DISS ETH NO. 15874
RttlOlp, provides a novel link between ubiquitination and DNA replication restart
The cullin,
A dissertation submitted to the
SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH for the
degree
of
Doctor of Natural Sciences
Presented
by
Brian Luke
Bsc. Honours,
Queen's University, Kingston, Ontario, Canada Born
May 28,1976
Citizen of Canada and
Accepted on
Belgium
the recommendation of
Prof. Matthias Peter- examiner Prof. Wilhelm Krek- co-examiner Dr.
Philippe
Pasero- co-examiner
2
Table of Contents
ZUSAMMENFASSUNG
5
SUMMARY
6
Opening
7
Remark
CHAPTER 1
-
8
GENERAL INTRODUCTION-1
Novel roles for cullin dependent ligases
8
Protein degradation by the Ubiquitin/Proteasome system
8 9
Ubiquitin and Ubiquitin Conjugation E3 Ligases DNA
replication of
11
19
and
repair replication origins
20
Regulation Replication slow zones Sensing DNA damage Checkpoints Repairing Double Stranded Breaks
22 24
-
The role of
ubiquitin in
DNA
25
28
replication and Repair and Replication
28
Ubiquitin conjugation/degradation Ubiquitin and Repair CHAPTER-II THE YEAST
30
CULLIN, RTT101P, IS INVOLVED IN GENOMIC 32
INTEGRITY AND DNA REPAIR
33
Introduction
36
Results rttlOl A cells have
a
checkpoint dependent G2/M delay
and accumulate with short
pre-anaphase 36
spindles rttlOlA cells exhibit
a
slower S
phase
than wild type cells and have fewer
firing RttlOlp is synthetically-lethal with the replication helicase Rrm3p overlapping functions rttlOlA cells are highly sensitive to the damaging agents MMS and sensitive to HU, and not sensitive to
an
firing replication origins 38
,
but
they
do not share
40 CPT but
only moderately
HO induced cut
phase checkpoint replication following MMS treatment but not HU treatment is impaired Cells lacking RttlOlp have increased DSBs and genomic instability
44 47
49
Discussion G2/M cell
cycle delay due to a defect during DNA-replication involved in the replication of proteinaceous chromatin regions not required for the Rad53p-checkpoint or DSB-repair, but specifically functions at
rttlOlA cells exhibit
a
RttlOlp is RttlOlp is collapsed replication forks RttlOlp is required to restart collapsed but not stalled replication forks The ability to restart collapsed replication forks is important to ensure genome stability Does the cullin RttlOlp function as an E3-ubiquitin ligase to restart collapsed replication forks?
Strains constructions and
manipulations
49 50 51 52 53 54
56
Material and Methods
DNA
42
is activated and executed in rttlOlA cells, however re-initiation of
The intra-S
genetic manipulations
56
56
3
56
Microscopy
57
with the Gal HA-RTT101 strains
Arrest/release
experiments analysis and BrdU detection 2D gel analysis and ERC isolation Dynamic molecular combing
57
PFGE
58 58
CHAPTER-III OTHER COMPONENTS OF THE RTT101P PATHWAY approaches to find other components of the RttlOl pathway Ubc4p and Ubc5p, interact genetically and physically with RttlOlp The N-terminal region of RttlOlp is essential for function indication a conserved role in adaptor binding A genomic wide screen to find other RttlO lp interactors Modification and regulation of the cullin RttlOlp
Using
60 60
Genetic
60
The redundant E2s,
substrate
CHAPTER-IV GENERAL DISCUSSION
cycle
71
71
contribution of RttlOlp
Checkpoints Checkpoint
65 69
71
Overview
The cell
64
and cell
cycle
71
arrest
inactivation to reveal
pathway replication initiation? DNA repair? a
involved in
Is the cullin,
RttlOlp, play direct role in So what is RttlO lp doing? Is RttlOl functioning as a ubiquitin ligase? Are there other components? Is RttlOlp regulated? Is this a conserved process? RttlOlp and the cancer connection Does RttlO lp
72
73 75 78 79 81 81 82 83
Remarks and Outlook
Concluding
72
CHAPTER- V CONTROL OF NUTRIENT-SENSITIVE TRANSCRIPTION
PROGRAMS BY THE UNCONVENTIONAL PREFOLDIN URI
84
Abstract
85
Introduction
85
Results
85
Discussion
90
Figures
92
References
97
98
SUPPORTING ONLINE MATERIAL
98
Materials and Methods
107
Supporting Figures
115
APPENDIX I
115
Materials and Methods Determination of DNA content DNA prep for 2D 2D gel
Preparation
by
FACS
REFERENCES
115 115
gel analysis
of BrdU-substituted DNA
PFGE
analysi s
116
plugs
117 117
118
4
ACKNOWLEDGMENTS
129
CURRICULUM VITAE
130
5
Zusammenfassung
Zuverlässige Rephkation
der DNS ist
essentieller
von
Bedeutung
für den
und somit für das
Überleben eines Organismus. Ist die Rephkation
beeinträchtigt,
können Fehler entstehen. Solche Mutationen führen
so
Instabilität und dadurch
zu
zu
genetischer
für Schäden, die
chemischen, mutagenen Substanzen, y-Strahlung, aber auch durch zelleigene
von
Rephkation
oxidative Metaboliten oder fehlerhafte
entstehen können. Eine Zelle
deshalb in der
Lage sein, solche Schäden reparieren
Verdoppelung
der DNS sicherzustellen.
Mittels der
Phase
Sprosshefe
Hefeprotein
dass das
um
der DNS
anfällig
Krankheiten wie Krebs. DNS ist
Zellzyklus
zu
lösen.
in sich
S.cerevisae als
der DNS
Replikationsgabeln
konnten wir
nicht
wiederhergestellt,
Doppelstrangbrüche reparieren;
Replizieren
von
beschleunigen (Michel
eine
aber
E3-Ligase Diese
doppelsträngigen
zu
können
von
Weiter haben wir bewiesen,
'checkpoints'
sog.
Wirkung
von
RttlOlp
et
für
Ubiquitin
oder
das
Hypothese
bei in sich
wird
von
E2-Ubiquitin Enzymen Ubc4p von
Ubiquitin-Ketten in
genetischen und
Cullin-Familie das Fortsetzen der
angehalten wurde, gewährleistet.
von
Zellextrakten anzutreffen. Dies
DNS-Replikation nachdem
eine
E3-Ligase
der
Replikationsgabel
Höchstwahrscheinlich existiert ein Protein, dass
Doppelstrangbrüchen ubiquitiniert hat.
Ubc5p
vitro
mit den obenerwähnten Daten, darauf hin, dass eine
Folge
und
ai, 2003a).
chromatinhaltigen Fraktionen
zusammen
zur
die
DNS wieder aufnehmen können.
gestützt. Zudem kann RttlOlp die Bildung
Synthese
RttlOlp benötigt wird,
reparieren, damit
führt dies oft
angehalten,
zwar
biochemischen Interaktionen mit den
weist,
zu
dass
RttlOlp-Aktivität aufweisen,
zusammengefallenen Replikationsgabeln.
ist in
konnten wir feststellen,
dies weist darauf hin, dass dieses Cullin keine direkte
RttlOlp ist höchstwahrscheinlich
RttlOlp
zeigen,
stabilisiert werden, auch ohne die
Kontrollpunkten
eine fehlerfreie
dazu dient, Probleme in der S-
generellen DNS-Reparaturmechanismen spielt.
Replikationsgabeln, die
um
werden kann. Werden solche defekte
fortgesetzt
Brüchen in der DNS. Hefen die keine
dass
RttlOlp,
zusammengefallene Replikationsgabeln
Rephkation
Rolle in
Modellorganismus,
der 'Cullin'-Familie,
Genaugenommen,
können,
zu
muss
wird und damit die Wiederaufnahme der DNS-
an
6
Summary Accurate DNA
organismal
replication
survival. If the
which may lead to
replication machinery
compromised,
and diseases like
genetic instability
attack from exogenous agents
is
including y-irradiation
errors can occur
DNA is vulnerable to
cancer.
and chemical mutagens,
however the accumulation of endogenous oxidative metabolites, and
occurring DNA replication with such
equipped to deal error
free
also result in DNA
errors can
replicative
and
and
cycle progression
is essential for cell
damage.
naturally
A cell must be
induced stress in order to
damage
ensure
genomic duplication.
Using
the
budding yeast,
is
protein, RttlOlp,
that the yeast cullin
problems. Specifically,
we
collapsed replication forks, deleted for
S. cerevisiae,
RttlOlp,
are
to
repair
is
Furthermore,
have demonstrated that
replication
we
efficiently
to
result in
a
a
we
have found
cope with S
phase
in the re-start of
double stranded break. Cells
double stranded break
intact and the cullin does
repair machinery
organism,
RttlOlp is involved
frequently
competent
fully
required
have shown that which
model
as a
directly
function
indicating
that
during repair.
stalled, but checkpoint stabilized
a
fork is able to restart in the absence of
RttlOlp.
As
well, RttlOlp, is
localized to the chromatin fraction of cell extracts.
RttlOlp collapsed forks,
is most as
it shows
ubiquitin conjugating RttlOlp
can
most
is
resumption
enzymes,
together
required
likely
genetic
as an
E3
ubiquitin conjugating
ligase
Ubc4p
at
a
for
these data
replication
and
Ubc5p.
It has been shown
provide
the first evidence that
fork re-start, and suggests that
double stranded break site, that becomes
of DNA
synthesis.
at
and biochemical interactions with the E2
previously
enhance the formation of ubiquitin chains in vitro (Michel
Taken
ligase
likely acting
a
a
et
al.
that
2003).
cullin based E3
target protein exists,
ubiquitinated to
allow the
7
Opening
Remark
The work that I have done which
are
thesis
was
during
my PhD thesis
very different in terms of
devoted to
determining
topic.
The
was
split
minority
the role of the novel
into two different
of
(approximately 1/4)
prefoldin protein, URI,
sensing pathway, through
transcription factor, Gcn4p.
