Wie kontrolliert p53 die genom' Stabilitt

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Unit 2 October 10, 2001 DNA repair Apoptosis R
epair Genes
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OUTLINE

• How do cells die from ionizing radiation?
• DNA damage and its repair
• DNA repair hierarchy
• Importance of double-strand breaks
• Homologous recombination (HR) and
  non-homologous end-joining (NHEJ)
• Protein complexes supporting HR, NHEJ, or both
• Genetic instability and DNA repair

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DEATH FROM IONIZING RADIATION

• post-mitotic death
• loss of genetic material
• metabolic death
• delayed apoptotic death
• genetic instability
• delayed apoptotic death
• interphase death
• immediate (rapid) apoptosis
• reproductive death
• long-term arrest (exit cell cycle, senesce,
  differentiate)
• remain metabolically viable for long time

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WHAT CAN BE REPAIRED?

• Loss of genetic material
• DNA repair
• Genetic instability
• ?with difficulty - mutations in regulatory genes
• Immediate apoptosis
• modulators of apoptosis
• Long-term arrest
• ?reverse checkpoint ?de-differentiate

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DNA REPAIR HIERARCHY

• REVERSAL OF DAMAGE
• SPECIFIC ENZYMES FOR SPECIFIC (common) DAMAGE
• e.g. alkyl transferase
• BASE EXCISION REPAIR
• SINGLE NUCLEOTIDE (APURINIC/APYRIMIDINIC)
• MIS-MATCH REPAIR
• Template editing mismatches/loops
• ?mechanism of strand incision
• NUCLEOTIDE EXCISION REPAIR (NER)
• PATCH EXCISION
• DSB REPAIR
• NON-HOMOLOGOUS END JOINING e.g. VDJ
• HOMOLOGOUS RECOMBINATION

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Genes and Cancer Risk
Xeroderma pigmentosum
Ataxia telangiectasia
XP
AT
HOMOZYGOUS - Cancer Prone HETEROZYGOUS - ?
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DIRECT REVERSAL OF DAMAGE
P
S
P
Base
Base
S
S
Base
Base
P
P
S
P
P
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BASE EXCISION REPAIR
P
P
S
S
glycosylase
Base
Base
Base
S
S
P
P
P
P
BASE DAMAGE
A-P SITE - apurinic - apyrimidinic
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BASE EXCISION REPAIR
P
P
S
a-p endonuclease
Base
Base
S
S
A-P SITE
strand break
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BASE EXCISION REPAIR
P
P
polymerase ligase
S
Base
Base
Base
S
S
P
P
strand break
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BULKY LESIONS (gt1 BASE)
UV DAMAGE INVOLVES 2 PYRIMIDINES CYCLOBUTANE
DIMERS/ 6-4 PHOTOPRODUCTS DIFUNCTIONAL
ALKYLATING AGENTS e.g. cisplatin SINGLE STRAND
GAP e.g. base and sugar damage cluster GAP is
produced by endonucleases
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NUCLEOTIDE EXCISION REPAIR

• Bacteria have four genes/proteins to complete the
  incision step of NER
• Human at least 10 genes 7 X-P, 2 CS, HHR23
• YEAST-HAMSTER-MAN (evolution conserved proteins)
• RAD3 ERCC2 XP-D
• RAD25ERCC3 XP-B
• RAD2 ERCC5 XP-G
• PROTEIN COMPLEXES
• Biochemical Purification/ Two Hybrid Assay/
  Protein interaction
• IN VITRO REPAIR
• can now FULLY reconstitute repair with purified
  proteins

