Excision Repair: Takes Advantage of The Double-Stranded (Double Information) Nature of The DNA Molecule

Excision Repair
Takes advantage of the double-stranded (double

information) nature of the DNA molecule.
* Mismatch repair
* Base excision repair
* Nucleotide excision repair
http://asajj.roswellpark.org/huberman/DNA_Repair/DNA_Repair.htm
Nucleotide Excision Repair
• Extremely flexible
• Corrects any damage that distorts the DNA molecule and that alters normal
DNA chemistry
• 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
Some of the Proteins Required for Eukaryotic Nucleotide Excision Repair
Human protein Probable Function
DDB1 Binds damaged DNA (with XPE); component of E3 ubiquitin ligase (E3UL)
XPE (DDB2) Binds damaged DNA (with DDB1); partner with DDB1 in some E3ULs
XPC Binds damaged DNA (with HR23B); recruits other NER proteins
HR23B Binds damaged DNA (with XPC); recruits other NER proteins
XPB 3' to 5' helicase; early and late DNA unwinding
p62 (GTF2H1) ?
p44 (GTF2H2) Regulation of XPD
p34 (GTF2H3) ?
p52 (GTF2H4) Regulation of XPB
p8 ( GTF2H5; TTD-A) Stimulates early unwinding by XPB
XPD 5' to 3' helicase; late DNA unwinding
MNAT1 CDK assembly factor; transcription only
Cdk7 CDK; C-terminal domain kinase; CAK; transcription only
CCNH Cyclin; transcription only
XPA Binds, stabilizes open complex; confirms damage; recruits RPA, ERCC1
RPA1, 2, 3 Binds undamaged strand in open complex
XPG Endonuclease (3' incision); stabilizes full open complex
XPF Part of endonuclease (5' incision)
ERCC1 Part of endonuclease (5' incision)
Early Stages of Global Genome NER
XPE-DDB1
XPE
DDB1
XPC-HR23B XPE-DDB1
HR23B
XPC
TFIIH
XPB XPD
ATP
ADP
XPB XPD
XPA
RPA
XPC-HR23B XPG
XPD
XPG
XPB XPA
RPA
Initial Steps of Transcription-Coupled NER
CSA
RNA polymerase holoenzyme CSB
ATP TFIIH
ADP RNA polymerase, RNA, CSA, CSB
XPB XPD
XPA
RPA
XPG
XPG XPD
XPB XPA
RPA
Final Steps of Eukaryotic NER
XPGXPD
XPB XPA
RPA
XPF-ERCC1
XPF
ERCC1 XPGXPD
XPB XPA
RPA
Double incision
15-24 nucleotides from lesion XPF 2-8 nucleotides from lesion

ERCC1 XPGXPD
XPB XPA
RPA
Excision
XPA, TFIIH
RFC, PCNA
DNA polymerase δ/ε
DNA ligase I XPG, XPF, ERCC1
Polδ/ε
Ligase I
RFC PCNA
RPA
NER and Human Genetic Diseases
• Xeroderma pigmentosum
1. Severe light sensitivity
2. Frequent neurological defects
Cancer 3. Severe pigmentation irregularities
4. Early onset of skin cancer at high incidence
5. Elevated frequency of other forms of cancer
• Cockayne’s syndrome
1. Light sensitivity in some cases
2. Neurological abnormalities
Aging 3. Premature aging of some tissues
4. Facial and limb abnormalities
5. Dwarfism
6. Early death due to neurodegeneration
• Cockayne’s syndrome
2. Neurological abnormalities
Aging 3. Premature aging of some tissues
4. Facial and limb abnormalities
5. Dwarfism
6. Early death due to neurodegeneration
• Trichothiodystrophy
2. Premature aging of some tissues
Aging 3. Facial abnormalities
4. Short stature
5. Ichthyosis (fish-like scales on the skin)
6. Sulfur-deficient brittle hair
Human protein
Some TTD and CS patients carry DDB1
mutations in genes involved in or XPE (DDB2)

XPC
related to NER HR23B
XPB
p62 (GTF2H1)
Trichothiodystrophy Complementation Groups p44 (GTF2H2)
• TTD-A (component of TFIIH)
p34 (GTF2H3)
• XPB/TTD
• XPD/TTD p52 (GTF2H4)
p8 ( GTF2H5; TTD-A)
XPD
MNAT1
Cdk7
CCNH
XPA
RPA1, 2, 3
XPG
XPF
http://asajj.roswellpark.org/huberman/DNA_Repair/DNA_Repair.htm ERCC1
Human protein
Some TTD and CS patients carry DDB1
mutations in genes involved in or XPE (DDB2)