This work is not introduced in the
the role of the cullin,
Chapter
RttlOlp,
to efficient and accurate cell
potential
cycle progression,
in the
in the
general introduction,
V. The remainder of my thesis work
and how this
of my
the translational control of the
activation of the amino acid
however it is covered in
projects,
E3
was
ligase component
budding yeast,
focused
on
contributes
S. cerevisiae.
8
1- General lntroduction-1
Chapter
Novel roles for cullin The
dependent ligases of proteins
regulated degradation
sequence
interrogate
RttlOlp.
the role of the novel and
We
is
only recently begun
has
mainly
to be elucidated.
budding yeast, Saccharomyces cerevisiae,
employed
about the role of
RttlOlp
ligases
homology it has long been known that other cullin proteins exist, their
cellular functions have We used the
cullin-based E3
(Cull) containing complex. Although
been addressed in the context of the Cullin 1
by
by
both
RttlOlp
previously
genetic
in the cell.
model system to
uncharacterized cullin
and biochemical
approaches
My studies have led
replication
involved in both DNA
intimately
as a
us to
to
protein,
try to learn
more
the conclusion that
and DNA
repair following
damage. Therefore, the following introduction will provide background information both
of cullin based
ubiquitin ligases, focusing
on
ligases.
I will also introduce
aspects of DNA replication and repair and finally the last part of the introduction will describe what is
currently know
about the role of
ubiquitination
with
regards
DNA
to
metabolism.
Protein
degradation by
The cell
activity, signals,
which
can
is driven
in turn
by
Ubiquitin/Proteasome system
oscillations of
cycle
until the
on, however
be shut off to
ensure
whereas STOP
that
signals
stage. The best way
irreversibility.
they
to
cyclin dependent kinase (CDK)
phosphorylate targets, thereby generating
which promote entry into the next cell
slow the cell
signals
cycle
the
previous stage
also need an
to
abolish
a
completed.
be turned off. For
event is not
need to be
is
cycle stage,
repeated
destroyed
signal
is to
to
both "GO"
and "STOP"
CDK
activity
example
twice within the
signals
can
a, GO same
signal, cell
allow entry into the next cell
destroy it, thereby ensuring
The controlled destruction of
protein signals
within
a
turn
cell is
which these must
cycle,
cycle
9
accomplished with great This system is based
on
accuracy and
the
principal
target, it becomes recognized
that when
that
degraded become polyubiquitinated, resulting spatial
ensure
addition to
and
temporal protein
degradation
of mis-folded
Ubiquitin
and
Ubiquitin,
76 amino acid
a
to
ubiquitin
in
an
ATP a
which is
al. 1984).
protein.
highly
Many regulatory proteins to
a
ubiquitin
chain. The
generically
conserved
are
cycle.
In
the
conjugation
to the
to an
glycine
et al.
active-site
responsible for recruiting an
a
thio-ester bond
residue
cysteine
on an
by
an
E-amino group
E2, ubiquitin
E3
1.
on
ubiquitin
or
can
the
conjugated to
is recruited to the 26S proteasome which
Figure
(G76) of
1981). Ubiquitin is then
rounds of this reaction the substrate
This reaction is outlined in
ubiquitin
E3, which
the substrate to the E2, which
accepting lysine
of
targeted substrate (Hershko
The last step of the reaction is carried out
repeated
protein
subject to rapid
termed El, E2 and
dependent manner (Ciechanover
trans-thioesterification
polyubiquitin chain, the
most
conjugate ubiquitin
subsequently transfer ubiquitin to After
is the
residue in the El and the C-terminal
conjugating enzyme.
.
cell
single
1998). The El, ubiquitin activating enzyme, forms
cysteine
transferred via
protein
a
three amino acids substitutions from yeast to human
activate and then
and Ciechanover
ligase,
protein,
the action of three enzymes,
a
distribution within
key
This system
"targeted degradation".
in
is
that should be
proteins
endocytosis, receptor down-regulation and
proteolysis following conjugation
between
those
Specificity
Ubiquitin Conjugation
ubiquitin (Ozkaynak et
cooperate
only
enzymatic
via
peptides
proteins.
eukaryotes, with only
requires
to a
playing a fundamental role in cell cycle progression, ubiquitination has
been shown to be involved in
among
ubiquitin chain is conjugated
a
substrate for the 26S proteasome, which
ubiquitin/proteasome system, ensuring
to
ubiquitin/proteasome system.
al. 1983; Hershko and Ciechanover 1998).
proteolysis (Hershko et
helps
the
the target into small amino acid
consequently degrades
to the
as a
efficiency by
targeted
a
enzymatically degrades
10
A. Ub-Ub-Ub
J
.
Substrate
B.
AMP+PPj
ATP
°
^A
9
2 ^» Ei~S-C-Ub—-"* E2-S-C-Ub
Ub-C-01O
Protein
Ë2-SH
ATP
ADP+Pj
Ub
Vl^
h
Ub-peptides-*
Protein—(Ub)n
26S Proteasome
Peptides
Ub
I8 Amino acids
Figure
1. A. A schematic of the
reactions
as
Hershko and
For
chain
poly-ubiquitin
a
for
and Rechsteiner
proteolysis.
1986), whereas
conjugates
more
et al.
1984b).
26S proteasome.
Rad6p/Ubc2p,
as
K48
Although
formation, they do
not
well
shown
to be
as
linkage
which
can
linkages
other
than
most
a
lysine residues
and
al. 1995), which has been shown to
Ubc5p
play
a
are
ubiquitin.
Hough
may result in
something
other than
mono-ubiquitinate
a
substrate but is
proteolysis (Hershko a
have
substrate with
an
within
targeted degradation.
Ubc4p
are
al. 1984b;
substrate with
likely
there
et
biochemically that
degraded
result in
manners as
ubiquitin chains primarily
1984a; Hershko
does not result in substrate
was
likely
et al.
different
a
(methyl ubiquitin),
1985). Furthermore it
(Hershko
be formed in many different
degradation (Hershko
Me-Ub
unable to form
is
can
it has become clear that K48-G76 linked
protein
conjugate
implicated in the ubiquitin system. B. The conjugation of ubiquitin to a substrate, from
within the 76 amino acids that make up
potential acceptor lysines
example,
mark
enzymes
Ciechanover, 1998.
The seven
generic
described in the text of the activation and
a
short
increased
affinity
for the
used in chain
example, the
involved in K63
long ubiquitin
conjugate
ubiquitin are
For
a
and Heller
E2 enzyme
linkages (Spence
fundamental role in DNA
repair (see
et
11
When K63R
below). are
dependent
the E2s
on
the structure of the
Ubc4p
expressed
1987)
and
ubiquitin chain
differentially placed lysines Kumar et al.
is
repair defective. K29
rendered DNA
also
ubiquitin
as can
seen
linked
only
of
source
conjugates
Ubc5p (Spence
drastically
in
Figure
are
et al.
ubiquitin,
different
the cells
also formed and this is
1995). It is
most
conjugate
as
structures
likely the
(Vijay-
2.
KW
K«J
Figure
the
that determines the fate of the substrate
leads to
be
as
Ubiquitin. The center and right images are two right turns about the y-axis. sticking up towards the top of the page and the chain forming lysines are accordingly. Taken from, Ubiquitin, and the biology of the cell, chapter 2,1998.
2. The structure of
The C-terminal G76 is
marked
There is
activating
only
one
El
the total cellular
protein
pool
of
in both yeast and
ubiquitin.
E2s, 13 in yeast (Hochstrasser 1996), and hundreds. It is the E3 substrate will be
E3
which
provides
However there
far
an
the
are
there
are
many
unknown number of E3s,
specificity
more
possibly
and determines which
degraded.
Ligases E3
trying
to
into the
ligases
identify
come
can
multi-subunit
in various forms, which has
provided
the
them. The fundamental function of the E3 is to
proximity
substrate. E3s a
ligase,
so
human, which is responsible for
of the E2 enzyme be
composed
complex.
of
a
Some E3s
thereby promoting ubiquitin single protein, however,
participate directly
by transiently accepting ubiquitin before
it is
in the
conjugated to
can
major hurdle bring
in
the substrate
transfer to the also be made up of
ubiquitination reaction
the substrate, whereas
12
others
form
never
E3-Ub intermediate. The different types of E3s will be
an
described below with
emphasis
on
the SCF and its role in cell
cycle integrity.
HECT domain HECT domain
C-terminal
on a
This is
containing
lysine
dependent
on
E3
proteins
are
unique
within the conserved HECT domain
the action of
an
E2
they accept ubiquitin
in that
(Scheffner
et al.
1995).
enzyme, which
ubiquitin conjugating
promotes the formation of a Ub-HECT intermediate. Ubiquitin is subsequently the substrate from the E3, rather than from the E2
passed onto
genome encodes for
five
(Huibregtse
et
a
al.
large family of HECT
domain
1995). HECT proteins
can
proteins,
bind
The human
directly.
whereas yeast has
directly
to
the
only
targeted
substrate; for example the yeast Rsp5p HECT containing protein interacts with the
large
subunit of RNA
destruction
polymerase after
(Huibregtse
substrate via
an
p53 through
the
et al.
1997).
as
which marks it for
damage
However
adaptor intermediate,
adaptor E6-AP,
DNA
is the
and results in
some
case
HECT
proteins
with human
proteasomal
interact with the
E6, which binds
to
al.
p53 degradation (Scheffner et
1993). In contrast to
ubiquitination via
ubiquitin conjugation permease,
Gaplp,
is
does not
always
ubiquitinated
media conditions. This
in
the N-end rule
lead
proteolysis.
et al.
HECT
The yeast
dependent
general
Rsp5p dependent manner when
an
ubiquitination results
endocytosis pathway (Hein
pathway,
in the internalization of
amino acid
grown in rich
Gaplp
via the
1995).
