Transcript transcription

Nucleotide Excision Repair（NER）
核苷酸切除修理
• Nucleotide excision repair（NER）operates by a
cut-and-patch mechanism that removes a variety
of bulky lesions，including pyrimidine dimers
（嘧啶二聚体）and nucleotides to which various
chemical groups have become attached.
In all organisms，NER involves the
following steps：
• 1、Damage recognition
• 2、Binding of a multi-protein complex at the
damaged site
• 3、Double incision of the damaged strand several
nucleotides away from the damaged site，on both the
5' and 3' sides
• 4、Removal of the damage-containing
oligonucleotide from between the two nicks
• 5、Filling in of the resulting gap by a DNA
polymerase
• 6、Ligation
S. cerevisiae protein
Human protein
Probable function
GGR（also required for TC-NER in yeast）；works with
HR23B；binds damaged DNA；recruits other NER proteins
GGR；cooperates with XPC（see above）；contains
ubiquitin domain；interacts with proteasome and XPC
Binds and stabilizes open complex；checks for damage
XPCRequired for Eukaryotic
Proteins
Rad23
HR23B
Nucleotide
Excision Repair
Rad4
Rad14
Ssl2（Rad25）
XPA
RPAp70，p32，
p14
XPB
Tfb1
GTF2H1
?
Tfb2
GTF2H4
?
Ssl1
GTF2H2
Zn finger；DNA binding?
Tfb4
GTF2H3
Ring finger；DNA binding?
Tfb5
TFB5；TTD-A
Stabilization of TFIIH
Rad3
XPD
5' to 3' helicase
Tfb3/Rig2
MAT1
CDK assembly factor
Kin28
Cdk7
CDK；C-terminal domain kinase；CAK
Ccl1
CycH
Cyclin
Rad2
XPG
Endonuclease（3' incision）；stabilizes full open complex
Rad1
XPF
Part of endonuclease（5' incision）
Rad10
ERCC1
Part of endonuclease（5' incision）
Rpa1，2，3
Stabilizes open complex（with Rad14/XPA）
3' to 5' helicase
Particularly noteworthy is the 10-protein complex called
TFIIH（background color = green），which is essential for
DNA repair and transcription（it stimulates promoter
clearing by RNA polymerase II）.
Ssl2（Rad25）
XPB
3' to 5' helicase
Tfb1
GTF2H1
?
Tfb2
GTF2H4
?
Ssl1
GTF2H2
Zn finger；DNA binding?
Tfb4
GTF2H3
Ring finger；DNA binding?
Tfb5
TFB5；TTD-A
Stabilization of TFIIH
Rad3
XPD
5' to 3' helicase
Tfb3/Rig2
MAT1
CDK assembly factor
Kin28
Cdk7
CDK；C-terminal domain kinase；CAK
Ccl1
CycH
Cyclin
Two distinct NER pathways can be
distinguished
 global genome repair（GGR）
 transcription-coupled NER（TC-NER）
global genome repair（GGR）
• The initial steps depend on whether the damage is
in the actively transcribed strand of a gene or
elsewhere in the genome. If the damage is not in
the actively transcribed strand of a gene，then the
damage is recognized and bound by a heterodimer
consisting of the XPC and HR23B proteins. The
binding of XPC and HR23B initiates the process
of "global genome repair"（GGR），which
simply means repair anywhere in the genome.
• The XPC/HR23B dimer
appears to recognize
damaged DNA based on
the extent of distortion of
the normal helical DNA
structure caused by the
damage.
• In the process of binding
to the damaged region，
XPC/HR23B is thought
to further increase the
extent of structural
distortion
damaged site
• The increased distortion
produced by XPC/HR23B
permits the entry and
binding of the general
transcription factor TFIIH
• Two of these subunits
（XPB and XPD）are
helicases，which bind to
the damaged strand and
use the energy of ATP to
unwind a stretch of 20-30
nucleotides including the
damaged site.
• Three additional proteins
then bind to and stabilize
the open complex：
• The precise role of XPA
is unclear，but evidence
suggests that it checks to
confirm that damage is
present in the opened
region and assists in
stabilizing the open
complex.
• RPA is the major
eukaryotic singlestranded-DNA-binding
protein. It is a
heterotrimer，and it
binds to and protects both
of the separated strands in
the open complex.
• XPG is a structurespecific nuclease.
