Transcriptional Activators Stimulate DNA Repair

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Molecular Cell, Vol 10, 1391-1401, December 2002
Transcriptional Activators Stimulate DNA
Repair Philippe Frit , Kyungrim Kwon , Frédéric
Coin, Jérôme Auriol, Sandy Dubaele, Bernard
Salles , and Jean-Marc Egly
Lab of  Biochem and Mol Biol ?? 1? ? ? ?
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Molecular mechanism of nucleotide excision repair
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GG-NER

• 
  
• repair of lesions over the entire genome,
  referred to as global genome NER
• GG-NER is dependent on the activity of all
  factors mentioned above, including the
  GG-NER-specific complex XPC-hHR23B
• 2. The rate of repair for GG-NER strongly
  depends on the type of lesion.
• Ex) 6-4PPs are removed much faster from
  the genome than CPDs,
• probably because of differences in
  affinity of the damage sensor XPC-hHR23B.
• 3. The location (accessibility) of a lesion
  influences the removal rate in vivo.

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XPC-hHR23B

• the XPC-hHR23B complex is the first NER factor to
  detect a lesion and recruit the rest of the
  repair machinery to the damaged site in GG-NER
• 2. The complex has affinity for a variety of
  NER lesions including UV-induced injury and
  chemical damage, such as cisplatin and
  N-acetyl-acetoxyaminofluorene (AAF) adducts.
• XPC is the subunit responsible for discerning
  right from wrong' in DNA, but
• at this moment it is unclear how this
  protein senses the wide range of structurally
  unrelated lesions in a vast excess of normal DNA.

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• 
  TC-NER
• repair of transcription-blocking lesions
  present in transcribed DNA strands,
• hence called transcription-coupled NER
• Damage is detected by the elongating RNA
  polymerase II complex when it encounters a
  lesion.
• - Elongating RNA Pol II is blocked by
  many lesions in the transcribed strand.
• This makes it an efficient damage
  sensor (Donahue et al. 1994 Hanawalt and Mellon
  1993 )
• 2. Interestingly, a distinct disorder,
  Cockayne syndrome (CS), is associated with a
  specific defect in transcription-coupled repair.
• 3. The identification of two complementation
  groups (CS-A and CS-B) shows that at least two
  gene products are specifically needed for fast
  and efficient repair of transcribed strands.

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Molecular model for the incision stage of NER
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object

• role of the transcriptional activators
  in DNA repair
• Gal4-VP16 and RAR transcriptional activators
  stimulate nucleotide excision repair (NER)
• Using a reconstituted dual incision assay, we
  show that binding of activators to their cognate
  sequences induces a local chromatin remodeling
  mediated by ATP-driven chromatin remodeling and
  acetyltransferase activities to facilitate DNA
  repair.

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platinated GTG site
NdeI (157)
EcoRI (61)
standard template
TATAA
TCGAG

• Figure 1
• The pBluescript phagemid was modified by
  inserting first, the adenovirus major late
• promoter, second, either the GAL4 binding
  sites or a DR5-type retinoic acid response
  element (RARE) (Dilworth et al., 2000 ) at
  position -85, and third, a single GTG
  1,3-intrastrand cisplatin DNA adduct (at position
  105 on the transcribed strand).

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The GTG cisplatin adduct inhibits transcription
elongation.

• Figure 1
• Untreated or platinated 105.TS, as well as
  platinated 101.NTS. templates, was linearized by
  XhoI at position 128 for a run-off transcription
  assay with HeLa NE (Gerard et al., 1991 ).
• RNAs were resolved on 8 urea PAGE.