N-end rule E3s The human E3a based
on
protein's to
protein
their N-terminal amino acid
and the yeast
with the E2,
and then becomes
Ubc2/Rad6p,
ubiquitinated
or
bulky
and
and
hydrophobic
degraded.
it
a
can
bind
This E3 also interacts
which is also conserved from yeast to
1989;Dohmenetal. 1991).
substrates
(Hershko and Ciechanover 1992). When
N-terminal amino acid is either basic
E3a/Ubrlp,
Ubrlp, recognize
man
(Reiss
et
al.
13
APC/C
-
Anaphase promoting complex/Cyclosome
The APC
B-type cyclins
discovered
was
immunoprecipitations APC is
sequence domains bear
a
in
screens
Xenopus
(Zachariae
resemblance to
are
dependent
manner.
The mitotic
Pdslp/securin contain 1996; Ciosk
et al.
them at the
cohesin
complex
anaphase
onset,
After correct
dependent
and
the APC via
It
al. 1995;
of
found
was
King
et
al.
1995). The
striking primary
no
subunits, Apc2p and
two
Rbxlp,
can no
a
which
are core
components
conserved nine amino-acid motif
mutagenesis
or
of
a
substrate's destruction
longer be ubiquitinated
B-type cyclins
destruction boxes and
as
are
well
the
as
in
an
APC
inhibitor
anaphase
targets of the APC (Cohen-Fix
et al.
1998).
Once cells have
align
it
1995).
stability
Nasmyth 1999).
called the destruction box. The deletion as
the
regulate
many of which have
Cullp/Cdc53p
recognized by
box results in its stabilization,
et
1998). However
of the SCF (see below) (Zachariae and Substrates
(Irniger
extracts
al.
et
et al.
to
in yeast, and biochemical
subunits,
of at least 12
composed
Apcl lp,
(King
upon the exit from mitosis
independently through genetic
ability
account of its
on
replicated
their DNA
metaphase plate. in order to
manner
condense their chromosomes and
At this time chromosomes
ensure
correct
thereby minimizing
spindle
they
bipolar spindle
are
the chances of chromosome
degrades Pdslp/Securin (Visintin
together by the
attachment before
mis-segregation.
is established the APC becomes activated in
and
held
et
a
Cdc20p
al. 1997;
Harper et
al.
2002). The destruction of Pdslp/Securin results in the release of Esplp/separase, which cleaves the
(Uhlmann
et
al.
Scclp
subunit of
onset
1999) (Figure 3). The mitotic cyclin, Clb2p, is degraded
the exit from mitosis while at the
origins
cohesin, thereby allowing anaphase
for the next round of S
same
time
allowing
phase (see below).
accessory/activating factor, Cdhlp,
rather than
the
This is
to stimulate
licencing of replication
accomplished by
Cdc20p (Visintin
et al.
the
1997).
14
Inartne
c>f«)|»!asm
nucleus
Securin
^^
Following phosphorylation ot Pdslp/Securin by Cdk I/Cdc28p it is degraded by the APCrill2
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>
'iMt'X
Figure 12. Taken from Lisby et al, 2004. choreography of DSB protein recruitment. A-B MRX complex, Tellp and Sae2 are recruited, followed by C MRX dependent strand resection, the ssDNA is coated by D RP-A which recruits Ddc2/Mecl, Rad24/Rfc2-5, and Ddcl/Radl7/Mec3. E-F. Once cells have entered S phase, RadSl and Rad52 are recnnted, which promotes sister chromatid strand
invasion.
recruited to
Hydroxyurea (HU)
stabilized due to the activation of the
Rad53p checkpoint.
Interestingly, Rad51p stalled forks, which However Rad5
(eg.
30
lp
are
and
and
Rad52p,
maintain
a
and
a
rad53A
recombination,
as
activity
it promotes 5'
prolonged checkpoint activation. is
up-regulated
in
fork. The
-
is
early
required at
of
et
al.
2004).
HU exposure
they
also
inability
to
well
as
proteins
collapse.
for efficient
as
It has
recently
homologous
the break site, which
In the absence of CDKl
compensation (Ira
due to the
response
independent
3' resection
prolonged
be maintained, moreover,
background again
form at HU stalled forks
been demonstrated that CDKl
not
longer
can no
stably paused replication Mec3p,
are
foci do form in response to
hours), when fork stability
form in the presence of HU in
Ddclp
Rad52p
activity
ensures
the NHEJ
pathway
28
When the NHEJ well
as
the Mrel
pathway
Ip/Rad50p/Xrs2p complex
stranded break. After strand
lp complex,
Mrel
activated, the Ku proteins, Ku70p and Ku80p,
is
are
targeted
as
the site of the double
to
resection, perhaps via the endonuclease activity of the
the DSB ends
are
ligated in
a
unspecific
sequence
manner
(van
Gent et al. 2001).
The role of
As discussed above, mono
and
in DNA
ubiquitin
ubiquitination
poly-ubiquitination,
linkage. Recently
in all forms
a
regards
to
fundamental role in S
replication
and
phosphorylated by CDKl,
have
already
it is
ubiquitin
chain
and DNA
ubiquitin
and
including variable
linkage is also
ubiquitination conjugation In the
repair.
following
modifications
ubiquitin-like
repair.
pre-RC components
and further re-activation of
Repair
be detected in many forms
phase
and
Ubiquitin conjugation/degradation The
and
it has been shown that
section I will outline what is known about
with
can
and the type of
from K48 to K63
plays
replication
a
Replication
complex regulation
fired
origins
of
to
replication.
prevent the re-loading
Cdc6p
Once
is
targeted for ubiquitination and degradation by the
SCFCdc4 E3 ligase, and then further prevented from pre-RC formation by interaction with Clb2, which is then itself
1995; Drury to
be
et al.
regulated
in
following origin
degraded
a
very similar way to
initiation
and
Cdc6p
degradation by
the APC/C
et al.
licencing
2003; Hu
et
al.
(Piatti
et
al.
is
ubiquitinated
by
(Figure 13).
DDB1
degraded
(Feng
and
Geminin, which is subsequently
the APC/C
Yoshida et al.
and
2004). As Cdtlp levels rise, it remains
via interaction with
Lygerou 2002;
in yeast
Cdc6p. Cdtlp
by the Cul-4A based ligase containing
upon the exit from mitosis
Nishitani and
for
1997). In human cells the licencing factor, Cdtlp, has been shown
Kipreos 2003; Zhong inhibited from
targeted
(Wohlschlegel
2004), in
an
et
al. 2000;
analogous fashion
to
Clb2
29
AK/C
[M'i
/A t D11
f
f
""*!i^^?J**^^^!^""^"^"'"'"^*
WK*
S [I've
G
Mi'i't
CDK*
a? rflitf>-
i
Figure 13 Taken from Li and Blow, 2003 At the end of mitosis the inhibitor of Cdtlp, Geminin, degraded by the APC/C leaving Cdtlp competent for the licencing reaction Once a licenced ongin has fired, Cdtlp, is degraded in a Cul-4 dependent manner.
In contrast the S. via
cerevisiae, Cdtlp is stable throughout the cell cycle and is regulated
sub-cellular localization rather than
fact that the cells
is
regulates
these
degradation (Tanaka
replication
events
and
Diffley 2002).
The
through protein degradation
augments the importance of irreversibility of these reactions within the cell cycle, thus
ensuring
that
licencing
As cells enter S
the
production
of
factors do not become competent for
phase
one
of the basic
deoxyribonucleotides (dNTPs)
a
reduction reaction
,
are
regulated
in
a
cell
degradation, whereby of S
is
Rnrlp
Spdlp (Sp)/Smllp(Sc) (Woollard
et
proteins
are
to
initiate
incorporated
negatively regulated by
manner
via
an
replication,
into the
converts NTPs
new
interacting protein and
specifically targeted et
for destruction
at
the onset
al. 2001 ; Bondar et al.
2004). In S. pombe, the Cul4p/Pcu4p and the substrate specific adaptor Ddbl et
al. 2003; Bondar et al.
cerevisiae, the E3 ligase has not yet been identified. So far has not yet been identified in the human genome.
Smllp
proteasomal dependent
phase, thereby de-repressmg Rnrlp activity (Zhao
required for Spdlp degradation (Liu
is
into dNTPs
1999). Spdlp
al. 1996; Chabes et al.
cycle dependent the
to be
protein, Rnrlp,
strand. The ribonucleotide reductase
through
requirements
re-licencing.
a
2004), whereas
protein
are
in
S.
inhibitor of RNR
30
and
Ubiquitin The
Repair cell nuclear
proliferating
in yeast. PCNA, which forms RNA-DNA
primed
trimeric
a
by
the POL30 gene
around DNA, is loaded onto Pola
clamp
clamp loader, replication factor
the
primers by
is encoded
antigen (PCNA),
C
(RF-C)
(Tsurimoto and Stillman 1989; Tsurimoto and Stillman 1991). PCNA interacts with Polo and promotes
directly
translationally
modified
(K164) (Hoege
et al.
replicative processivity.
both SUMO and
by
as
sizlA cells, which
the
are
for
impaired
sumo
hypersensitive
damaging agent, indicating
independent
phase.
the was
same
lysine
ligase
function
to DNA
that K164 is
a
important
required
replication defect,
(Hoege
damage
residue
shown that PCNA
However sumolation is not
K164R mutated PCNA does not have
contrast K164R cells are or
on
2002). Using synchronized cells it
becomes sumolated at the onset of S PCNA function
ubiquitin
PCNA is post-
et
al.
UV
by
damage repair
moving replication
fork encounters
damage along
the
template
strand, the E2 enzyme, Rad6p, along with the ring finger protein, Radl 8p, add
single ubiquitin
to the
to
as was
and
trans-lesion
Ubcl3p
replicate past
then add
PCNA is not
PCNA there appears to be
Figure
14.
does not affect the
polymerase, polr^ (Kannouche
a
promote template switching
ubiquitinated
mono-ubiquitination
demonstrated in cultured human cells, promotes
which is necessary to
Mms2p
The
onto PCNA.
PCNA however,
2004),
light
of sumolation.
