Download MGMT Methylation damage reversal

Methylation damage reversal
O
N
O
N
O
O
O
N
NH
N
CH3
O
NH2
Me
N
+
MGMT
O
N
N
O
MGMT: Methylguanine methyltransferase
Cancer Chemotherapy: temozolomide, streptozotocin,
dacarbazine, procarbazine, etc.
NH2
Mechlorethamine
Alkylators
Dacarbazine
Generate substrates for
MGMT and MMR
Temozolomide
MGMT levels and Cancer Therapy
•
Many tumours do not express MGMT (ideal scenario)
– Treatment with methylating agents may be effective even for colon cancer (?)
•
Tumours expressing high levels of MGMT
– No point in treating these with methylating agents (side effects, secondary
tumours)
•
BUT: MMR-deficient cells are extremely resistant to alkylators
Biochemistry of MMR
5'
3'
5'
3'
G
T
hMSH6
hMSH2
3'
5'
5'
3'
3'
G
C
5'
DNA polymerase  / PCNA
DNA ligase
3'
5'
5'
3'
G
3'
ADP
5'
ATP
ATP
ADP
3'
hMSH6
hMSH2
3'
5'
ATP
ADP
hMLH1/hPMS2
PCNA ?
5'
3'
hMLH1
hPMS2
G
T
G
T
5'
5'-3' exonuclease
DNA helicase (?)
3'
5'
Biochemistry of MMR
5'
Me
3'
5'
3'
3'
G
T
5'
hMSH6
hMSH2
Replication fork arrest
3'
5'
5'
Me
3'
G
3'
ATP
ATP
Me
5'
3'
ADP
Me
G
T
hMSH6
hMSH2
5'-3' exonuclease
DNA helicase (?)
3'
5'
ATP
ADP
hMLH1/hPMS2
PCNA ?
5'
3'
hMLH1
G
T
hPMS2
ADP
5'
3'
5'
Replication
blockades
Replication fork
arrest/collapse
mRNA
RAD50
NBS1
MRE11
BRCA1
BLM?
BRCA2
BLOOM’S SYNDROME CELL LINE CHARACTERISTICS
Phill North
- elevated spontaneous chromosome aberrations
Phillip.North
- elevated spontaneous Sister Chromatid Exchanges
- prolonged S-phase/abnormal replication intermediates
Metaphase spread of a
These abnormalities are presumed to arise from failure
BS cell showing
abnormally high numbers
in the processing of DNA replication intermediates
of sister chromatid
exchanges i.e.
exchanges between
dark/light staining
regions.
FORK REGRESSION - A PROCESS THAT REQUIRES THE
MULTIPLE ACTIVITIES OF BLM
Strand
displacement
Unwinding of
nascent strands
Strand
Annealing
Formation of
regressed arm
Branch
migration
Stabilisation and
extension
of regressed arm
Lesions that cause replication forks to stall can result in the
formation of “chicken foot” structures
Access to lesion
to allow repair
Polymerase
uncoupling
Restoration of
replication fork
Fork regression to
form “chicken foot”
Template
switching
Restoration of
replication fork
ssDNA gap
Rad51-mediated
strand invasion
Copying of genetic
information from
homologous sequence
DOUBLE HOLLIDAY JUNCTION
Endonucleolytic cleavage
and rejoining of junctions
Endonucleolytic cleavage
and rejoining of junctions
in opposite orientations
Gene Conversion
Gene Conversion
with crossing over
[SCE]
BLM
Fork
regression
Resetting of
replication fork


