Learning outcomes (Lecture 2a)
Understanding:
• Detailed mechanism of NER in E. coli
• The defect in NER in xeroderma
pigmentosum (XP)
• The genetic heterogeneity of XP
• Global genome repair and transcriptioncoupled
DNA repair
2.1
Removal of damage
Base excision repair (BER)
Removes damaged base by breaking
bond between base and sugar
Nucleotide excision repair (NER)
Removes damaged base(s) as part of
an oligonucleotide fragment
(A2, A7)
2.2
UvrA protein
Action of UvrA2B
2.3
UvrA is a
DNA-binding
protein
UvrB is a
helicase/
translocase
Friedberg et al, 2005
DNA Repair and Mutagenesis
Incision by UvrC
2.4 Friedberg et al, 2005
DNA Repair and Mutagenesis
Late steps of NER
2.5 Friedberg et al, 2005
DNA Repair and Mutagenesis
UV-sensitive mutants
Epistasis analysis in yeast
Rad3 epistasis group is involved in NER.
More than 10 genes.
UV dose
wt
Survival
rad1
rad3rad1rad3
UV dose
wt
rad1
rad6
rad1rad6
Damage Repair
Rad1 Rad3
Damage Repair
Rad1
Rad6
Xeroderma Pigmentosum
Patients
XP-C
XP-V
XP-D
Robbins et al
1974
Clinical symptoms of repair-deficient
diseases
1. Xeroderma pigmentosum (XP). Sensitivity of skin to
sunlight: freckles, pigmentation abnormalities, thickening of
the skin, multiple skin cancers. In some case neurological
abnormalities and mental retardation.
2. Trichothiodystrophy (TTD). Sulphur deficient brittle hair,
mental and physical retardation, ichthyosis (fish-like scaly
skin), sun sensitivity, no cancer.
3. Cockayne Syndrome (CS). Dwarfism, loss of adipose
tissue, beaked nose, sunken eyes, premature ageing, skeletal
abnormalities, severe mental retardation, retinal atrophy, sun
sensitivity, no cancer.
4. HNPCC. Familial non-polyposis colon carcinoma.
5. Familial breast cancer.
6. Immunodeficiency (Ligase IV syndrome) Microcephaly,
mental retardation, unusual facial features, immune
deficiencies (cellular and humoral)
7. Nijmegen Breakage Syndome (NBS). Microcephaly, mental
retardation, unusual facial features, immune deficiencies
(cellular and humoral), frequent infections, lymphomas,
radiation sensitive. Chromosome aberrations.
8. Ataxia-telangiectasia (A-T). Progressive cerebellar ataxia,
telangiectasia, immune deficiencies (cellular and humoral),
lymphoreticular cancers and carcinomas, radiation-sensitive.
Chromosome aberrations.
9. Fanconi anaemia. Bone marrow deficiency, abnormal
pigmentation, skeletal deformities especially of radius and
thumb, leukemias, and other cancers. Chromosome
aberrations.
10. Bloom Syndrome. Small size, sun-sensitive rash, immune
deficiencies – infections, leukemias. Chromosome
aberrations and SCE’s.
2.7
UV-sensitive mutants
UV dose
wt
XP
Epistasis analysis in yeast
Rad3 epistasis group is involved in NER.
More than 10 genes.
2.6
UV dose
wt
Survival
rad1
rad3rad1rad3
UV dose
wt
rad1
rad6
rad1rad6
Survival
Human cells
Damage Repair
Rad1 Rad3
Damage Repair
Rad1
Rad6
Unscheduled DNA Synthesis (UDS)
No UV
+ UV
2.8
S phase nuclei
UDS in non-S phase nuclei
Absent in XP cells
Most XP patients are defective in NER
Diagnostic test for UDS
UV (Jm-2)
0 2 4 6 8 10 12
UDS
0
20
40
60
80
100
120
140
160
180
Normal
XP1
XP2
2.8a
Properties of XP, CS and TTD
complementation groups
Clinical features Repair characteristics
Group Skin
Cancer
Neurological
abnormalities
Relative
frequency of
occurrence
UV-
sensitivity
Residual
UDS*
Remarks
XP-A + ++ high +++ <5
XP-B +/- +++/+ very rare ++ <10 Combined XP/CS or TTD
XP-C + - high + 15-30 Deficient in ‘global genome’
repair.
