated, yeast cells were grown at 30uC in
minimal media containing glucose (SD) to late log phase
(A600,1.0), washed with ice-cold H2O and resuspended in ice-
cold 2% glucose. For analyses of repair of UV-induced CPDs, the
cell suspension was irradiated with 80 J/m2of 254 nm UV. For
analyses of UV-induced sumoylation and degradation of Rpb1
and phosphorylation of Rad53, the cell suspension was irradiated
with 240 J/m2of 254 nm UV. The irradiated cell suspension was
added with one-tenth volume of a stock solution containing 10%
yeast extract and 20% peptone and incubated in the dark at 30uC.
Aliquots were removed from the cultures at different times of the
incubation and the cells of the aliquots were harvested.
For analyses of the role of Ubc9 in UV-induced Rpb1
sumoylation, cells expressing native Ubc9 and those expressing
degron-myc tagged Ubc9 and transformed with plasmid pKL142
were grown in minimal medium containing 2% raffinose and
1 mM of CuSO4at the permissive temperature (24uC) to late log
Sumoylation of Rpb1
PLoS ONE | www.plosone.org9 April 2009 | Volume 4 | Issue 4 | e5267
phase (A600,1.0). Half of each of the cultures continued to be
incubated at 24uC. The other half of each was washed with H2O,
resuspended in pre-warmed (at 37uC) minimal medium containing
galactose (SG) (without CuSO4) and incubated at 37uC for 2 hrs.
The 24uC and 37uC cultures were irradiated with 240 J/m2of
254 nm UV, and the cells were harvested after 1 hr of further
incubation at the respective temperatures.
Genomic DNA was isolated from the harvested cells that had
been irradiated with 80 J/m2of UV and incubated in the repair
medium for different lengths of time, as described previously .
The gene fragments of interest were 39-end labeled with [a-
32P]dATP using a procedure described previously [69,70]. Briefly,
1 mg of total genomic DNA was digested with restriction enzyme(s)
to release the fragments of interest and incised at CPD sites with
an excess amount of purified T4 endonuclease V (Epicentre).
Excess copies of biotinylated oligonucleotides, which are comple-
mentary to the 39 end of the fragments to be labeled, were mixed
with the sample. The mixture was heated at 95uC for 5 min to
denature the DNA and then cooled to an annealing temperature
of around 50uC. The annealed fragments were attached to
streptavidin-conjugated magnetic beads (Invitrogen), and the other
fragments were removed by washing the beads at the annealing
[a-32P]dATP (Perkin-Elmer) and resolved on sequencing gels.
Table 1. Yeast strains used in this study.
BJ5465MATa ura3-52 trp1 leu2D1 his3D200 pep4::HIS3 prb1D1.6R can1
BY4741 MATa his3D1 leu2D0 met15D0 ura3D0 Open Biosystems
GHY498MATa his3D200 his4-912d lys2-128d leu2D1 ura3-52 rpb1D187::HIS3 [pRP112]
JD47-13cMATa leu2-D1 trp1-D63 his3-D200 ura3-52 lys2-801 ade2-101
JKM179 ho hml::ADE1 MATa hmr::ADE1 ade1-100 leu2-3,112 trp1::hisG lys5 ura3-52 ade3::GAL::HO 
MHY501 MATa his3-D200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2
Y452 MATa ura3-52 his3-1 leu2-3 leu2-112L. Prakash
2412as BY4741, but Siz2::KanMXOpen Biosystems
4245as BY4741, but Siz1::KanMXOpen Biosystems
CR14as BJ5465, but rad14::URA3This study
CR105 as BJ5465, but elc1::KanMX This study
CR109 as Y452, but rad16::hisG degron-mycUBC9 This study
CR191 as GHY498, but [pRP112] replaced with [ pJS670-K330R RPB1]This study
CR192as GHY498, but [pRP112] replaced with [ pJS670-K695R RPB1]This study
CX79 as GHY498, but [pRP112] replaced with [ pJS670-K1487R RPB1]This study
CX84 as GHY498, but [pRP112] replaced with [ pJS670]This study
CX85as Y452, but rad16::hisG rpb1::KanMX [ pJS670] This study
CX87as Y452, but rad16::hisG rpb1::KanMX [ pJS670-K1487R RPB1] This study