Template
switching

Polymerase
uncoupling
Replication
fork cleavage
(Mus81?)
Recombinationmediated
replication re-start
Holliday junction
resolution
Sister chromatid
exchange
Figure 16.24a The Biology of Cancer (© Garland Science 2007)
Figure 16.24b The Biology of Cancer (© Garland Science 2007)
Gleevec/Glivec (Novartis)
Structural model of the eukaryotic DNA
replication fork
Aminopterin
Methotrexate
5-Fluorouracil
Inhibitors of DHFR
(Dihydrofolate reductase)
QuickTime™ and a
decompressor
are needed to see this picture.
6-Mercaptopurine
Inhibitors of ribonucleotide reductase
(RNR)
Fludarabine
QuickTime™ and a
decompressor
are needed to see this picture.
Cisplatin
Inhibitors of DNA synthesis
5-Fluorouracil
Fludarabine
ROLES FOR HR IN REPAIR OF LAGGING STRAND TEMPLATE DAMAGE
Post-replication
gap filling
Lagging strand adduct
Double Holliday
junction
Irinotecan
Topoisomerase I/III
Inhibitor
Doxorubicin
Topoisomerase II
Non-homologous end-joining
Endonuclease
Ligase
Figure 12.1 The Biology of Cancer (© Garland Science 2007)
Figure 12.3c The Biology of Cancer (© Garland Science 2007)
Figure 12.3a The Biology of Cancer (© Garland Science 2007)
Figure 12.5a The Biology of Cancer (© Garland Science 2007)
Figure 12.2a The Biology of Cancer (© Garland Science 2007)
Figure 12.2c The Biology of Cancer (© Garland Science 2007)
Figure 12.3b The Biology of Cancer (© Garland Science 2007)
Figure 12.5c The Biology of Cancer (© Garland Science 2007)
Figure 12.5d The Biology of Cancer (© Garland Science 2007)
Figure 12.2b The Biology of Cancer (© Garland Science 2007)
Figure 12.4 The Biology of Cancer (© Garland Science 2007)
Figure 16.30 The Biology of Cancer (© Garland Science 2007)
Severe Combined Immune Deficiency
(SCID)
V(D)J recombination allows B and T cells to generate a variety of antigen receptors
and thereby recognize many different pathogens. During B- and T-cell development
the genes encoding antigen receptors are assembled from individual gene
segments (pale yellow boxes). a, DNA is cut precisely at the border of the gene
segments by the lymphocyte-specific RAG proteins. The excised DNA fragment is
circularized, while the gene segments are protected by hairpin structures. b,
Artemis–DNA-PKcs complex (green ellipses) opens these hairpin seals at different
positions near their tips. c, The ends of the gene segments are processed —
presumably also in part by the Artemis–DNA-PKcs complex — and joined.
Diversification of antibodies
Model of AID involvement in SHM and CSR
AID
activation-induced
deaminase
Somatic
Hypermutation
Switch recombination
PKA
AID
RPA
C
Phase 1
MSH2/6
Phase 2
UNG
PKA
AID
RPA
UNG
MSH2/6
UNG
cytosine
BER
Replication
Replication
Replication
BER
BER
BER
H2O
DNA
error-proneDNA
DNA
DNAsynthesis
synthesis by
by error-prone
polymerase(s)
polymerase(s)
U
IgM
IgM
IgG
IgG (IgA,
(IgA,IgE)
IgE)
uracil
Alt + Storb, Current opinion in immunology, 2006
DNA-Replication
•
•
•
DNA-Replication
requires 3 DNApolymerases: Pol- and
Pol- (see figure).
Pol- are unable to bypass damaged sites in the
template DNA. When
they come to such a site,
they tend to dissociate.
Ubiquitylation of PCNA
at stalled replication forks
probably results in the
loading of one of several
“by-pass” polymerases.
To date, more than ten
“by-pass” polymerases
have been identified:
REV-1, Pol-, Pol-, Pol, Pol-, Pol-, Pol-µ..
NHEJ
MODEL FOR NON-ENDONUCLEOLYTIC RESUOLUTION OF DOUBLE
HOLLIDAY JUNCTIONS BY A HELICASE AND TOPOISOMERASE
Branch migration
BLM (and Topo III?)
Double Holliday Junction
(Hemicatenane)
Topo III (and RMI1?)
Double Holliday
junction dissolution;
Wu and Hickson, Nature
426, 870, 2003
Tutorial
•
•
•
•
•
•
Stem cells/cancer stem cells
Parp inhibitor and BRCA1/2
MGMT/MMR and temozolomide
cisplatin/mitomycin C and NER/FA
Camptothecin and DSB repair
5FU and replication/BER/MMR
Vincristine
Figure 12.5b The Biology of Cancer (© Garland Science 2007)
Nucleotide Excision Repair (NER)
1. Helix-distorting lesion
4. Full helix opening and damage verification
TFIIH
XPA
XPB
2. Initial damage recognition
XPG
XPD
RPA
23B
XPC
5. Dual incision
TFIIH
23B
XPC
ERCC1
XPA
XPF

3. Recruitment of TFIIH and partial opening

XPG
RPA
6. Repair synthesis