When the
switch,
do
In
2002).
induced either for
nor
for
the
K63-1 inked
to
the
area.
2004; Vidal
competition
poly-ubiquitin chain,
which is
polymerase et
al.
ubiquitination
predicted
The multi-
this process. Therefore in the
between
stability of
The E2 enzymes
undamaged sister chromatid.
degraded during a
damaged
et al.
a
a
case
of
and sumolation.
31
K127 K164
Ubiquit.nat.on
•AW/*/
Multi-K63 linked
Mono
K,164
,v#f Lnor-free DNA repair
F
x
%^
PCNA
SUMO modification
Figure 14. SUMO and ubiquitin compete for the same lysine During replication PCNA is ubiqumated on K164, however becomes ubiquitinated and then further poly-ubiquinated after damage induction.
Other targets
including
are
also
degraded during
the above mentioned
and enhance
repair. Cdtlp
DNA
Spdlp (Bondar
is also
degraded
et
damage
expressing
after exposure to UV irradiation
likely
to
are
not as
MMS
to DNA
(Chang
et
damage.
one or
multiple targets
al.
as
ligase components
In addition,
the
single
most
RttlOlp, is
likely
also
al. 2002), and further it has been shown to possess
ubiquitin conjugation activity (Michel
has
et
factors has been
sensitive to MMS
ubc!3A cells, indication that these
targets in response
hypersensitive in vitro
or
(Hu
targets probably exist. For example, cells
more
the PCNA K164R mutation
radô, mms2A, have other
it is clear that
stress
al. 2004), to increase dNTP levels
2004). Although the regulation of many repair and replication
extensively studied,
replication
or
DNA
in the DNA
et
al. 2003),
damage
revealing
response.
that it also most
32
Chapter-ll The yeast cullin, RttlOlp, genomic integrity and DNA repair The work in this
chapter
was
is involved in
submitted for publication
RttlOlp, is required for recovery after damage and replication stress
The S. cerevisiae cullin, DNA
Brian
Luke1,
Lionel
1
Gwennaelle
Versini2,
Malika
Jaquenoud1'3, Thimo Kurz1,
Pintard1, Philippe Wiget1, Philippe Pasero2&,
and Matthias
Peter1*
Swiss Federal Institure of Technology Zurich (ETH)
Biochemistry Hoenggerberg HPM
Institute of ETH
G 10.0
8093 Zurich Switzerland Tel: 441 1 632 31334 / Fax: +41 1 632 1269
2
Institute of Human Genetics, CNRS
141 Rue de la Cardonille
34396
Montpellier
France 3
Present address:
Department of Biochemistry University of Fribourg Switzerland &
To whom
correspondence
should be addressed:
[email protected] [email protected]
Running Title: The S. cerevisiae cullin, RttlOlp, instability following replication collapse
is
required
to
prevent genomic
Key words: ubiquitin, E3-ligase, Cullin, DNA damage, RttlOl, Cul8
33
Introduction Accurate
replication
complex signaling high fidelity
and
network that
precision.
cycle progression
and maintenance of
that DNA
ensures
organism's genome requires and
duplication
onset of
DNA-segregation during
a
repair occur with orderly
These processes must be coordinated with
prevent the
to
an
cell
mitosis before
DNA-replication and repair are completed. stress, cells activate
Upon genotoxic arrest
the cell
activation of
cycle
until the lesion is removed
DNA-checkpoints
in either S
metaphase/anaphase
transition
2001 ;
2003). Some of the
Agarwal
et
al.
by
replication/damage checkpoints kinases
Meclp/ATR
kinases
are
and
and
responsible
or
or
repaired.
most
Rad53p/Chk2 (Gardner
an
arrest at
Pdslp (Clarke
the
et al.
upstream components of the
partially overlapping PI3-kinase-related Rouse and Jackson
phosphorylation of two
et al.
or
budding yeast,
G2 phase results in
Tellp/ATM (Carr 2002;
for the
In
stabilization of the securin
include the
to slow down
checkpoint pathways
1999; Sanchez
et
2002). These
downstream kinases,
al. 1999), which in
turn
Chklp activate
targets involved in DNA-metabolism and repair. Chklp and Rad53p have also been shown to
directly phosphorylate Pdslp,
which prevents its
Anaphase Promoting Complex0*20 (APCCdc20) (Gardner et 1999; Wang
et
(Agarwal
et al.
pathways
can
al.
2001),
most
likely by inhibiting
degradation by
al. 1999; Sanchez et al.
the interaction with
2003). Mutations that affect faithful DNA-replication
lead to
genomic instability,
as seen
the
Cdc20p
or
checkpoint
in many human diseases
including
cancer.
Genomic
integrity
checkpoint signaling
is not
and DNA
only dependent
replication
modification of these enzymes to the
the APC
or
cullin-based
checkpoint pathways.
complex via
that
right place. Regulated ubiquitinylation
triggered by and
ensure
an
Cullins
characterized based
adaptors (Pintard
on
et al.
and
they
and
provide
a
the enzymes
repair, are
but also
requires
activated at the
control many cell
scaffolding
covalent
right
time and
of substrates
cycle
transitions
function within the E3-
ubiquitin-conjugating
Pagano 2004).
directly involved in
subsequent degradation
E3-ligases
and interact both with the E2
adaptor (Cardozo
and
on
enzyme and the substrate
Different cullin families have been
their N-terminal interactions with different substrate
specific
2004). The budding yeast genome encodes for three cullin
34
proteins, Cullp/Cdc53, Cul3p Cullp
and its role in the SCF
and
Rttl01p/Cul8p (RttlOlp
from here on). Whereas have been
(Skpl,Çullin, F-Box) complex
studied, little is known about the role and subunits of the other
two
extensively
cullin
proteins
in
S. cerevisiae.
RttlOlp
can
support ubiquitination in vitro, and like other cullins, interacts via
its C-terminal domain with the
2003). Cells deleted for RTT101
with
preferential
are
viable, but display
of the retrotransposon
transposition rate
insertion
occurring
Ty 1 (rö
UV
damage (Chang
delay
in
the cullin
gene RAD9
RttlOlp
replication
or
In the
are
which is et al.
(Michel
is involved in
400
CPT and IR, but
al. 2003).
Finally,
are
2001).
not sensitive to
rttlOlA cells show
aspect of DNA metabolism,
most
a
DNA
likely
involves
DNA
replication
replication origins (Raghuraman
et al.
2001; Wyrick
are
timing
likely replicative helicase, mitosis and
the
distributed all
along
the
et
al.
2001),
replicative
the chromosomes
Inactivation of cyclin-dependent kinase
promotes the
licensing of replication origins
and
While the
is bound to
allows the
efficiency during
regions.
replication (Noton
recognition complex (ORC) activity
and
physically
at the end of mitosis
the next round of DNA
kinase
et al.
suppressed by deleting the intra-S-phase
S. cerevisiae,
Replication origins
(Cdklp) activity
phase
(Scholes
complete
budding yeast
from the telomeric to the centromeric
couples
transposition)
2003). Taken together, these results indicate that
some
activated with different
low CDKl
et
of Xy 1
regulation
repair.
approximately
process.
2002; Michel
anaphase progression,
checkpoint
which
et al.
60-fold increase in the
deletions has revealed that ml 01A cells
genotoxic agents MMS, HU,
sensitive to the
=
a
in front of tRNA genes
High-throughput screening of viable yeast are
et al.
ring-finger containing protein, Rbxlp (Michel
assembly
Diffley 2000).
origin
replication origins throughout the of the
cell
cycle,
licensing factors: Cdc6p, Ctdlp
MCM-complex (Diffley 2004).
followed
by
and the
This mechanism
DNA-replication. Subsequent reactivation of CDKl
Cdc7p/Dbf4p triggers origin firing
DNA
and the S-
replication (Lei
al. 1997; Zou and Stillman 1998).
Recently through specific
it has become clear that chromosomal
regions
timely
and accurate
of the genome
DNA-replication
including telomeric,
subtelomeric, centromeric, tRNA genes, rDNA and silenced regions requires
specialized factors (Ivessa
et
al.
2002;
Ivessa et al.
for
2003). These non-nucleosomal
et
35
form stable
proteinaceous regions
sites. For
replication pause
DNA-protein complexes, resulting
example,
in natural
Rrm3p (Bessler et
the Pif-like DNA helicase,
al.
2001), is only required for the efficient replication of these genomic regions while the bulk of
replication
localize to these
is
independent
al. 2000; Ivessa et al.
et
replication pausing
at these
significant pausing
was
survival of rrm3A cells
specific
found
sites is
throughout
depends
stabilize the
to
al. 2002).
et
combined with many as
on an
dramatically increased,
well
as
and
replication (eg. cdc45ts)
when
Rrm3p
replication machinery of
regions
known to be
an
mutants
order to
in rrm3A
cells, these cells require that
are
a
background
DSB upon
al., 2004), suggesting chromosomal
we
a
regions.
specific replication
found that rttlOlA is
(eg.
stalled forks
collapse (Ivessa
occur
with
a
et al.
higher
able to restart in
role of this
In this
putative E3-ligase
study,
we
stress or
DNA
checkpoint
arrest in
a
is
promoting
required restart
damage
pause sites.
or
damage,
timely
an
increased
rttlOlA cells
manner.
for accurate and
to
cell
cycle
with rrm3A
(Tong
et
replicate specialized
demonstrate that rttlOlA cells indeed exhibit
defect that results in
replication
ligases during
synthetic-lethal
breaks and gross chromosomal rearrangements.
DNA
as
replication fork is
interested in the function of cullin-based
progression. Recently,
RttlOlp
a
forks
complete replication.
We
a
replication
The double-stranded break
2000; Cha and Kleckner 2002). Since DSBs and fork stalls
frequency
(eg. radSOA,
compromised to
cannot be
rrm3A
unstable and often result in
highly
is absent. In
2004). These results suggest that
high protein density.