• Concomitant with the
binding of XPA，RPA
and XPG，XPC and
HR23B are released.
These two proteins are
then free to recycle to
other damaged sites
where the repair process
has not yet been initiated.
transcription-coupled NER（TC-NER）
• Numerous experiments have demonstrated that
damage within the transcribed strands of genes is
usually repaired more rapidly than damage in the
non-transcribed strand or damage in non-gene
regions. In general，the less structural distortion
produced by the damage，the greater the ratio of
rate of repair in transcribed strands to rate of
repair elsewhere. In humans TC-NER requires all
of the proteins needed for GGR except for XPC
and HR23B，suggesting that a different
mechanism（not requiring XPC）is involved in
recognizing damage in transcribed strands.
• Defects in either of the
two proteins shown
associated with RNA
polymerase in the right
diagram，CSA and CSB，
can lead to the human
genetic disease，
Cockayne's syndrome，
which I'll discuss in more
detail below.
• RNA polymerase’s
function is important for
TC-NER，presumably in
helping to recruit TFIIH
to the damaged site and in
helping to displace RNA
polymerase and the
nascent transcript so that
TFIIH can access the
damaged region. As in the
case of GGR（above），after
recruitment TFIIH unwinds a 20-30
nucleotide stretch of DNA including
the damaged region. Presumably the
partially unwound region produced
by the stalled polymerase assists in
providing access to TFIIH.
• Additional evidence，some of
which is discussed below，
suggests that the XPB and
XPD helicase subunits of
TFIIH，the TTD-A subunit of
TFIIH，and also the XPG
nuclease，play special roles
in TC-NER--roles that go
beyond their roles in GGR. It
may be that these three
proteins assist in the removal
of RNA polymerase and RNA.
• The next step in the repair
process，for both GGR and
TC-NER，is recruitment of
another structure-specific
endonuclease，the XPFERCC1 heterodimer：
• Both XPG and XPF-ERCC1
are specific for junctions
between single- and doublestranded DNA.
• XPG，which is closely
related to the FEN-1 nuclease
that participates in base
excision repair，cuts on the 3'
side of such a junction，while
ERCC1/XPF（a
heterodimeric protein
complex）cuts on the 5' side.
• The cut made by XPG is 2-8
nucleotides from the lesion，
and the cut made by
ERCC1/XPF is 15-24
nucleotides away. These
distances are paired with each
other（probably as a
consequence of the structure
of the multiprotein complex）
in such a way that the
damage-containing
oligonucleotide between the
cuts averages 27 nucleotides
（range 24-32 nucleotides）.
XPG
• The damage-containing
oligonucleotide is displaced
concomitant with the binding
of replicative gap-repair
proteins（RFC，PCNA，
DNA polymeraseδ/ε），with
the displacement of TFIIH，
XPA，XPG，and XPFERCC1，and with new DNA
synthesis that fills the gap.
The final nick is sealed by
DNA ligase I.
NER and human genetic diseases
• The genes encoding many of the human NER proteins
（see the table above）were first identified in genetic
complementation studies of the human DNA repair disease，
Xeroderma pigmentosum（XP），which suggested that
mutations in any of 7 genes（XPA-XPG；Note：XPE is
not listed here due to its relatively minor phenotype and
continuing uncertainty about its role）could give rise to
the disease. In addition to XP，two other human genetic
diseases involve defects in or related to NER. These two
additional diseases are Cockayne's syndrome（CS）and
trichothiodystrophy（TTD）.
Although all 3 diseases are associated with repair
defects，they have strikingly different clinical
manifestations：
• Patients suffering from XP have
–
–
–
–
–
severe light sensitivity
severe pigmentation irregularities
frequent neurological defects
early onset of skin cancer at high incidence
elevated frequency of other forms of cancer
• Cockayne's syndrome gives rise to
–
–
–
–
–
–
premature aging of some tissues
Dwarfism
light sensitivity in some cases
facial and limb abnormalities
neurological abnormalities
early death due to neurodegeneration
• Trichothiodystrophy patients display
– premature aging of some tissues
– sulfur deficient brittle hair
– facial abnormalities
– short stature
– ichthyosis（fish-like scales on the skin）
– light sensitivity in some cases
• Note that the clinical symptoms of XP on the one hand and
CS or TTD on the other are largely non-overlapping. In
particular，CS and TTD patients do not have elevated
rates of cancer，while XP patients do not display
premature aging.
Thank you !