CX105as GHY498, but [pRP112] replaced with [ pJS670-K1487R K431R RPB1] This study
CX106as GHY498, but [pRP112] replaced with [ pJS670-K1487R K1221R RPB1] This study
CX108 as GHY498, but [pRP112] replaced with [ pJS670-K1487R K1286R RPB1]This study
CX110 as GHY498, but [pRP112] replaced with [ pJS670-K1487R K1720R RPB1]This study
CX111 as GHY498, but [pRP112] replaced with [ pJS670-K1487R K1725R RPB1]This study
CX112 as Y452, but rad16::hisG rad26::URA rpb1::KanMX [ pJS670] This study
CX113as Y452, but rad16::hisG rad26::URA rpb1::KanMX [ pJS670-K1487R RPB1] This study
MHY3861hex3::KanMX4 slx8::KanMX4 (generated from a cross between the two single mutants)
SL81as Y452, but rad26::URA3 rpb9::LEU2
SL128as Y452, but def1::URA3 This study
SL212as BJ5465, but rad7D
SL221 as BJ5465, but rad7D rpb9D
SL243 as BJ5465, but rad7D rpb9D rad26::URA3 spt4:LEU2 This study
SL244 as BJ5465, but rad7D rpb9D rad26D
Y542-16 as Y452, but rad16::hisGThis study
YAA25 as JKM179, but sml1::KAN mec1::NAT
YFD756as JKM179, but sml1::KAN 
YKU116as JD47-13c, but Smt3-R11, 15, 19
aPlasmid contained in a strain is shown in a bracket.
Sumoylation of Rpb1
PLoS ONE | www.plosone.org 10 April 2009 | Volume 4 | Issue 4 | e5267
The gels were dried and exposed against a Phosphorimager screen
Treatments of cells with transcription inhibitors
Yeast cells were grown in SD medium at 30uC to late log phase
(A600,1.0). 1, 10-phenanthroline, thiolutin and mycophenolic
acid (MPA) were added to the cultures to final concentrations of
200 mg/ml,10 mg/ml,and 200 mg/ml,
1.5 hours of further incubation, the cells were harvested.
Whole cell extract preparation
Whole cell extracts were prepared using a TCA method, as
described previously . Briefly, harvested cells were resus-
pended in 15% TCA and broken by vortexing them with acid-
washed glass beads (Sigma, #G9268). The proteins in the cell
lysates were pelleted by centrifugation at 20,0006g for 15 min.
The protein pellet was washed with ice-cold 80% acetone, and
dissolved in 26SDS-PAGE gel loading buffer .
Yeast cells harvested from 25 ml of culture were washed once
with IP buffer (10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 0.4 mM Na4VO3, 10 mM Na4P2O7,
10 mM NaF, 0.5% NP-40, 1% Triton X-100, 0.1% SDS, 0.2 mM
PMSF and protease inhibitors cocktail) and resuspended in 0.5 ml
of the same IP buffer. The cells were disrupted by vortexing with
acid-washed glass beads and the cell lysates were cleared by
centrifugation twice at 14,000 rpm for 5 minutes at 4uC. Eight mg
of 8WG16 (Neoclone, WP011), which specifically recognizes the
C-terminal heptapeptide repeats of Rpb1 , or anti-SUMO
(Rockland, 200-401-428) antibody were added to the cell lysate
and the mixture was incubated at 4uC overnight with gentle
rotation. Protein A-coated agarose beads (Millipore) were added to
the mixture and incubated at 4uC for 3 hours with gentle rotation.
The beads were washed four times with IP buffer. Bound proteins
were eluted by boiling the beads in 26 SDS-PAGE gel loading
Proteins in the whole cell extracts or immunoprecipitated
samples were resolved on SDS-PAGE gels and transferred onto
PVDF membranes (Immobilon-P, Millipore). Proteins of interest
on the blots were probed with specific antibodies. The antibodies
against the myc tag, tubulin and Rad53 were from Sigma
(M4439), GeneTex (GTX76511) and Santa Cruz (sc-6749),
respectively. Blots were incubated with SuperSignalH West Femto
Maximum Sensitivity Substrate (Pierce), and the protein bands
were detected using a chemiluminescence scanner (Fluorchem
8800, Alpha Innotech). As indicated, band intensities on some
Western blots were quantified using AlphaEaseFC 4.0 software.