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NUCLEOTIDE EXCISION REPAIR
IN VITRO REPAIR e.g. Wood, Sancar,
Prakash TFIIH XP-B XP-D p62, p44, p34
(helicase) (cyclin H/cdk7 in complex, not needed
for NER) XP-A damage recognition XP-F 5
cut XP-G 3 cut XP-C global genome repair, not
TCR RP-A replication and repair factor
essential XP-E (not needed for in vitro
repair) ?role damage binding/ chromatin unwinding
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NUCLEOTIDE EXCISION REPAIR
damage
patch removal 30bp
unwinding by helicases (XP-B, XP-D in TFIIH)
hel
hel
polymerase e RF-C/ PCNA
XP-A/ XP-C (binds to damage)
re-synthesis
hel
hel
re-ligation
strand incision 3 XP-G
strand incision 5 XP-F
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TRANSCRIPTION COUPLED REPAIR (TCR)
REPAIR IS FASTER IN TRANSCRIBED GENES REPAIR IS
FASTER IN TRANSCRIBED STRAND
REPAIROSOME
EXON 1
XP-F
TFIIH Pol II
XP-A
damage
XP-G
EXON 2
REQUIRES ALL XP GENES EXCEPT XP-C, XP-E
In TCR, damage is recognized by the polymerase
reaching a bulky single-strand lesion
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COMPLEMENTATION
A
B-
AA-
FUSE CELLS
BB-
A-
B
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YEAST TO MAMMALIAN CELLS
HUMAN XP-A XP-B XP-C XP-D XP-E XP-F XP-G CS-A CS-
C ? ? ? HHR23
RODENT ? ERCC3 ? ERCC2 ? ERCC1 ERCC5 ? ERCC6 ERCC
4 ? ? ?
YEAST RAD14 RAD25 (SSL2) RAD4 RAD3 ? RAD10? RAD2
? ? RAD1 RAD7 RAD16 RAD23
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MIS-MATCH REPAIRCONSEQUENCES OF MIS-MATCH REPAIR
DEFICIENCY
NO REPAIR
T
T
C
C
CACACACACACA
(A) or (CA) n-/1/2/3
MICRO-SATELLITE INSTABILITY
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FUNCTIONS OF MISMATCH REPAIR

• REMOVAL OF MISMATCHED BASES
• TEMPLATE EDITING - (C/A)n DELETIONS
• NEW STRAND EDITING - (C/A)n INSERTIONS
• EDITING OF RECOMBINATION (heteroduplex)
• NORMALLY SERVES TO PREVENT RECOMBINATION

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MISMATCH REPAIR
mis-match
patch removal
e.g. MSH6 / MSH2 binds to mis-match
unwound DNA
re-synthesis
??strand incision
??strand incision
hMSH6
hMSH2
mut H?
mut H?
re-ligation
pms 2
hMLH1
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DOUBLE-STRAND DNA DAMAGEIONIZING RADIATION
cluster of ionizations
NER
GAP
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THE NUMBER AND TYPE OF IONIZING RADIATION INDUCED
LESIONS IN DNA
TYPE OF LESION NUMBER /Gy /diploid cell
double strand break single strand break base
damage sugar damage DNA-DNA crosslinks DNA-prot
ein crosslinks
40 500-1000 1000-2000 800-1600 30
150
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Single strand repair in Radiation Sensitivity?

• Base-excision repair
• knockouts lethal essential maintenance
• Mismatch repair minor role at most
• at low-dose rate add back gene - resistant
• Nucleotide excision repair
• XP mutants not X-ray sensitive

But, all components of repair are necessary to
repair X-ray damage
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DNA double-strand-break repair
DNA double-strand-break
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IR-Sensitive Mutants 2 groups

• DSBR-defective
• Fragmented DNA remains
• V(D)J rejoining is defective
• NHEJ defect
• Ku70/Ku80/DNA-Pkcs/XRCC4/ligase IV

• DSBR-competent
• Fragmented DNA resolves
• Increased chromosomal aberrations
• HR defect
• Rad51/Rad52/Rad51 homologues/XRCC2/ XRCC3/Rad54/BR
  CA1/2

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IONIZING RADIATION REPAIR GENES DOUBLE STRAND
BREAK REPAIR AND VDJ DEFICIENT