XPC
related to NER HR23B
XPB
p62 (GTF2H1)
Trichothiodystrophy Complementation Groups p44 (GTF2H2)
• TTD-A (component of TFIIH)
p34 (GTF2H3)
• XPB/TTD
• XPD/TTD p52 (GTF2H4)
p8 ( GTF2H5; TTD-A)
Cockayne’s Syndrome Complementation Groups XPD

• CS-A (encodes a protein with multiple WD-40 MNAT1
repeats; E3UL component, similar in this way to XPE) Cdk7
• CS-B (encodes a DNA-dependent ATPase of the CCNH
SNF2 family; substrate of CSA-containing E3UL) XPA
• XPB/CS
RPA1, 2, 3
• XPD/CS
• XPG/CS XPG
XPF
http://asajj.roswellpark.org/huberman/DNA_Repair/DNA_Repair.htm ERCC1
Why does XP cause cancer, while CS and
TTD cause premature aging?
• The symptoms of TTD and CS cannot be simply due to failure to repair transcribed
strands of genes, since XPA and XPF mutant individuals, who cannot carry out any
NER (including repair of transcribed strands), suffer from XP, not TTD or CS.
• XPA and XPF are either downstream of the RNA polymerase removal step of TC-
NER (as shown in the diagram) or downstream of the steps needed to ensure proper
resumption of transcription at the gene. Thus, in XPA or XPF mutant cells, the
functional early steps of TC-NER may permit eventual resumption of transcription.
• Stalled or non-functional transcription may activate the p53 signaling pathway,

leading to apoptosis or cell senescence. Dead or non-proliferating cells can’t cause
cancer, but a sufficient number of them will cause premature tissue aging.
• Stalled or non-functional transcription may activate the p53 signaling pathway,

leading to apoptosis or cell senescence. Dead or non-proliferating cells can’t cause
cancer, but a sufficient number of them will cause premature tissue aging.
• Failure of NER, in contrast, will cause mutations, and accumulating mutations can
lead to cancer. Thus DNA repair failures may be at the root of both cancer (as a
result of damage-induced mutagenesis in proliferating cells) and aging (as a result
of damage-induced cellular senescence or apoptosis).
Strand-break repair
Strand-break repair
• Usually essential for cell survival
Strand-break repair
• Many pathways, whose relative importance varies between and within

organisms
Strand-break repair

organisms
* Double-strand break repair by homologous recombination (HR)
Strand-break repair

organisms
* Double-strand break repair by non-homologous end joining (NHEJ)
Strand-break repair

organisms
* Double-strand break repair by non-homologous end joining (NHEJ)
* Single-strand break repair (SSBR)
Homologous Recombination is Based on the Ability of
Single DNA Strands to Find Regions of Near-Perfect
Homology Elsewhere in the Genome
Facilitation of Homology Searching by RecA and its

Eukaryotic Homologs
RecA
(Rad51)
+
Facilitation of Homology Searching by RecA and its

Eukaryotic Homologs
RecA
(Rad51)
+
• Eukaryotic proteins important in this process include Rad 51,

Rad52, Rad54, Rad55, Rad57 and Rad59. BRCA1 and BRCA2
interact with Rad51 and may regulate it.
Synthesis-dependent strand annealing
Double-strand break
Double-strand break
5' to 3' resection
Double-strand break
5' to 3' resection
Strand invasion
Double-strand break
5' to 3' resection
Strand invasion
New DNA synthesis
Double-strand break
5' to 3' resection
Strand invasion
New DNA synthesis
Unwinding from template; Annealing
Double-strand break
5' to 3' resection
Strand invasion
New DNA synthesis

Note “conservative” DNA synthesis
Double-strand break
5' to 3' resection
Strand invasion
New DNA synthesis

Ligation
Synthesis-dependent strand annealing Single-strand Annealing
Double-strand break
5' to 3' resection
Strand invasion
New DNA synthesis

Ligation
Double-strand break
5' to 3' resection
Strand invasion Annealing
New DNA synthesis

Ligation
Double-strand break
5' to 3' resection
Flap removal;
New DNA synthesis
Ligation