(DSB) repair machinery becomes essential in are
repair
et al.
in the absence of Rrm3p the traverse
2004; Torres
the
inviable when
are
with mutants involved in re-start of stalled et al.
no
phase checkpoint, which is
intact intra-S
paused replication complex
while
Interestingly,
the rest of the genome.
top3A, sgslA, srs2A) (Tong
successfully
found to
was
2002). In the absence of Rrm3p,
addition, synthetic-lethal analysis revealed that rrm3A cells
mrel 1 A),
Rrm3p
proteinaceous regions by chromatin immunoprecipitation (CHIP)
experiments (Ivessa
thought
of Rrm3p (Ivessa
Taken
DNA
of replication forks that have
unable to
were
our
data
have been unable to be maintained in
a
a
recover
most
likely by
result of
stable
from the
strongly suggest that
replication,
collapsed as
of double stranded
upon induction of
Importantly,
together,
complete
frequency
manner
encountering at
natural
36
Results
checkpoint dependent G2/M delay and accumulate with short pre-anaphase spindles rtt101A cells have a
To
investigate
progression al.
2003),
of
wild-type
the bud neck,
at
RttlOlp,
compared
we
a
short
spindle
during
et
mitosis with the nucleus
unseparated
and
cycle
cell
(Michel
and rttlOlA cells. Consistent with recent results
found that rttlOlA cells accumulate
we
positioned
the function of the yeast cullin
sister chromatids (data
not
shown). Importantly, time-lapse movies of single wild-type and rttlOlA cells
expressing
tubulin-GFP revealed that the
severely delayed
as
spindle elongation
onset
of
anaphase
in rttlOlA cells is
compared
starts at 120'
to 10 minutes in wild-
type cells (Fig. 1A and supplementary movie). We counted time 0'
pre-anaphase spindle
the short
until
elongation.
rttlOlA cells
The average
(n=5) and 7 minutes
was
migrated
half way into the bud
quite significant.
properly attached mitotic
moved to the mother/bud neck and measured the time
elongation time
variation
spindle,
in wt cells
The short on
subsequent
anaphase transition,
events
spindle
the cell-to-cell
dynamic
in rttlOlA cells is very
suggesting
including cytokinesis,
that the
cell
spindle
which results in chromosomal a severe
is
separation and exit
damage.
delay
and
elongation of the
that the cells do not
timing, indicating
at the
undergo
Taken
an
together,
metaphase
to
transition.
To determine when in the cell
functional
HA-Rttl01p
(Figure 1C).
Cells
hour. The culture
expression
of
were
was
protein.
grown in 2%
immunoblotting
in the
was
galactose
divided, and 2% glucose After three hours,
was
followed by
of marker
were
required,
expressed fully
and arrested in Gl with ct-factor for 1 was
added to
HA-Rttl01p
released
that
half to inhibit the
was no
by washing
was not
one
longer detectable
HA-Rttl01p is
morphological criteria,
proteins. RttlOlp
we
regulatable GAL/,70-promoter
glucose culture, indicating
The two cultures
cycle progression
cycle RttlOlp
in rttlOlA cells from the
HA-Rttl01p.
by immunoblotting unstable
100 minutes in
(n=4) (Fig. IB), although
these results suggest that rttlOlA cells exhibit
anaphase
approximately
several occasions,
from mitosis occurred with normal aberrant
was
to chromosomes and the cortex. After the sudden
the
the time when
as
out
FACS
required
a
rather
a-factor, and cell
analysis
and
for bud emergence
or
37
the onset of the bulk part of DNA
lacking RttlOlp of
Pdslp-myc
of
accumulated at the
and
RttlOlp-activity
or
Clb2p
in
Pdslp-myc
demonstrating
that
a
not
shown). However, cells
anaphase-metaphase transition,
significantly delayed.
was
is necessary
RttlOlp results
Clb2p
replication (data
during
every S-
specific delay prior
or
is not
required
G2-phase of the cell cycle, and lack
to the onset of
to
degradation
These results suggest that
anaphase. Interestingly,
accumulated in the Gl-arrested cells after
RttlOlp
and
depletion
maintain APC-Cdhl
of
RttlOlp,
activity.
r*V*{t-1
JiUJ^
Fig.
Figure
1.
rttlOlA cells have
a
Meclp dependent
G2/M
no
1
delay
pre-anaphase spindle. Tubulin-GFP (A) delay was used to visualize the spindle via live cell fluorescence imaging (supplementary figure (SI)). We noticed that the spindle was delayed in the onset of elongation from the time it reached the bud-neck (time 0) until the length of the spindle spanned the length of the two cells, although the timing was quite variable from cell to cell (B). (C) Cells with a Gal regulatable HA-RttlOlp were synchronized in Gl with alpha factor and released into media containing either glucose or galactose. The degradation of Pdslp-myc and Clb2p were delayed in the glucose culture where RttlOlp was shut off. (D) Logarithmic DAPI stained cells were scored for bud-neck localization (butterfly nuclei) As expected rttlOlA showed a G2/M delay that was suppressed by deleting the checkpoint protein Mec lp but not rttJOJA cells have
a
G2/M
and
accumulate with
a
short
Mad2p.
I
38
Degradation
of
the APC is inhibited
Pdslp by
in
DNA-damage checkpoint
unreplicated
checkpoint
mitotic
Mad2-dependent
of
case
by either the ATR/Mecl-dependent
in
case
of
activation of
of these
one
the mitotic index of
at
the mother/bud neck,
this G2/M
delay
was
compared
phase checkpoint kinase,
Mad2p
had
no
rttWIA meclA
effect
on
8% in
to
to
wild-type
the
cells
S
Rad9p
also suppress the mitotic
can
the ATR/Mecl S
spindle assembly checkpoint
slower S
phase fewer firing replication origins firing To determine if kinetics of
a
RttlOlp indeed
DNA-replication
Herpes simplex thymidine
cycle
2002), The cells
in
wild-type
and ml01A
kinase to allow BrdU
were
arrested in Gl
pulse-field gel electrophoresis (PFGE), chromosomes entered the
and visualized
by
approximately 45 rttlOlA cells, 50% BrdU
as
an
gels.
than wild
during S-phase,
functions
by
in the presence of BrdU. Genomic DNA
It has
delay
inhibitor
previously
of ml01A occurs
so
that
type cells and have
we
first
incorporation
2A.
was
then
prepared
and
migrated
via
only completely replicated were
Interestingly,
after 45 minutes, BrdU
DNA-replication.
and
a-factor and released into the cell
transferred onto was
minutes after release from the Gl-arrest in both
Figure
the
strains, which express the
antibody against BrdU. BrdU detection
shown in
compared
incorporation (Lengronne
The chromosomes
significantly delayed (lower graph). efficient
to measure
phase.
rtt101Acells exhibit
Schwob
caused
(Fig. 1D). Importantly,
cells, suggesting that the G2/M delay may result from DNA damage that
during
the
rttlOlA macïlA cells.
the mitotic accumulation of ml 01A cells.
been shown that deletion of
was
staining
by inactivating
wild type levels
deleting
while
or
by
unattached
or
of rttlOlA cells
used DAPI
or
23% of ml 01A cells accumulated with their DNA
delay,
suppressed
we
DNA-damage,
misfunction
delay
checkpoint pathways,
wild-type, rttlOlA,
Consistent with the mitotic
or
spindle
chromosomes. To determine whether the mitotic
by
DNA
while
a
membrane
possible wild-type and
wild-type cells showed
incorporation in ml01A cells
These data suggest that
was
RttlOlp is required
for
39
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2. S
Figure
Wild
BrdU.
PFGE
wild type bromide
1b2
jrtj
n
^0
di^lincü
^4
_ft
kb
Fig.
is slow and less
phase
origins
type and rttlOlA cells expressing TK
analysis
revealed that rttlOlA cells chromosomes
replicated staining and (upper) as
10 *>)
were
are
were
were
2
active in rttlOlA cells arrested
delayed
m
Gl and released in the presence of
completion of S phase compared gel (A) as judged by efhidium
in
slower to re-enter the
to
incorporation (lower). (B) Cells released from Gl into BrdU
and BrdU
were prepared and aligned linearly onto coverslips, followed by staining with anti-BrdU (green) and anti-DNA (red). Measurements between origins (IOD) and replication track length were made (upper panel). (C) The average IOD in rttlOlA cells was greater (68.8 kb) than the corresponding wt (54.2 kb). (D) BrdU track length is 21.7 kb m rttlOlA cells
were
arrested with HU and chromosomes
compared
The slower S
required
corresponding wild type.
to 14.2 kb in the
phase progression
for efficient initiation
between these two
or
possibilities,
chromosomes
using
chromosomes
were
DNA
first
in rttlOl A cells suggests that
elongation
we
was
DNA fibers
significantly
To discriminate
replication
combing (Herrick and Bensimon 1999).
prepared
from
linearly arranged
antibodies to visualize
synthesis.
have monitored the
wild-type
after release from a-factor into 0.2M HU
2003). The DNA
of DNA
replicated
(Figure 2B). Interestingly, reduced in cells
on a
DNA and
and rttlOl A cells
described
as
glass
anti-guanosine
the number of
lacking RttlOlp,
of individual For this purpose,
pulsed
previously (Versini
slide and
is either
RttlOlp
probed
with BrdU et
al,
with BrdU
antibodies to counterstain
firing replication origins
was
and the average distance between
40
increased to 68.8 Kb
replicating origins 2C).