We are grateful to Drs. R. Jurgen Dohmen, James Haber, Grant Hartzog,
Mark Hochstrasser, Jeff Strathern, and Toshio Tsukiyama for providing
yeast strains and plasmids.
Conceived and designed the experiments: XC SL. Performed the
experiments: XC BD DL CR. Wrote the paper: XC SL.
1. Ljungman M, Lane DP (2004) Transcription - guarding the genome by sensing
DNA damage. Nat Rev Cancer 4: 727–737.
2. Harrison JC, Haber JE (2006) Surviving the breakup: the DNA damage
checkpoint. Annu Rev Genet 40: 209–235.
3. Friedberg EC, Walker GC, Siede W, Wood RD, Schultz RA, et al. (2006) DNA
Repair and Mutagenesis. Washington D.C.: ASM Press.
4. Venema J, van Hoffen A, Karcagi V, Natarajan AT, van Zeeland AA, et al.
(1991) Xeroderma pigmentosum complementation group C cells remove
pyrimidine dimers selectively from the transcribed strand of active genes. Mol
Cell Biol 11: 4128–4134.
5. Verhage R, Zeeman AM, de Groot N, Gleig F, Bang DD, et al. (1994) The
RAD7 and RAD16 genes, which are essential for pyrimidine dimer removal
from the silent mating type loci, are also required for repair of the
nontranscribed strand of an active gene in Saccharomyces cerevisiae. Mol Cell
Biol 14: 6135–6142.
6. Troelstra C, van Gool A, de Wit J, Vermeulen W, Bootsma D, et al. (1992)
ERCC6, a member of a subfamily of putative helicases, is involved in
Cockayne’s syndrome and preferential repair of active genes. Cell 71: 939–953.
7. van Hoffen A, Natarajan AT, Mayne LV, van Zeeland AA, Mullenders LH, et
al. (1993) Deficient repair of the transcribed strand of active genes in Cockayne’s
syndrome cells. Nucleic Acids Res 21: 5890–5895.
8. Venema J, Mullenders LH, Natarajan AT, van Zeeland AA, Mayne LV (1990)
The genetic defect in Cockayne syndrome is associated with a defect in repair of
UV-induced DNA damage in transcriptionally active DNA. Proc Natl Acad
Sci U S A 87: 4707–4711.
9. van Gool AJ, Verhage R, Swagemakers SM, van de Putte P, Brouwer J, et al.
(1994) RAD26, the functional S. cerevisiae homolog of the Cockayne syndrome
B gene ERCC6. Embo J 13: 5361–5369.
10. Li S, Smerdon MJ (2002) Rpb4 and Rpb9 mediate subpathways of transcription-
coupled DNA repair in Saccharomyces cerevisiae. Embo J 21: 5921–5929.
11. Li S, Smerdon MJ (2004) Dissecting transcription-coupled and global genomic
repair in the chromatin of yeast GAL1-10 genes. J Biol Chem 279:
12. Laine JP, Egly JM (2006) When transcription and repair meet: a complex
system. Trends Genet 22: 430–436.
13. Beaudenon SL, Huacani MR, Wang G, McDonnell DP, Huibregtse JM (1999)
Rsp5 ubiquitin-protein ligase mediates DNA damage-induced degradation of
the large subunit of RNA polymerase II in Saccharomyces cerevisiae. Mol Cell
Biol 19: 6972–6979.
14. Bregman DB, Halaban R, van Gool AJ, Henning KA, Friedberg EC, et al.
(1996) UV-induced ubiquitination of RNA polymerase II: a novel modification
deficient in Cockayne syndrome cells. Proc Natl Acad Sci U S A 93:
15. Chen X, Ruggiero C, Li S (2007) Yeast Rpb9 plays an important role in
ubiquitylation and degradation of Rpb1 in response to UV-induced DNA
damage. Mol Cell Biol 27: 4617–4625.