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Drosophila embryo extracts as a source for
chromatin assembly factors and purified core
histones
untreated
TFIIH-immunodepleted HeLa nuclear extracts
(purified HeLa TFIIH)
(1 nM)
3 4 5 6 7 8 9 10 11
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347
Figure 1 (c) DNA repair a) HeLa NE is able to
repair the damaged 105.TS template ( lane 7) b)
DmEE extract alone exhibits no repair activity
(lanes 34) c) DNA repair is increased upon
addition of the Gal4-VP16 activator (lanes 78
and 1112) d) using TFIIH-immunodepleted NE in
the context of preincubation with DmEE, there is
no repair unless TFIIH is added (lanes 912)
e) in the absence of cisplatin damage, there is
no signal (lane 6)
EcoRI-NdeI fragment containing the GTG site
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DNA Repair Is Stimulated by Transcriptional
Activators
(chromatinized 105.TS-RARE template)
0.5
0.25, 0.5, 1 nM
1 nM
10-6 M
0.1, 0.25, 0.5, 0.75,
1 nM

• Figure 2 Gal4-VP16 and RARa Activate DNA
  Repair In Vitro
• Transcription activity was analyzed by S1
  nuclease protection assay leading to a 93-mer
• fragment.
• (C) To rule out the possibility that NER
  activation relied on a peculiar feature of
  Gal4-VP16, Tested the effect of RAR, the
  retinoic acid nuclear receptor.

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• Figure 3 Both Domains of Gal4-VP16 Are Required
  for DNA Repair
• The oligo(GAL4) oligonucleotide competitor,
  containing a GAL4 binding site, specifically
  inhibits the GAL4-VP16-activated DNA repair of
  the chromatinized 105.TS, as compared to
  oligo(-GAL4), which lacks the Gal4 binding sites
  (lanes 4 and 2, respectively)
• (B) When the platinated 105.TS. GAL4 template
  lacking the GAL4 binding sites was used,
  Gal4-VP16 did not stimulate the DNA repair
  activity

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• Figure 3 Both Domains of Gal4-VP16 Are Required
  for DNA Repair
• Purified Gal4-DBD, VP16-AD proteins, as well as
  Gal4-VP16 were first tested in a gel shift assay.
• a) Both Gal4-DBD and Gal4-VP16 are able
  to form nucleoprotein complexes (NC) with labeled
  oligo (GAL4), whereas VP16-AD does not .
• b) Only oligo(GAL4), and not
  oligo(-GAL4), can compete for the formation of
  the NC complexes, showing the specificity of the
  interaction (lanes 5, 6, 10, and 11).

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(1 nM)
0.5 1 nM

• Figure 3 Both Domains of Gal4-VP16 Are Required
  for DNA Repair
• Neither VP16-AD nor Gal4-DBD proteins stimulate
  DNA repair
• ( compare lane 2 with lanes 6, 10, and
  11)
• both domains of Gal4-VP16 are required for DNA
  repair stimulation
• Gal4-VP16 stimulates repair resynthesis when the
  cisplatin adduct is located in the vicinity of
  its binding site, at positions 16, 37, and 105
  (lanes 16)
• its stimulatory effect is strongly
  reduced or even disappears when the cisplatin
  lesion switches from position 105 to position
  487 or 1068 (lanes 510).

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how transcription activators could be involved in
DNA repair?
(5 ng/µl)
Figure 4. Activation of DNA Repair Occurs
Independently of Transcription First, addition
of -amanitin, which inhibits RNA pol II, did not
prevent the stimulation of DNA repair
by Gal4-VP16 Second, chromatinized template,
either 105.TS.TATAmut that abolish RNA synthesis
or lacking the TATA box (105.TS.
TATA) allowed NER activation (lanes
36), suggesting that the proper assembly of a
preinitiation transcription
complex is not a prerequisite for DNA repair
activation. Third, when the damage was located
on 101.NTS-GAL4, Gal4-VP16 also stimulated DNA
repair (lanes 1, 2, 7, and
8). Finally, since activators interact with basal
transcription factors such as TBP/TFIID, TFIIB,
or TFIIH their role in mediating DNA
repair cannot be ruled out.
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• Figure 4. Activation of DNA Repair Occurs
  Independently of Transcription
• (C) The TBP/TFIID-immunodepleted (ID-TBP)
  extract was unable to support RNA
• synthesis (lanes 14), but still
  retained its ability to activate DNA repair
  (lanes 14).
• Addition of either TBP or TFIID, which restored
  the transcription activity (lanes 5 and 6), had
  no significant effect on the repair activity
  (lanes 5 and 6).
• As a control, an XPG-immunodepleted NE
  (XPG being an essential NER factor), which works
  in transcription (lanes 710), lost its DNA
  repair activity. This activity can be restored
  upon addition of XPG (C, lanes 912).