In order to
measure
cells
exponentially-growing tracks
the
was
analyzed by of
length
replicated
which is reminiscent of
RttlOlp
is either
Although
the
the rate of were
DNA
pulse-labeled
sgslA
(Versini
mutants
indirectly
cells have
54.2 Kb in
~50%
longer al.
et
needed for efficient
increased
replication
Rtt101p is synthetically-lethal with but they do not share overlapping
proteinaceous
replication pausing
areas
throughout
which often leads to unstable
restart
mutants,
as
the
a
at
combined with rrm3A
(Fig. 3A),
determine if the cullin,
RttlOlp,
region, CEN4, found that
zones, we
that
pausing
however there
fork
speed,
pausing
intermediates that
mutants, DSB
can
repair
was
also involved in
2D
synthetic-lethality
mutants,
also
et al.
preventing
require Rrm3p
was
increased in the rrm3A deletion strain
difference in the amount of
pausing
or
replication
repaired
synthetically
shown
to
,
be converted into
to
and
lethal when
2004). To
extensive
gel electrophoresis analysis
previously
in
likely
is enhanced in rrm3A cells
previously reported (Tong
was
performed
(open arrows).
most
intermediates need to be stabilized,
as
program is
replication origins.
was
was no
and rttlOl A cells
replication
many non-nucleosomal
restarted, respectively. We confirmed that rttlOlA
proteinaceous
DNA-replication.
result rrm3A mutants confer
checkpoint
replication
We conclude that
functions
occur
replication
(Fig, 2D),
replication helicase Rrm3p
the
the genome. The
toxic double stranded breaks. As when combined with either
sites
of BrdU
Surprisingly,
in rttlOl A cells
2003).
order to compensate for the decreased number of active
Natural
length
with BrdU and the
is weak in rttlOl A cells, the an
wild-type cells (Fig.
in wild type and rttlOl A cells. was
or
to
the level of individual molecules,
at
DNA stretches
directly
as
elongation
combing
replication delay
clearly perturbed
compared
on a
pausing
at
centromeric
prevent pausing. We
(Fig. 3B,
black
arrow),
between wild type cells
41
B
H
ÏSftSs'
W0:
fl> :.ï:iii;,ï5 IIS SUSI: §ij#ïS $;$$$
Sffilsi ffl: @;llï
%-
i
;
fi '
P-value
sequence
observed
IGACTCAT[1]
25/32
1 67E-06
ATGAGTCAfJI
24/32
2.45E-06
Gcn4p-myc
TGACTCGfH
27/32
0.009
a-actin
TGACTCA
13/32
0 01
120 —
ut
c
J8 £
wt pORF-lacZ
QuriA
MORF4-onfy-lacZ
100 ,uv
t
I
80
gcn4 A nun'A/gcn4A
60 40
20 0
100 riM
rapamycin
min
0
4p-myc a-actin
30 >
—
aa-starvation
uriA
wt
60
«*
—-
0
30
»>#| —|
uriA
wt
60 It'
-
hrs
Gcn4p-myc elF2a
2
0
0
2
„
'
»f^>#-
scUrip represses GC/V4-dependent transcription. (A) Conserved motifs within the consensus site for Gcn4p is underlined. mismatches. P-values correspond of allowed Numbers in parenthesis refer to the number Fig.
4.
promoter region of scUrip-dependent genes. The
to
the false positive probabilities for each motif. (B) Extracts from indicated strains were analyzed by Western blotting using anti-myc antibody 9E10. (C) Indicated strains harbouring or the mutant, GCN4uORF4only-lacZ, were analyzed for lacZ activity. (D) Standardized mRNA expression values of GAP1, GCN4, GLN3 and GAT1 in the indicated strains relative to wild-type were obtained from genome wide transcription analysis. (E) Wild-
GCN4uORF-lacZ
type and uriA mutant cells were analyzed for Gcn4p-myc expression following 100 nm rapamycin treatment. (F) Wild-type and uriA mutant cells were analyzed for Gcn4p-myc expression following
amino
acid starvation.
96
B
PPase
+
Hrs: 0 -URI-P
+
1
0
8
:ÉiL-ÉfflMx ;jmWMm mP ^P „
"URI
+
insulin
Jtamtii,
insulin,
rapamycin 12
4
8 -
tfwwm
^f WE
ÜI
^
*ääm
URI-P
-URI
D +
vector
+insulin. rapamycin +
-
HA-mTOR(WT) HA-mTOR (RR)
-
-
-
-
-
-
+
-
•ÊltËj^À
+
-
àaut .aaatfiaE,. _o—,1
-URI-P
-URI
i«w»
URI HA-mTOR
-
0
30
1.5
60 1.0
0
30 60 120
0.5
.I
Ctr
120
m "»
o -o x o"0 CD 30 W I- 33
-H » W O
BS OD
i»n
^ i o r- tj s*
Z X ^i
siURI
w
S-
M b
ut
3'
O
Fig.5. URI participates in mTOR signaling. (A) HEK 293 cells were serum-starved, treated insulin, processed for lambda phosphatase treatment and analyzed by Western blotting for URI. (B) Serum-starved HEK293 cells were induced for the indicated time with insulin in the absence or presence of rapamycin and analyzed by Western blotting for URL (C) Untransfected HEK293 cells or cells transfected with HA-mTOR(wt) or rapamycin resistant mutant HA-mTOR(RR) plasmids were serum-starved, treated with insulin and rapamycin and with
analyzed by immunoblotting. (D) pSuper
or
pSuper-si-URI
profile of pSuper-HeLa Silencing
were
analyzed by
cells and
of URI affected the
Cell extracts of HeLa cells
pSuper-si-URI
rapamycin
stably transfected with either (E) Gene expression
immunoblotting for URI. cell lines
following rapamycin
treatment.
response of 28 genes shown in the cluster
analysis.
97
References
Sonenberg, Proc.
1.
Raught, A. C. Gingras, 98,7037(2001).
2.
T. Schmelzle, M. N. Hall, Cell 103, 253
3.
T.
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B.
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Natl. Acad. Sei. USA
(2000).
Peng, T. R. Golub, D. M. Sabatini, Mol. Cell. Biol. 22, 5575 (2002).
Shamji, S. USA96,14866 (1999).
Schreiber, Proc. Natl. Acad. Sei.
Tong,
A. F.
5.
J. Lisztwan et al., EMBO J.
6.
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7.
Siegert, M. 621 (2000).
8.
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9.
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10.
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11.
Woychik, 313(1990).
12.
M. Kanemaki et al., Biochem.
13.
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14.
D.
15.
Y. Murakami et al., Nat. Genet.
16.
T. Ito et al., Proc. Natl. Acad. Sei.
USA9S, 4569 (2001 ).
17.
C. J. Gimeno, P. O.
A.
Sutterlüty
Vainberg
Siegers
17, 368 (1998).
al., Nature Cell Biology 1, 207 (1999).
et
R.
L.
R. Leroux, C. Scheufler, F. U. Hartl, I. Moarefi, Cell 103,
et al., Cell
93, 863 (1998).
et al., EMBO J. 18, 75
S. M. Liao, P. A.
N. A.
(1999). (2002).
Kolodziej,
Biophys
R. A.
Young,
Genes Dev. 4,
Res. Commun. 235, 64
(1997).
Wood, S. B. McMahon, M. D. Cole, Mol. Cell 5, 321 (2000).
Dorjsuren
et
al., Mol. Cell. Biol. 18, 7546 (1998).
10, 261 (1995).
Ljungdahl, C.
Styles,
G. R. Fink, Cell
68,1077
(1992). Krön, N. A. Gow, Curr. Opin. Cell. Biol. 7, 845 (1995).
18.
S. J.
19.
A. P. Gasch et
20.
K.
21.
V. A.
Natarajan
al., Mol. Biol. Cell 11, 4241 (2000).
et al., Mol. Cell. Biol. 21, 4347
Cherkasova,
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98
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(1995).
Acknowledgments:
23.
laboratory for discussions and Drs. A. G. Hinnebusch for myc-GCN4 and lacZ-reporter plasmids, G. Thomas for mTOR plasmids and R. Agami for the pSUPER vector. Special thanks goes to Drs. J. Paszkowski, U. Müller and G. Thomas for critical reading of the manuscript. M. V. acknowledges support We thank all members of
our
from the Association pour la Recherche sur le Cancer. This work is supported by the Swiss Cancer League, the Novartis Research Foundation and an SNF grant to M. P.
SUPPORTING ONLINE MATERIAL Materials and Methods
Yeast
two-hybrid
screen
carried out with the
yeast reporter
(pGAD-GH plasmid; Clontech) 113-435 of human SKP2 clones
were
sequence construct STAP1 in STAP1
analyzed by
encoding
cloned into
digested
A standard
Y153, using
two-hybrid
a
Gal4-fused SKP2
a
direct DNA
sequencing.
as a
bait. Positive
an
was
untagged
used to version of
generate pcDNA3-HA-STAP1, pcDNA3-
with BamHI and EcoRl and the
HA-tag previously
an
acids
All were found to contain a
expression plasmid encoding
pcDNA3 containing
library
encoding amino
ORF of 157 amino acids. One
To
screen was
HeLa cDNA
expressed from pAS2-1 plasmid
pcDNA3 (InVitrogen).
was
strain
and
predicted
a
mammalian
a
plasmids.
and
resulting fragment introduced at the
Hindlll/BamHI site.
URI to
peptide sequences obtained from MS/MS sequencing,
identify overlapping
EST sequences and human
encoding full-length URI. Based and used in PCR reactions to
from
a
human
amplify
the first 426
lymphoid library (Strubin
product was digested
et al.
used
genomic sequences
this information,
on
were
primers
were
designed,
bp of URI coding sequence
1995).
The
resulting
with BamHI/EcoRI and subcloned into
PCR
pcDNA3.
The
commercially available cDNA clone IMAGE clone 3609351, containing the 3' part of the URI cDNA,
was
digested
with EcoRl and the
resulting fragment
99
into the EcoRl site of
ligated
region of plasmid
pcDNA3 containing
resulting plasmid is referred
URI. The
Flag-epitope tagged
a
version
was
426bp fragemnt of the 5'
the
to
pcDNA3-URI.
as
from
HeLa cDNA
a
cDNAs
encoding
amplified
from
human cells
yeast proteins scPfd6, scRpb5
the
cDNA libraries
pGEX
we
(Brummelkamp
were
PCR
amplified
library (Clontech) and subcloned into pGEX and pcDNA3.
yeast
subcloned into
in the
generated, resulting
plasmid pcDNA3-FT-URI. Human RPB5 and p17 cDNAs
From this
and
a
For stable
following oligonucleotides
2002)
to obtain
scUrip
were
one-step PCR protocol and
pBS-KS, respectively.
cloned the et al.
using
and
into
silencing of URI in
pSuper
pSuper-siURI:
forward:
GATCCCCATGGAGAAGATACGACATCTTCAAGAGAGATGTCGTATCTTCT
CCATTTTTTGGAAA; reverse:
AGCTTTTCCAAAAAATGGAGAAGATACGACATCTCTCTTGAAGATGTCGT ATCTTCTCCATGGG.