• XRCC-4 (XR-1 mutant of CHO) _at_Ch5
• cloned and corrects dsbr and VDJ
• cell cycle dependence sensitive in G1
• associates with DNA ligase IV
• XRCC-5 (xrs mutants of CHO-K1) _at_Ch2
• Ku 80/86 (antigen)
• DNA end-binding activity
• XRCC-6
• Ku 70
• DNA end-binding heterodimer with Ku 80/86
• XRCC-7 (scid/ V3 mutant of V79)
• DNA-PK (catalytic subunit) 460kD protein
• physiologic substrate not yet defined
• ?signal damage ?directly facilitate repair

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Repair Homologous vs Non-homologous

• non-homologous recombination involves
• Ku70/ Ku80/86/ DNA-PKcs
• XRCC4/ ligase IV
• mre11/ rad50/ NBS
• ?overlap between two pathways of repair?
• distinguish between presentation of lesion and
  repair of lesion
• must involve a signaling pathway
• HR vs NHR may be site / context /cell cycle
  dependent
• ataxia-telangiectasia?
• HR defects
• ?NHR defects

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Repair Homologous vs Non-homologous

• XRCC 4,5,6,7 all involved in non-homologous
  recombination including V(D)J recombination
• homologous recombination involves
• a lot of proteins -? in one recombinosome
• rad 51 and homologues (rad51A,B,C,D) strand
  pairing
• rad 52 strand exchange
• rad 54 associated with 51 and 52,
• rad 55, 57, 59 functions not yet established
• RP-A
• ?XRCC 2,3
• BRCA1/2

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Recombinational Repair
A



dsb
strand exchange
resolution patch
single strand gap fill
mis-match repair of heteroduplex DNA
B
C
single strand annealing
interstitial deletion
interstitial deletion
dsb
dsb
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Rad50/Mre11/NBS1

• preserved protein complex from yeast to man
• Rad50 and Mre11 are vital in mammalian cells
• NBS Nijmegen Breakage syndrome
• radiation sensitive
• defect in double-strand break repair
• short telomeres
• radio-resistant DNA synthesis

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Multiple functions of Rad50/Mre11/NBS1



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Radioresistance Genes
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ATAXIA TELANGIECTASIA
LACK OF CELL CYCLE ARREST TO DAMAGE (P53 absent
or delayed)
?LACK OF DAMAGE SENSING
X-RAY
ILLEGITIMATE RECOMBINATION
INDUCTION OF APOPTOSIS
(?P53 DEPENDENT)
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ATAXIA TELANGIECTASIA
July 1995 GENE CLONED - LARGE - 65 EXONS RELATED
TO THE DNA-PK GENE FAMILY ATM HAS LIPID (PI-3)
KINASE DOMAIN - ?protein kinase HAS HOMOLOGY TO
RAD3/ MEC GENES (CELL CYCLE CHECKPOINT
GENE) ANTIBODIES TO PROTEIN LOCALIZE in
NUCLEUS ASSOCIATE WITH p53/ DNA-PK/ c-abl Kinase
is activated by DNA DAMAGE NF-kB pathway may be
related A-T phenotype suppressed by NF/I-KB gene
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Radiation-Induced Apoptosis
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Lymphocyte Identification using
Immunofluorescence
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Apoptosis Detection Live/Dead
Mitochondrial Stain Di06
Propidium Iodide
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Apoptosis Detection Tunnel Assay
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Cancer Genes - Rad51, p53
p53 is the guardian of the genome protein and
controls apoptosis
p53 protein plays a major role in cancer risk
Human Papillomavirus (HPV) causes cervical cancer
(Sexually Transmitted Cancer) HPV makes E6
oncoprotein which attacks and inactivates p53,
preventing apoptosis and causing cancer
When p53 is genetically inactivated Knocked Out
in the cells of a mouse, the mouse has a higher
spontaneous cancer risk and is also more prone to
radiation-induced cancer
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p53 Knockout Mice
Kemp et al. Nature Genetics 866-69, 1994
Normal p53 Two good copies Homozygous
No p53 Two Bad Copies Knockout
One bad p53 copy One good copy Heterozygous
gt 100 Weeks no tumours
21 weeks tumors
70 weeks tumours
4 Gy
1 Gy
4 Gy
gt 100 Weeks no tumours
14 Weeks tumours
40 Weeks tumours