Ligation
Double-strand break
5' to 3' resection
Flap removal;
New DNA synthesis
Ligation
Note deletion
Ligation
Two Sets of Proteins Important for Non-Homologous End Joining
Genes and Proteins Important for NHEJ

Yeast gene name Human gene name Protein
LIG4 LIGASE IV Ligase IV
LIF1 XRCC4 XRCC4; in collaboration with KU, targets
DNA ligase IV to DNA ends
HDF2 XRCC5 Ku80
HDF1 XRCC6 Ku70
Not in yeast XRCC7 DNA-PKcs
Artemis; nuclease regulated by DNA-PKcs;
Not in yeast ARTEMIS important for preparing DNA ends to make
them ligatable
Identified by:

HDF2 XRCC5 Ku80
HDF1 XRCC6 Ku70
them ligatable
Identified by:
• Biochemical fractionation, in vitro end joining assays

HDF2 XRCC5 Ku80
HDF1 XRCC6 Ku70
them ligatable
Identified by:
• Genetic and biochemical analysis of V(D)J recombination

HDF2 XRCC5 Ku80
HDF1 XRCC6 Ku70
them ligatable
Identified by:
• Genetic analyses in yeast, rodent and human cell lines

HDF2 XRCC5 Ku80
HDF1 XRCC6 Ku70
them ligatable
Identified by:
• Genetic analyses in yeast, rodent and human cell lines
• Gene mapping and cloning

HDF2 XRCC5 Ku80
HDF1 XRCC6 Ku70
them ligatable
The Basic Steps of Non-Homologous End Joining
DNA break;
“Synapsis”
Ku binds to ends
Ku recruits DNA-PKcs
Juxtaposition of ends
Nuclease, polymerase,
polynucleotide kinase;
XRCC4, Ligase IV binding
Ligation by DNA ligase IV

and XRCC4
The Basic Steps of Single-Strand Break Repair
PARP binds to break
XRCC1 and Ligase 3

bind to break
End processing by PNK;

Gap filling by Polβ
OH P
Ligation by Ligase 3
Important, but varying, roles of the MRN (MRX)
complex in double-strand break repair
• Mre11, Rad50, Nbs1 (mammals; fission yeast)

Mre11, Rad50, Xrs2 (budding yeast)

• Mammalian cells, fission yeast: major role in DS break repair by

HR; minor or redundant role in NHEJ, if any


• Budding yeast: major role in NHEJ; minor in mitotic HR


• Checkpoint signaling
Nijmegen breakage syndrome (NBS)
Ataxia-telangiectasia-like disease (ATLD)


• Checkpoint signaling
Nijmegen breakage syndrome (NBS)
Ataxia-telangiectasia-like disease (ATLD)
• Possible roles in “synapsis” and end processing
The Mre11-Rad50 complex may tether DNA molecules together
A M CC H CC M B
Rad 50 protein
(not to scale)
Mre11 nuclease
may be involved in
M processing DNA ends.
A+B
MR monomer
(not found)
MR tetramer
Based on Hopfner et al., Nature 418:562-566, 2002.
Chromatin Alterations around Double-Strand Breaks
Mre11
Relative ChIP
Data from Shroff et al.
Current Biology 14: 1703-1711, 2004
Value γ-H2AX
-30 -20 -10 0 10 20 30

Distance from DSB (kb)
Mre11
Relative ChIP
Value γ-H2AX
-30 -20 -10 0 10 20 30

• The position of Mre11 at the DSB is consistent with a potential role in
tethering ends together.
Mre11
Relative ChIP
Value γ-H2AX
-30 -20 -10 0 10 20 30

• The DNA repair protein, Rad51, colocalizes with Mre11.
Mre11
Relative ChIP
Value γ-H2AX
-30 -20 -10 0 10 20 30

• Other proteins are brought to the region by γ-H2AX.
Mre11
Relative ChIP
Value γ-H2AX
-30 -20 -10 0 10 20 30

Cohesins presumably keep the recombination target close by.
Mre11
Relative ChIP
Value γ-H2AX
-30 -20 -10 0 10 20 30

Ino80 chromatin remodeling complex
Mre11
Relative ChIP
Value γ-H2AX
-30 -20 -10 0 10 20 30