Antibodies. Mouse monoclonal antibodies 4H7 and 8WG16 directed
respectively,
RPB5 and RPB1, al. 1989;
Nguyen
and CUL1
were
et al.
(105.72
and
105.128)
previously (Lisztwan
et al.
and URI
(179.30
and
179.63)
human
proteins expressed
(GST)-fusion protein.
Rabbit
polyclonal
a
as
antibodies
were
Mouse
raised
glutathione
recognizing
against
the
S-transferase STAP1
against URI(179.63.1,179.58.1)
against full-length human proteins expressed
Polyclonal
1998).
were
maltose-binding protein (MBP)-fusion protein.
Monoclonal antibodies directed raised
et
recognizing SKP1 (90.122b), SKP2 (95.60.2), STAP1
respective full-length
generated against
previously (Thompson
have been described
1996). Polyclonal antibodies directed against SKP2
described
monoclonal antibodies
against
antibodies
recognizing
URI
were
as
have been
GST-fusion
proteins.
provided by Dr. Seishi Murakami
100
(Dorjsuren were
et al.
raised
1998).
Rabbit
polyclonal
anti-TIP48 and TIP49-antibodies
TIP49
(TIP48 peptide: MKEYQDAFLFNELKGETMDTS;
LFYDAKSSAKILADQQDKYMK). Peptides hemocyanin by glutaraldehyde coupling polyclonal number:
antibodies
Q9NUG6)
specific
were
peptide:
into rabbits. Rabbit
injected
and
incubation with first
a
against bacterially produced
GST-affinity-column
followed
protein column, prepared by covalently cross-linking
the
a
achieved
sera was
GST-fusion
1999).
described
(Lisztwan
et al.
Anti-peptide
were
affinity-purified
as
Monoclonal antibodies HA-11, 12CA5, 9E10 and anti-actin mAb
purchased
form Babco,
International
Boehringer Mannheim, Santa Cruz
respectively. Anti-elF2a antibodies
were
to
respective proteins and Lane
with
1998).
by
GST-fusion
dimethylpimelimidate (Harlow
glutathione-Sepharose antibodies
Accession
p17 (PFD4r,
protein. Affinity-purification of the different polyclonal rabbit
by
C-termini
coupled to keyhole limpet
were
for human PFD3 and
raised
respective
to their
against peptides corresponding
were
and Chemicon
kindly provided by
Dr.
Ron Wek.
Immunoprecipiation, immunoblotting, gel filtration, phosphatase assay. Immunoprecipitations and immunoblotting described
(Lisztwan
et al.
membranes has been
lysis
buffer
Ponceau S
as
of western blot
procedures of the
109
HeLa cells
[50 mM Tris-HCI (pH 8.0), 150
mM
NaCI, 1% Triton X-100,1
Superose
for 30 min
6
gel-filtration
on
ice, lysates
min at 4°C at 3000 g and filtered
Superose
(Sigma) staining
to the
performed according
3-4
mM DTT, 0.5 mM PMSF, 20 mM NaF and 1
(Roche)]
performed essentially
x
manufacturer. For 10 ml
1998).
were
6 column
0.5 ml/min flow rate
were
through
(FPLC; Pharmacia). using gel-filtration
x
were
lysed
in
protease inhibitor cocktail
pre-cleared by centrifugation for 30 a
0.45
urn
filter
Gel filtration
buffer
[50
mM
prior
was
to
loading
performed
on a
at 4°C at
Hepes (pH 7.5), 150 mM
NaCI, 10 mM MgCfe and 0.5 mM DTT] and 4 ml fractions
were
collected. Blue
101
Dextran 2000
(232 kDa) For
(158 kDa)
and aldolase
phosphatase experiments
DMEM w/o
buffer %
(2 MDa), thyroglobuline (669 kDa), ferritin (440 kDa),
[50
NP40]
serum
used
as
for 30 min
(NEB)
0.1 mM EDTA, 5 mM
(pH 7.5),
on
ice. 200 ug of
in 50
pi
Reactions have been
standards.
HEK293 cells have been starved for 24h in
prior
to
lysis
DTT, 250 mM NaCI. 0.5
1x X-PPase buffer
[50 mM Tris-Hcl (pH 7.5), 0.1 for 30 min at 30° C.
Brij 35]
stopped by adding 20 ul Laemmli-buffer and boiling for
loading
6% SDS-PAGE
on
gels.
Purification of STAP1-associated proteins and
purification
of STAP1-associated
mass
proteins,
spectrometric 4 ml of
packed
analysis.
For
cells
were
lysed for 30 min
7.5),
250 mM NaCI, 5 mM EDTA, 0.5% NP-40, 50 mM NaF, 0.2 mM
(DTT),
1 mM dithiothreitol
ug/ml aprotinin], lysates
cleared
in PL
protein extracts have incubated with 400
mM EDTA, 5 mM DTT, 250 mM NaCI, 0.01 %
10 min before
molecular-weight
and induced with 10uM insulin for 1h
mM Tris-Hcl
U of X-PPase
were
catalase
dounce
on
ice in 50 ml TNN-buffer
1 mM
by centrifugation
fluoride
phenylmethylsulfonyl
homogenized (10 at 18000
strokes with x
mM Tris-HCI
[50
a
B
g for 20 min.
(pH
Na3V04,
(PMSF),
pestle)
HeLa
10
and
Supernatants
were
collected and incubated for 2 hrs at 4°C with anti-STAP1 mAb 105.128
covalently coupled to protein A-Sepharose using
coupling procedure. Immunobeads proteins eluted with 300 K2HPO4, concentrated a
6%-15%
protein
ul 0.2 M
on a
were
washed 4
were
analysis. Peptides
dimethylpimelimidate x
with TNN-buffer,
glycine (pH 2.5), neutralized with 100 ul 1M
Centricon concentrator
gradient SDS-polyacrylamide gel.
bands
a
The
(Amicon)
gel
was
and resolved
on
silver stained,
excised, digested with trypsin and subjected to sequence
were
sequenced using nano-electrospray tandem
spectrometry (NanoESI-MSMS)
on a
API 300
Toronto, Ontario, Canada) and identified Identities of STAP1-associated
proteins
as
mass
spectrometer (PE Sciex,
described
were
mass
(Mann
confirmed in
an
and Wilm
1994).
independent
102
experiment using
MALDI-TOF
analysis
on a
TofSpec
2E
(Micromass,
Manchester, UK) according the direction of the manufacturer. The full dataset of this
analysis
In vitro
is available upon request.
binding
expressed
reactions.
GST-fusion
as
STAP1, hRPB5, scRpb5
proteins
in E.
coli, purified
Sepharose beads and subsequently incubated vitro translation
(Promega) analyzed
as
on
products
previously
described
on
scPfd6
glutathione-
35S-methionine-labeled
(Krek
et al.
1994).
Bound
in
proteins
were
by fluorography.
have been used
techniques. The following yeast strains
study: yBM80 (W303):
were
scUrip generated by the TNT system
or
SDS-PAGE and detected
Yeast strains and in this
of either URI
with
or
uri::HIS3 ade2-1
trp1-1
can1-100
Ieu2-3,112
his3-11,15 ura3 psi+. yBM79 (W303):ade2-1 trp1-1 canMOO Ieu2-3,112 his311,15 ura3 psi+. yBL45 (K699): ade2-1, trp1-1, can1-100, leu2-3,112, his311,15, ura3, psi+, ssd1-d2 bar- uri::Uri-13myc His3. yBL284 (W303): ade2-1,
trp1-1, can1-100, leu2-3, 112, his3-11,15, ura3, psi+ gcn4::kan. yBL285
(W303): ade2-1, trp1-1, canMOO, leu2-3, 112, his3-11,15, ura3, psi+, gcn4::kan uri::HIS3.
YBL295
(W303): ade2-1, trp1-1, can1-100, leu2-3,112,
his3-11,15, ura3, psi+, ssd1-d2, uri::HIS gcn2::kan. yBL282 (W303): ade2-1,
trp1-1, can1-100, leu2-3,112, his3-11, 15, ura3, psi+, uri::Uri-13myc-HIS3. yBL225 (2):ura3. yBL234 (2):ura3-52
uri::KAN.
yMJ205 (W303): cdc34-2
ade2-1, trp1-1, can1-100, leu2-3,112, his3-11, 15, ura3, psi+, ssd1-d2. Details about strain constructions will be otherwise standard used
(Guthrie
at 25 °C until
yeast growth
and Fink
1991b).
early-log phase
in
provided upon request. If
conditions and
For
protein
not stated
genetic manipulations
half-life
analyis
synthetic media. Cells
cells
were
were
were
grown
then shifted to
a
non-permissive temperature of 37 °C for 3 hours. At time 0 cyclohexamide was
added to
a
final concentration of 50
ug/ml
and
samples
were
taken at the
103
extracts
indicated times,
protein
intensities
analysed
were
with
were
analyzed by
Quantity
One
was
as a
ratio
(intensity
always normalized and
set to
For
a
where time 0
value of 100. Standard deviations
experiments
and
plotted
on a
scatter
performed
graph.
Hinnebusch).