16. Woudstra EC, Gilbert C, Fellows J, Jansen L, Brouwer J, et al. (2002) A Rad26-
Def1 complex coordinates repair and RNA pol II proteolysis in response to
DNA damage. Nature 415: 929–933.
17. Anindya R, Aygun O, Svejstrup JQ (2007) Damage-induced ubiquitylation of
human RNA polymerase II by the ubiquitin ligase Nedd4, but not Cockayne
syndrome proteins or BRCA1. Mol Cell 28: 386–397.
18. Ribar B, Prakash L, Prakash S (2006) Requirement of ELC1 for RNA
polymerase II polyubiquitylation and degradation in response to DNA damage
in Saccharomyces cerevisiae. Mol Cell Biol 26: 3999–4005.
19. Lommel L, Bucheli ME, Sweder KS (2000) Transcription-coupled repair in
yeast is independent from ubiquitylation of RNA pol II: implications for
Cockayne’s syndrome. Proc Natl Acad Sci U S A 97: 9088–9092.
20. Lejeune D, Chen X, Ruggiero C, Berryhill S, Ding B, et al. (2008) Yeast Elc1
plays an important role in global genomic repair but not in transcription coupled
repair. DNA Repair (Amst).
21. Li S, Ding B, Chen R, Ruggiero C, Chen X (2006) Evidence that the
transcription elongation function of Rpb9 is involved in transcription-coupled
DNA repair in Saccharomyces cerevisiae. Mol Cell Biol 26: 9430–9441.
22. Gill G (2004) SUMO and ubiquitin in the nucleus: different functions, similar
mechanisms? Genes Dev 18: 2046–2059.
23. Hay RT (2005) SUMO: a history of modification. Mol Cell 18: 1–12.
24. Hay RT (2007) SUMO-specific proteases: a twist in the tail. Trends Cell Biol 17:
25. Thompson NE, Steinberg TH, Aronson DB, Burgess RR (1989) Inhibition of in
vivo and in vitro transcription by monoclonal antibodies prepared against wheat
germ RNA polymerase II that react with the heptapeptide repeat of eukaryotic
RNA polymerase II. J Biol Chem 264: 11511–11520.
26. Su TT (2006) Cellular responses to DNA damage: one signal, multiple choices.
Annu Rev Genet 40: 187–208.
27. Wood JL, Chen J (2007) DNA damage sensing and signaling. In: Wei Q, Li L,
Chen DJ, eds. DNA Repair, Genetic Instability, and Cancer. 1 ed: World
Scientific Publishing. pp 1–22.
28. Desany BA, Alcasabas AA, Bachant JB, Elledge SJ (1998) Recovery from DNA
replicational stress is the essential function of the S-phase checkpoint pathway.
Genes Dev 12: 2956–2970.
Sumoylation of Rpb1
PLoS ONE | www.plosone.org 11 April 2009 | Volume 4 | Issue 4 | e5267
29. Zhao X, Rothstein R (2002) The Dun1 checkpoint kinase phosphorylates and Download full-text
regulates the ribonucleotide reductase inhibitor Sml1. Proc Natl Acad Sci U S A
30. Lopes M, Cotta-Ramusino C, Pellicioli A, Liberi G, Plevani P, et al. (2001) The
DNA replication checkpoint response stabilizes stalled replication forks. Nature
31. Tercero JA, Diffley JF (2001) Regulation of DNA replication fork progression
through damaged DNA by the Mec1/Rad53 checkpoint. Nature 412: 553–557.
32. Zhao X, Muller EG, Rothstein R (1998) A suppressor of two essential
checkpoint genes identifies a novel protein that negatively affects dNTP pools.
Mol Cell 2: 329–340.
33. Tornaletti S, Reines D, Hanawalt PC (1999) Structural characterization of RNA
polymerase II complexes arrested by a cyclobutane pyrimidine dimer in the
transcribed strand of template DNA. J Biol Chem 274: 24124–24130.
34. Grigull J, Mnaimneh S, Pootoolal J, Robinson MD, Hughes TR (2004)
Genome-wide analysis of mRNA stability using transcription inhibitors and
microarrays reveals posttranscriptional control of ribosome biogenesis factors.
Mol Cell Biol 24: 5534–5547.