Ino80 chromatin remodeling complex
✴ Important for resection of 5' ends at break
Bypass of DNA Damage
by Replication Forks
The DNA Damage Repair Epistasis
Groups of S. cerevisiae
RAD3 RAD52 RAD6
(Nucleotide Excision (Homologous (Damage Bypass)
Repair) Recombination)
RAD1 RAD50 RAD5
RAD2 RAD51 RAD6
RAD3 RAD52 RAD18
RAD4 RAD53 RAD30
RAD7 RAD54 REV1
RAD10 RAD55 REV3
RAD14 RAD56 REV7
RAD16 RAD57 MMS2
RAD23 RAD59 UBC13
POLδ
PCNA
SRS2
Mono- and poly-ubiquitylation control the RAD6 pathway
Homologous
Srs2 Recombination
PCNA
Rad18
RF
Rad6
164
K U Srs2
PCNA Rad18 Rad5
RF RF
Rad6 Ubc13
Bypass Synthesis Mms2
Error-Prone
Error-Free
164
K U K63 U K63 U
PCNA
Recombinational Bypass
(Template Switch; Error Free)
Possible Recombinational Bypass Mechanisms
In both of these hypothetical examples, information is read from the sister nascent strand.
Mutagenic and Non-Mutagenic Bypass Synthesis
Normal Replication Proteins
TT
Stalled at position of damaged DNA
Alternative options for translesion synthesis:

Polζ Rev7
Rev3/hRev3 Polη
+ Rad30 (POLH; XPV)
Rev1/hRev1
TT
TT
AA
or
N 1N 2
Restored replication fork movement
The Current Roster of DNA Polymerases
Greek Name Human Name Yeast Name Proposed Function
α POLA POL1 Replication
β POLB — BER; ss break repair
Mitochondrial
γ POLG MIP1 replication; Mt BER
δ POLD1 POL3 Replication
ε POLE POL2 Replication
ζ POLZ REV3 Bypass synthesis
η POLH RAD30 Bypass synthesis
θ POLQ — Bypass synthesis
ι POLI — Bypass synthesis (?)
κ POLK — Bypass synthesis
λ POLL POL4 NHEJ
μ POLM — NHEJ (?)
ν POLN — Bypass synthesis
— REV1 REV1 Bypass synthesis
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Questions?
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Excision Repair

Takes advantage of the double-stranded (double

* Base excision repair

* Nucleotide excision repair

• In all organisms, NER involves the following steps:

RNA polymerase holoenzyme CSB

15-24 nucleotides from lesion XPF 2-8 nucleotides from lesion

mutations in genes involved in or XPE (DDB2)

mutations in genes involved in or XPE (DDB2)

Cockayne’s Syndrome Complementation Groups XPD

• Stalled or non-functional transcription may activate the p53 signaling pathway,

• Stalled or non-functional transcription may activate the p53 signaling pathway,

• Many pathways, whose relative importance varies between and within

• Many pathways, whose relative importance varies between and within

* Double-strand break repair by homologous recombination (HR)

• Many pathways, whose relative importance varies between and within

* Double-strand break repair by homologous recombination (HR)

* Double-strand break repair by non-homologous end joining (NHEJ)

• Many pathways, whose relative importance varies between and within

* Double-strand break repair by homologous recombination (HR)

* Double-strand break repair by non-homologous end joining (NHEJ)

* Single-strand break repair (SSBR)

Facilitation of Homology Searching by RecA and its

Facilitation of Homology Searching by RecA and its

• Eukaryotic proteins important in this process include Rad 51,

5' to 3' resection

5' to 3' resection

5' to 3' resection

New DNA synthesis

5' to 3' resection

New DNA synthesis

Unwinding from template; Annealing

5' to 3' resection

New DNA synthesis

Unwinding from template; Annealing

5' to 3' resection

New DNA synthesis

Unwinding from template; Annealing

5' to 3' resection

New DNA synthesis

Unwinding from template; Annealing

5' to 3' resection

Strand invasion Annealing

New DNA synthesis

Unwinding from template; Annealing

5' to 3' resection

Strand invasion Annealing

Unwinding from template; Annealing

5' to 3' resection

Strand invasion Annealing

Genes and Proteins Important for NHEJ

Genes and Proteins Important for NHEJ

Genes and Proteins Important for NHEJ

Genes and Proteins Important for NHEJ

Genes and Proteins Important for NHEJ

Genes and Proteins Important for NHEJ

Ligation by DNA ligase IV