Normal transcription-coupled
repair
XP-D + ++/- intermediate ++ 15-50 Includes patients with TTD and
patients with XP/CS
XP-E +/- - rare + >50
XP-F +/- - rare/
intermediate
+ 15-30
XP-G +/- +++/+ rare ++ <10 Includes patients with XP/CS
XP-V + - high + 100 Defective in post-replication
repair. Normal NER
CS-A - ++ rare + 100 Defective in transcriptioncoupled
repair
‘Global genome’ repair normal
CS-B - ++ high + 100 Defective in transcriptioncoupled
repair
‘Global genome’ repair normal
TTD-A - + very rare + 15 TTD
*Unscheduled DNA synthesis as a percentage of wild-type activity
2.9
Cockayne
Syndrome
TTDXP
XP, TTD and CS
A. CLINICAL SYMPTOMS OF REPAIR-DEFICIENT DISEASES
1. Xeroderma pigmentosum (XP). Sensitivity of skin to
sunlight: freckles, pigmentation abnormalities, thickening of
the skin, multiple skin cancers. In some case neurological
abnormalities and mental retardation.
2. Trichothiodystrophy (TTD). Sulphur deficient brittle hair,
mental and physical retardation, ichthyosis (fish-like scaly
skin), sun sensitivity, no cancer.
3. Cockayne Syndrome (CS). Dwarfism, loss of adipose
tissue, beaked nose, sunken eyes, premature ageing, skeletal
abnormalities, severe mental retardation, retinal atrophy, sun
sensitivity, no cancer.
4. HNPCC. Familial non-polyposis colon carcinoma.
5. Familial breast cancer.
6. Immunodeficiency (Ligase IV syndrome) Microcephaly,
mental retardation, unusual facial features, immune
deficiencies (cellular and humoral)
7. Nijmegen Breakage Syndome (NBS). Microcephaly, mental
retardation, unusual facial features, immune deficiencies
(cellular and humoral), frequent infections, lymphomas,
radiation sensitive. Chromosome aberrations.
8. Ataxia-telangiectasia (A-T). Progressive cerebellar ataxia,
telangiectasia, immune deficiencies (cellular and humoral),
lymphoreticular cancers and carcinomas, radiation-sensitive.
Chromosome aberrations.
9. Fanconi anaemia. Bone marrow deficiency, abnormal
pigmentation, skeletal deformities especially of radius and
thumb, leukemias, and other cancers. Chromosome
aberrations.
10. Bloom Syndrome. Small size, sun-sensitive rash, immune
deficiencies – infections, leukemias. Chromosome
aberrations and SCE’s.
2.7
Properties of XP, CS and TTD
complementation groups
Clinical features Repair characteristics
Group Skin
Cancer
Neurological
abnormalities
Relative
frequency of
occurrence
UV-
sensitivity
Residual
UDS*
Remarks
XP-A + ++ high +++ <5
XP-B +/- +++/+ very rare ++ <10 Combined XP/CS or TTD
XP-C + - high + 15-30 Deficient in ‘global genome’
repair.
Normal transcription-coupled
repair
XP-D + ++/- intermediate ++ 15-50 Includes patients with TTD and
patients with XP/CS
XP-E +/- - rare + >50
XP-F +/- - rare/
intermediate
+ 15-30 Repair slow but prolonged
XP-G +/- +++/+ rare ++ <10 Includes patients with XP/CS
XP-V + - high + 100 Defective in post-replication
repair. Normal NER
CS-A - ++ rare + 100 Defective in transcriptioncoupled
repair
‘Global genome’ repair normal
CS-B - ++ high + 100 Defective in transcriptioncoupled
repair
‘Global genome’ repair normal
TTD-A - + very rare + 15 TTD
*Unscheduled DNA synthesis as a percentage of wild-type activity
2.9
Cockayne
Syndrome
TTDXP
XP, TTD and CS
A. CLINICAL SYMPTOMS OF REPAIR-DEFICIENT DISEASES
1. Xeroderma pigmentosum (XP). Sensitivity of skin to
sunlight: freckles, pigmentation abnormalities, thickening of
the skin, multiple skin cancers. In some case neurological
abnormalities and mental retardation.