(Mueller
under the
tap
1986),
and Hinnebusch
Nasmyth 1985). Yeast cells
were
water after
growth
on
were
(OD600
=
washed twice with H2O
in either amino acid starvation medium or
described
for two
in
urea
medium
(Breeden
growth by washing
days
at 30°C. In nutrient
cells grown in YPD
diluted to OD600 of 0.1 and grown in YPD to
0.4),
glucose),
were
yeast
experiments, overnight cultures of yeast
medium
as
tested for invasive
extract/peptone/dextrose (YPD) plates starvation
LacZ assays
where the ATGs of uORF1-3
GCN4uORF4only-lacZ,
or
have been mutated
yeast plates
Alan
yeast strains transformed with the reporter plasmids
on
GCN4uORF-lacZ
prewarmed
early log-phase
at 30°C and
resuspended
(3.4 g/l yeast nitrogen base,
(3.4 g/l yeast nitrogen base,
3%
5 uM urea, 3%
glucose, appropriate supplements for auxotrophs Ura, Leu, Trp, Ade). diauxic shift
0 hrs time total
experiments yeast
point)
protein
(yBM79) 30°C
and
were
were
grown to
early log phase
taken at different time
in YPD
(=
points thereafter for
expression analysis. Three colonies of wild-type strain
and uriA
on a
aliqots
cells
In
extraction.
Global gene
to
were
expression of GCN4p-myc yeast cells have been transformed with
plasmid p3330 (kindly provided by
and
version 4.2
determined and
were
GCN4-myc/control intensity),
of
determined from three individual
blotting. Band
quantitation software,
background
from Biorad. Intensities minus the lane
expressed
Western
(yBM80)
were
inocculated in 10 ml YPD, grown
overnight
at
shaker at 225 rpm, diluted to OD600 of 0.1 and continued to grow
early log phase (OD600=0.3).
Yeast cells
were
for 5 min at 3000 rpm and total RNA extracted
by
collected the
by centrifugation
acid-phenol method
104
from wt
(yBM79)
(yBM80)
In order to define
1990).
al.
and uriA
RNA from six
rapamycin
time-course
(ODeoo YPD.
of
0.3)
Aliquots
rapamycin
and
of 50 ml cell
points by centrifugation frozen in
yBM79
0.3),
and
collected
were
medium at
a
yBM80 yeast
cells
by centrifugation
30°C, (0.34
described above.
were
grown to
pellets
were
snap-
using
glucose). Aliqots
following oligonucleotide
extracted
to the was
were
performed
on
protocol
Ten ug of total RNA
recommended
transcript labelling
kit
protocol. The cRNA
cleaned
phase
In vitro
the Enzo
centrifugation
x
recommended
one
by Qiagen.
minute
by was
BioArray High Yield RNA the manufacturer's
using RNAeasy clean-up
g and then left for
removed
transcription
columns
improve the recovery from the columns, the elution water
the matrix at 27
by Affymetrix.
Double-stranded cDNA
(Enzo Diagnostics, USA) following
was
was
priming: 5'-
(Eppendorf).
template using
as
system for cDNA synthesis
and the aqueous
Phase-lock Gel
1 ug of cDNA
suspension
performed using YG_S98
Choice
used for
by phenol:chloroform
centrifugation through
of 50 ml cell
points for extraction of total RNA
SuperScript
the
temperature and washed
resuspending them in amino acid starvation
Microarray analyses
transcribed
early log phase (OD600 of
ggccagtgaattgtaatacgactcactatagggaggcgg-(t)24-3'.
To
early log-phase
independent replicate cultures
at 3000 rpm at room
% YNB, 3%
(Life Technologies) according
was
were
harvested at indicated time
were
GeneChips (Affymetrix, Santa Clara, USA).
The
URI,
final concentration of 100 nM in
amino acid starvation two
harvested at indicated time
reverse
loss of
yBM80
and
grown to
for 5 minutes at 3000 rpm and
twice with H2O at 30°C before
were
yBM79
by
experiments (Fig. 5B), two separate
added to
suspension
affected
et
liquid nitrogen before RNA isolation. For measuring global gene
expression following of
was
of
cells
replicate cultures of yBM79 and yBM80
microarray analysis (Schmitt
expression
gene
independent replicate cultures
For the
analyzed.
global
strains for
was
(Qiagen).
spun into
prior to the standard 8000 g
This low
speed wetting step gave
105
nearly
double the
cRNA
was
fragmented by heating
(pH 8.1),
acetate
of eluted RNA
yield
Affymetrix.
fragmented
Ten ug of
staining
protocol The
was
performed
standard in
were
Suite v4 for
AvgDiff
percentile
an
perform
The
values remained
negative
expression
calculations
were
or
based
on
AvgDiff values
whose absolute fold
the lower 90% confidence bound for fold
equal
provided
(Silicon Genetics)
used to
was
changes
Fig.
of human cells
respective
onto
a
10cm dish 24h
RNA was
prepared from
the TRIZOL the
prior
on
essentially
as
HG-U133
v5 and
assigned
Analysis
GeneSpring
5.0
of the
to functional
GeneSpring
4.2
for the presence of
global transcription analysis
HeLa cell line
samples
chips
from
was
were
seeded
rapamycin. Total
were
oligonucleotide microarray chip
described above.
MicroArraySuite
were
two 10cm dishes at the indicated time
RNA
and
and had P values for
change
4A. For
GeneChips
greater than 50, that
treatment of the cells with 20 nM
reagent (Life Technologies).
analyis
to account for
analyze promoter regions
million cells of the
50th
all arrays. Fold
at http://casiama.c8hi.ora/iian.
conserved sequence motifs shown in one
were
to 0.05. Yeast ORFs
groups using the tools
calculate fold
for those genes
between individual
passed
or
across
signals
only genes
smaller
(AvgDiff)
very low have been set to the
normalized
intensity
scanner.
are
average difference
include
change
(Affymetrix) using the
performed using the
values called absent
variations in overall fluorescence
fold
hours). Washing
16
Affymetrix GeneChip
determining
analysis.
mM Tris-
YG_S98
a
experiments performed
cluster
of the
hybridised to
was
The
recommended by
(www.biostat.harvard.edu/complab/dchip/) to
values and dCHIP v1.0 and to
as
procedures (45°C,
request. Chip analysis
Affymetrix Microarray
changes
MgOAc]
Fluidics Station 400
a
data sets for all array
communicated upon
change
cRNA
EukGE-WS2v4 and scanned in
complete
whose
fragmentation buffer [40
in 1x
100 mM KOAc, 30 mM
GeneChip (Affymetrix) using and
(E. 0., unpublished observation).
points using
processed for Affymetrix
performed using
(Silicon Genetics). Changes
in gene
106
expression
were
determined
that pass
replicates
signed
a
by looking
Wilcoxon rank test. The
was
identify
list of 194 genes that treatment in
rapamycin
Welch t-Test
(pwb[
2
3 4
WBf
I
^HA-STAP1 12
1
P
3
4
—>^HA-STAP1
Fig. S1. STAP1 is a member of the a class PFD family and part of a multiprotein complex. (A) Amino acid sequence alignment of STAP1 with members of the prefoldin protein family. Secondary structure elements were deduced from the crystal structure of M. thermoautotrophicum PFDcc subunit (Siegert
2000) and are represented as blue cylinders (a helices) and grey arrows (P strands). (B) Characterization of anti-STAP1 antibodies. Whole cell extracts of U2-OS cells (lanes 1 and 3) or U2-OS cells expressing untagged STAP1 (lanes 2 and 4) were analyzed by Western blotting with anti-STAP1 mAb 105-72 or affinity-purified polyclonal anti-STAP1 antibody. (C) Nuclear extracts of 293 cells were subjected to immunoprecipitation with control antibody (mouse IgG, lane 1) or anti-STAP1 mAb 105-128 (lane 2) coupled to protein A-Sepharose and the immunoblot probed with anti-SKP2 or antiSTAP1. (D) Top: Extracts from untransfected U2-OS cells (lanes 1 and 2) or from U2-OS cells transfected with an HA-STAP1 expression plasmid (lanes 3 and 4) were immunoprecipitated with control antibody (preimmune serum, lanes 1 and 3) or polyclonal anti-SKP2 antibody (lanes 2 and 4) followed by Western blotting using anti-HA antibody. Bottom: Extracts were immunoblotted with anti-HA antibody before immunoprecipitation. et al.
108
2MDa
Fraction: 1
5
440 kDa
670 kDa 15
10
20
158 kDa
25
30
35 STAP1
-!
-
PFD3
mi
URI RPB5
TIP49
TIP48
SKP2 CUL1
SKP1
t3
2MDa
2
440 kDa
670 kDa
6
14
18
22
26
158 kDa 30
34 STAP1 URI RPB5
TIP49 TIP48 STAP1 IP
Fig.
S2. STAP1 and URI
whole cell
lysate
are
part of
of HeLa cells
was
processed
for
~
WB
1MDa
complex in human cells. (A) A on a Superose 6 gel-filtration Western blotting using
fractionated
processed for proteins. (B) Fractions shown
column and individual fractions antibodies to the indicated
a
>
with anti-STAP1
immunoprecipitation immunoblotting with antibodies directed against right.
the
(A) were antibody followed by proteins indicated on in
the
109
complex by co-immunoprecipitation. (A) Aliquots immunoprecipitated using anti-URI polyclonal anti-TIP48 (lanes 2), polyclonal (lane 4) coupled to proteinA sepharose or control antibody (lanes 1 and 3) and analyzed by immunoblotting with antibodies directed against indicated proteins. (B) Prefoldin subunits STAP1 and PFD4r are stoichiometrically associated with URI. Whole cell extracts prepared from HEK293 cells were subjected to immunoprecipitation using monoclonal antibodies directed against human URL Lysates before (lane 1) and after sequential immunoprecipitations (lanes 2 and 4) and immunoprecipitates (lanes 3 and 5) were analyzed by Western blotting using antibodies to the proteins indicated.
Fig.
S3.
Analysis
of the URI
of HEK293 cell extracts
were
110
UH (hü) UR! St) UH Wra) URI
(EC)
URli&Cj Pf 05 (»C)
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123
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KBBTA3rWRNVBI8»ri3BS*lafiJ>ll»lJS*bBi6fn>KH
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