35. Hyle JW, Shaw RJ, Reines D (2003) Functional distinctions between IMP
dehydrogenase genes in providing mycophenolate resistance and guanine
prototrophy to yeast. J Biol Chem 278: 28470–28478.
36. Tipper DJ (1973) Inhibition of yeast ribonucleic acid polymerases by thiolutin.
J Bacteriol 116: 245–256.
37. Johnston JR (1994) Molecular genetics of yeast: a pratical approach. New York:
Oxford University Press.
38. Dohmen RJ, Varshavsky A (2005) Heat-inducible degron and the making of
conditional mutants. Methods Enzymol 399: 799–822.
39. Sanchez-Diaz A, Kanemaki M, Marchesi V, Labib K (2004) Rapid depletion of
budding yeast proteins by fusion to a heat-inducible degron. Sci STKE 2004:
40. Johnson ES, Gupta AA (2001) An E3-like factor that promotes SUMO
conjugation to the yeast septins. Cell 106: 735–744.
41. Takahashi Y, Kahyo T, Toh EA, Yasuda H, Kikuchi Y (2001) Yeast Ull1/Siz1
is a novel SUMO1/Smt3 ligase for septin components and functions as an
adaptor between conjugating enzyme and substrates. J Biol Chem 276:
42. Zhao X, Blobel G (2005) A SUMO ligase is part of a nuclear multiprotein
complex that affects DNA repair and chromosomal organization. Proc Natl
Acad Sci U S A 102: 4777–4782.
43. Cheng CH, Lo YH, Liang SS, Ti SC, Lin FM, et al. (2006) SUMO
modifications control assembly of synaptonemal complex and polycomplex in
meiosis of Saccharomyces cerevisiae. Genes Dev 20: 2067–2081.
44. Perry JJ, Tainer JA, Boddy MN (2008) A SIM-ultaneous role for SUMO and
ubiquitin. Trends Biochem Sci 33: 201–208.
45. Ii T, Fung J, Mullen JR, Brill SJ (2007) The yeast Slx5-Slx8 DNA integrity
complex displays ubiquitin ligase activity. Cell Cycle 6: 2800–2809.
46. Ii T, Mullen JR, Slagle CE, Brill SJ (2007) Stimulation of in vitro sumoylation by
Slx5–Slx8: evidence for a functional interaction with the SUMO pathway. DNA
Repair (Amst) 6: 1679–1691.
47. Wang Z, Jones GM, Prelich G (2006) Genetic analysis connects SLX5 and
SLX8 to the SUMO pathway in Saccharomyces cerevisiae. Genetics 172:
48. Xie Y, Kerscher O, Kroetz MB, McConchie HF, Sung P, et al. (2007) The yeast
Hex3.Slx8 heterodimer is a ubiquitin ligase stimulated by substrate sumoylation.
J Biol Chem 282: 34176–34184.
49. Prudden J, Pebernard S, Raffa G, Slavin DA, Perry JJ, et al. (2007) SUMO-
targeted ubiquitin ligases in genome stability. Embo J 26: 4089–4101.
50. Sun H, Leverson JD, Hunter T (2007) Conserved function of RNF4 family
proteins in eukaryotes: targeting a ubiquitin ligase to SUMOylated proteins.
Embo J 26: 4102–4112.
51. Mullen JR, Brill SJ (2008) Activation of the SLX5–SLX8 ubiquitin ligase by
poly-SUMO conjugates. J Biol Chem.
52. Uzunova K, Gottsche K, Miteva M, Weisshaar SR, Glanemann C, et al. (2007)
Ubiquitin-dependent proteolytic control of SUMO conjugates. J Biol Chem 282:
53. Bylebyl GR, Belichenko I, Johnson ES (2003) The SUMO isopeptidase Ulp2
prevents accumulation of SUMO chains in yeast. J Biol Chem 278:
54. Somesh BP, Sigurdsson S, Saeki H, Erdjument-Bromage H, Tempst P, et al.
(2007) Communication between distant sites in RNA polymerase II through
ubiquitylation factors and the polymerase CTD. Cell 129: 57–68.