2. Trichothiodystrophy (TTD). Sulphur deficient brittle hair,
mental and physical retardation, ichthyosis (fish-like scales),
sun sensitivity, no cancer.
3. Cockayne Syndrome (CS). Dwarfism, loss of adipose
tissue, beaked nose, sunken eyes, premature ageing, skeletal
abnormalities, severe mental retardation, retinal atrophy, sun
sensitivity, no cancer.
4. HNPCC. Familial non-polyposis colon carcinoma.
5. Familial breast cancer.
6. Immunodeficiency (Ligase IV syndrome) Microcephaly,
mental retardation, unusual facial features, immune
deficiencies (cellular and humoral)
7. Nijmegen Breakage Syndome (NBS). Microcephaly, mental
retardation, unusual facial features, immune deficiencies
(cellular and humoral), frequent infections, lymphomas,
radiation sensitive. Chromosome aberrations.
8. Ataxia-telangiectasia (A-T). Progressive cerebellar ataxia,
telangiectasia, immune deficiencies (cellular and humoral),
lymphoreticular cancers and carcinomas, radiation-sensitive.
Chromosome aberrations.
9. Fanconi anaemia. Bone marrow deficiency, abnormal
pigmentation, skeletal deformities especially of radius and
thumb, leukemias, and other cancers. Chromosome
aberrations.
10. Bloom Syndrome. Small size, sun-sensitive rash, immune
deficiencies – infections, leukemias. Chromosome
aberrations and SCE’s.
2.7
UV sensitivity of CS cells
2.10
UV sensitivity similar to XPs
But UDS is normal
Friedberg et al, 2005
DNA Repair and Mutagenesis
E822RNA
UV Jm-2
0 2 4 6 8 10 12 14 16
RNASynthesis(%ofunirradiated)
0
20
40
60
80
100
120
140
1BR3
CS2BR
CS15BR
CS14BR
Diagnostic test for CS
UV-irradiate cells. Incubate 24 h. Measure RNA synthesis.
2.11
Properties of XP, CS and TTD
complementation groups
Clinical features Repair characteristics
Group Skin
Cancer
Neurological
abnormalities
Relative
frequency of
occurrence
UV-
sensitivity
Residual
UDS*
Remarks
XP-A + ++ high +++ <5
XP-B +/- +++/+ very rare ++ <10 Combined XP/CS or TTD
XP-C + - high + 15-30 Deficient in ‘global genome’
repair.
Normal transcription-coupled
repair
XP-D + ++/- intermediate ++ 15-50 Includes patients with TTD and
patients with XP/CS
XP-E +/- - rare + >50
XP-F +/- - rare/
intermediate
+ 15-30 Repair slow but prolonged
XP-G +/- +++/+ rare ++ <10 Includes patients with XP/CS
XP-V + - high + 100 Defective in post-replication
repair. Normal NER
CS-A - ++ rare + 100 Defective in transcriptioncoupled
repair
‘Global genome’ repair normal
CS-B - ++ high + 100 Defective in transcriptioncoupled
repair
‘Global genome’ repair normal
TTD-A - + very rare + 15 TTD
*Unscheduled DNA synthesis as a percentage of wild-type activity
2.9
Measurement of gene-specific repair
Damage Endonuclease
2.12
Hamster dhfr gene
14kb Kpn1 fragment:
10Jm-2
58% repair in 8 h
Bulk DNA:
5 Jm-2
16% repair in 24 h
(alkaline gel)
Modified from
Friedberg et al, 2005
DNA Repair and Mutagenesis
(Adapted from Bohr, 1991)
with selected enzyme
Treat (or not) with damagespecific
endonuclease
• Rodent cells : CPD only repaired in transcribed
strand of active genes
• Human cells: CPD repaired much faster in
transcribed strands of active genes
• This is called Transcription-coupled repair (TCR)
• Repair of bulk DNA called global genome repair
(GGR)
Transcription-coupled repair
2.13
• CS cells defective in TCR
All repair at rate of GGR
• In contrast XP-C, not v. sensitive, but UDS low, 10-15%
XP-C cells only carry out TCR, defective in GGR
Summary (Lecture 2a)