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how activators regulate DNA repair ?
Figure 4. Chromatin Remodeling by Gal4-VP16,
Gal4-DBD, and RAR /RXR Upon addition of
Gal4-VP16, Southern blots revealed some changes
of the nucleosome structure proximal (105) to
its cognate DNA binding site (lanes 16, probe
a), but there was no distal (1068) disturbance
(lanes 16, probe b). In the presence of the
truncated Gal4-DBD, there is no obvious
nucleosomal rearrangement (compare lanes 79 with
lanes 13).
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Figure 4. restriction enzyme accessibility Indeed,
upon binding to its target site, RAR /RXR
facilitates the XhoI restriction enzyme cut
generating the PvuI-XhoI fragment detected by
probe a (lanes 2 and 3), evidencing some local
nucleosomal reorganization In contrast, neither
activator allowed ScaI digestion, the restriction
site of which is far away from the RARE and GAL4
responsive element (lanes 5 and 6 and lanes 11
and 12, respectively).
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p300 and ACF Chromatin Remodeling Activities Are
Involved in DNA Repair

• Figure 6
• relationship among ATP-dependent chromatin
  remodelling, histone acetyltransferase activities
• (that also play a role in transcription
  activation), and DNA repair
• Addition of either acetyl CoA or ATP together
  with HeLa NE and Gal4-VP16 does not significantly
  stimulate DNA repair, unless they are
  concomittantly added
• Moreover addition of H3-CoA-20, an
  inhibitor of histone acetyl transferase activity
  (HAT), prevents DNA repair activation ( lanes
  79).
• (B) Significant stimulation of DNA repair also
  occurred when both acetyl CoA and ATP were added
  together with RAR /RXR to a damaged 105.TS-RARE
  template

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Figure 6 Both ACF(ATP utilizing chromatin
assembly and remodeling factor) and p300(HAT) are
required for optimal activation of the dual
incision reaction triggered by either Gal4-VP16
or RAR /RXR. Note that ACF together with ATP
enhances the removal of the damaged fragment
(lanes 1 and 2) this stimulation is higher when
the activator is present (lane 5). In these
conditions, p300 also stimulates slightly, but
significantly, the removal of the damage in the
presence of the activators (lanes 4 and 7).
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In Vivo Stimulation of DNA Repair by Activators
Figure 7 (A) p(GAL4/RARE)-GTG(Pt) constructs
The pGAL4-GTG or pRARE-GTG plasmids contain
five GAL4 binding sites or a single RARE,
respectively, upstream of the SV40 promoter and a
cisplatinated or undamaged GTG site located on
the transcribed strand (TS) of the luciferase
gene in a region corresponding to the 5'- UTR of
the transcript.
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TCR-deficient
NER-deficient

• MRC5
• (SV40-transformed human fibroblats) ,
• XP12RO
• (from a patient suffering from XP group A),
• XP-At
• (derived from the XP-A cell line by
  transfection with a
• retroviral vector bearing the XPA cDNA),
• CS1AN cells
• (from a patient suffering from Cockayne
  syndrome
• group B)
• cells are cotransfected with pSG5-Gal4-VP16(B)
  and pG5-RAR expression vector(C).

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conclusion
Transcriptional activators stimulate the removal
of DNA damage in promoter regions. However,
contrary to what was expected, we show that the
DNA repair activation is not mediated by the
transcription machinery but rather results from a
specific function of activators in local
chromatin remodeling to give access to DNA repair
factors.
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• The fact that transcriptional activators
  stimulate NER might
• correspond to a role for TCR
• This hypothesis is unlikely since
• The DNA repair stimulation occurs on both the
  transcribed and nontranscribed strands and is
  seen in TCR-deficient CS-B cells upon
  overexpression of either Gal4-VP16 or RAR
• (2) The -amanitin RNA pol II inhibitor does not
  affect activator-mediated
• NER stimulation
• (3) The components of the basal transcription
  machinery are not required.