55. Prakash S, Prakash L (2000) Nucleotide excision repair in yeast. Mutat Res 451:
56. Jansen LE, den Dulk H, Brouns RM, de Ruijter M, Brandsma JA, et al. (2000)
Spt4 modulates Rad26 requirement in transcription-coupled nucleotide excision
repair. Embo J 19: 6498–6507.
57. Verhage RA, van Gool AJ, de Groot N, Hoeijmakers JH, van de Putte P, et al.
(1996) Double mutants of Saccharomyces cerevisiae with alterations in global
genome and transcription-coupled repair. Mol Cell Biol 16: 496–502.
58. Lloyd RS (2005) Investigations of pyrimidine dimer glycosylases–a paradigm for
DNA base excision repair enzymology. Mutat Res 577: 77–91.
59. Ljungman M, Zhang F (1996) Blockage of RNA polymerase as a possible trigger
for u.v. light-induced apoptosis. Oncogene 13: 823–831.
60. Derheimer FA, O’Hagan HM, Krueger HM, Hanasoge S, Paulsen MT, et al.
(2007) RPA and ATR link transcriptional stress to p53. Proc Natl Acad Sci U S A
61. Lindsey-Boltz LA, Sancar A (2007) RNA polymerase: the most specific damage
recognition protein in cellular responses to DNA damage? Proc Natl Acad
Sci U S A 104: 13213–13214.
62. Giannattasio M, Lazzaro F, Longhese MP, Plevani P, Muzi-Falconi M (2004)
Physical and functional interactions between nucleotide excision repair and
DNA damage checkpoint. Embo J 23: 429–438.
63. Zhang H, Taylor J, Siede W (2003) Checkpoint arrest signaling in response to
UV damage is independent of nucleotide excision repair in Saccharomyces
cerevisiae. J Biol Chem 278: 9382–9387.
64. Somesh BP, Reid J, Liu WF, Sogaard TM, Erdjument-Bromage H, et al. (2005)
Multiple mechanisms confining RNA polymerase II ubiquitylation to polymer-
ases undergoing transcriptional arrest. Cell 121: 913–923.
65. Johnson ES (2004) Protein modification by SUMO. Annu Rev Biochem 73:
66. Jiang G, Sancar A (2006) Recruitment of DNA damage checkpoint proteins to
damage in transcribed and nontranscribed sequences. Mol Cell Biol 26: 39–49.
67. Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains
designed for efficient manipulation of DNA in Saccharomyces cerevisiae.
Genetics 122: 19–27.
68. Boeke JD, LaCroute F, Fink GR (1984) A positive selection for mutants lacking
orotidine-59-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid
resistance. Mol Gen Genet 197: 345–346.
69. Li S, Waters R (1996) Nucleotide level detection of cyclobutane pyrimidine
dimers using oligonucleotides and magnetic beads to facilitate labelling of DNA
fragments incised at the dimers and chemical sequencing reference ladders.
Carcinogenesis 17: 1549–1552.
70. Li S, Waters R, Smerdon MJ (2000) Low- and high-resolution mapping of DNA
damage at specific sites. Methods 22: 170–179.
71. Sambrook J, Russell DW (2001) Molecular Cloning: A Laboratory Manual.
Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
72. Jones EW (1991) Tackling the protease problem in Saccharomyces cerevisiae.
Methods Enzymol 194: 428–453.
73. Lindstrom DL, Hartzog GA (2001) Genetic interactions of Spt4-Spt5 and TFIIS
with the RNA polymerase II CTD and CTD modifying enzymes in
Saccharomyces cerevisiae. Genetics 159: 487–497.
74. Dotiwala F, Haase J, Arbel-Eden A, Bloom K, Haber JE (2007) The yeast DNA
damage checkpoint proteins control a cytoplasmic response to DNA damage.
Proc Natl Acad Sci U S A 104: 11358–11363.
75. Chen P, Johnson P, Sommer T, Jentsch S, Hochstrasser M (1993) Multiple
ubiquitin-conjugating enzymes participate in the in vivo degradation of the yeast
MAT alpha 2 repressor. Cell 74: 357–369.
Sumoylation of Rpb1
PLoS ONE | www.plosone.org12 April 2009 | Volume 4 | Issue 4 | e5267