• NER in E. coli involves dual incisions and
can be reconstituted with 6 proteins
• XP cells are defective in NER
• There are 8 XP complementation groups
• TTD cells are also defective in NER, mainly
in XP-D group
• CS cells are specifically defective in TCR
• XP-C cells are specifically defective in GGR
2.14
Learning outcomes (Lecture 2b)
Understanding:
• Detailed mechanism of NER in human
cells
• The roles of the XP gene products
• The link via TFIIH between NER and
transcription
• The mechanism of TCR and the roles of
the CS proteins
2.15
HUMAN NER PROTEINS
Human protein Yeast protein AAs(MW - KD) Protein function
XPA Rad14 273 (31) Damage verification
XPC +HR23B Rad4 + Rad23 940 (106) + 409
(58)
Damage recognition
XPE (DDB2) 314 (48) UV-DNA binding
ERCC1 Rad10 297 (31) 5’ Nuclease subunit
XPF Rad1 1050 (115) 5’ Nuclease subunit
XPG Rad2 1186 (133) 3’ Nuclease
XPD Rad3 760 (87) TFIIH subunit,
helicase
XPB Rad25 782 (89) TFIIH subunit,
helicase
P62,P44, P52,
P34, P10(TTDA)
Tfb1, Ssl1, Tfb2,
Tfb3, Tfb5
548 (62), 395 (44),
464 (52), 303 (34),
71 (10)
TFIIH subunit
RPA(p70,p34,p14) Rfa1, Rfa2, Rfa3 616, 270, 121
(70, 34, 14)
SS-DNA binding
PCNA Pol30 261 (32) Polymerase clamp
DNA polδ or ε Pol3 or Pol2 DNA polymerase
LIG1 Cdc9 919 (102) DNA ligase
2.16
(A7)
,κ or ε
TFIIH recruitment
TFIIH
RPA
DNA helix unwinding
XPA-RPA recruitment
XPA
XPC-HR23B
Damage recognition
by XPC-HR23B and
XPE-DDB1
2.17
GGR in humans
• Recognition – must detect 1
damaged base in 1 million
undamaged bases
• XPC is a general damage
recognition factor
• CPD not well recognised by XPC.
6-4PP are.
• Both recognised by DDB
• DDB is a heterodimer of DDB1 and
DDB2
• DDB2 is the XPE protein
• DDB is part of large ubiquitin
ligase complex.
• It binds to damage, then
ubiquitinates and recruits XPC
(not degraded).
• Also self-ubiquitinates and
degrades itself.
• XPE not expressed in rodent cells.
Structures of DDB and XPC
• XPC binds flipped out bases
on the undamaged strand
• Explains broad specificity
XPE (DDB2) binds flipped
out 6-4PP in a pocket
Min and Pavletich, Nature 2007
Scrima et al, Cell 2008
2.18
WD40 proteins
7-bladed
propellor
domains
• XPD highly homologous to Rad3, well studied, ATPdependent
DNA helicase.
• Apart from helicase activity, also a separate essential
function, ie RAD3del lethal.
• Mutations found in XP-D and TTDs: mainly basechange
mutns.
• XPB is another helicase of opposite polarity
XPD, XPB and TFIIH
2.19
• TFIIH, basal transcription initiation factor - ten subunits.
• P89 isolated, helicase activity and gene cloned and
sequenced - it is XPB!, ie XPB is a subunit of TFIIH.
• XPD also.
• 10 subunits now cloned from yeast and humans.
• Products of these genes are therefore subunits of TFIIH,
which has 2 functions, in NER and in transcription
(essential function).
• XPB: part of TFIIH core and helicase activity is vital for
transcription. Required to open up promoter site.
Helicase activity not needed for NER. NB mutations can
affect repair, transcription or both.
• XPD: not as tightly associated – helicase activity not
needed for transcription, is needed for NER.
• TTDA (p8): not essential, only involved in NER.
TFIIH (A7)
• All three genes associated with TTD (XPD, XPB and
TTDA) encode subunits of TFIIH
• XP known to be a repair syndrome
• TTD proposed to be a “transcription syndrome”
• Slightly altered transcription must affect some proteins
specifically, eg hair and myelin.
What is the evidence?
Prediction: Sites of XPD mutations differ between two
syndromes
TFIIH, XP and TTD
2.20
IIIa III IV V VI
G47R
T76A
R112H
C259Y
D234N
R511Q
Y542C
S541R
R487G
A594P
R592P
R601L
R601W
G602D
R666W
D681N
R683W
R683Q
Q726am
G675R
R658H
R658C
C663R
D673G
G713R
R722W
A725P
F/S730
XP-D/CS TTDXP
Mutations in XPD gene (B1)
*
TTD
Mouse
(B2)
2.21
Structure of archaeal XPD (B3)
Red, XP mutations; purple, TTD mutations;
yellow, XP-CS mutations
Fan et al., Cell, 2008
2.22
TFIIH recruitment
TFIIH
RPA
DNA helix unwinding
XPA-RPA recruitment
XPA
XPC-HR23B
Damage recognition
by XPC-HR23B and
XPE-DDB1
2.23
GGR in humans
• CPD not well recognised by XPC.
6-4PP are.
• Both recognised by DDB
• DDB is a heterodimer of DDB1 and
DDB2
• DDB2 is the XPE protein
• DDB is part of large ubiquitin
ligase complex.
• It binds to damage then
ubiquitinates and recruits XPC
(not degraded).
• Also self-ubiquitinates and
degrades itself.
• XPE not expressed in rodent cells.
• Zinc finger protein, binds more strongly to UV-irradiated DNA,
homologous to yeast Rad14.
• Binding to UV-irradiated DNA increased by RPA.
• Originally thought to recognise damage. Now thought to verify
damage and position everything.
• XPA and XPC knockouts - mice sensitive to UV light and susceptible
to UV carcinogenesis. Gene not essential.
XPA
XPG, XPF-ERCC1
• XPG: structure specific nuclease, suitable for cutting 3' to
damage.
• ERCC1 and XPF protein heterodimer is a nuclease, cuts 5’
to damage.
• ERCC1 is not an XP gene. ERCC1 k/o mice died before
weaning with liver failure.
• More severe than XPA mice. Implies another function for
ERCC1.
2.24
Recruitment of NER proteins to damage:
XPC before XPA
From Volker et al., Mol Cell 2001 (B4)
UV-irradiate cells through a microfilter results in
localised damage in the nucleus
Sequential recruitment of NER proteins
to damage
Global Genome Repair pathway
(GGR)
Dual incisions (3', 5')
XPC-HR23BDamage recognition
by XPC-HR23B and
XPE-DDB1
RNA pol.
Transcription-Coupled Repair
(TCR)
+CSA
+CSB
2.25
NER in
humans
C
C
C
D B
TFIIH
TFIIH recruitment
D B
D
A
B
RPA
DNA helix unwinding
XPA
XPA-RPA recruitment A
XPG
XPG recruitment
G
D B
A
G
ERCC1-XPF
ERCC1-XPF recruitment.
Formation of incision
complex completed
D B
A G
F
F
Repair synthesis
needs pol δ, polε or
polκ, PCNA, RFC,
DNA ligase.
TRCF and NER (E. coli)
2.26
Transcription-repair
coupling factor --
Assembly of TCR complex (B5)
NER
proteins
Remodellers
2.27
Global Genome Repair pathway
(GGR)
Dual incisions (3', 5')
XPC-HR23BDamage recognition
by XPC-HR23B and
XPE-DDB1
RNA pol.
Transcription-Coupled Repair
(TCR)
+CSA
+CSB
2.25
TCR
C
C
C
D B
TFIIH
TFIIH recruitment
D B
D
A
B
RPA
DNA helix unwinding
XPA
XPA-RPA recruitment A
XPG
XPG recruitment
G
D B
A
G
ERCC1-XPF
ERCC1-XPF recruitment.
Formation of incision
complex completed
D B
A G
F
F
Repair synthesis
needs pol δ, polε or
polκ, PCNA, RFC,
DNA ligase.
Summary (Lecture 2b)
• XP proteins are involved in damage
recognition, unwinding of the DNA and
cutting on either side of the damage
• XPD, XPB and TTDA are subunits of TFIIH,
which has dual roles in NER and
transcription
• TTD is a transcription syndrome
• CS proteins are involved in recruiting NER
proteins and chromatin remodellers to
enable NER to take place at sites where
RNA polymerase is stalled at damage
2.28