Download Mantelstudium ``Biomedizinische Wissenschaften``

Mantelstudium/Jiricny
Sept. 2009
Mantelstudium ''Biomedizinische Wissenschaften''
Skript: DNA-Replikation und Krebs
Skriptunterlagen Prof. J. Jiricny
Literatur und Lehrbücher
Lodish H. et al. Molecular Cell Biology. 5. Edition
Chapter 4.6: DNA Replikation
Chapter 23.5: DNA Repair
Zusammenfassung des Themas
Mit Hilfe konkreter Fälle werden die Mechanismen der DNA Replikation sowie der DNA
Reparatur wiederholt. Konzepte, wie z.B. DNA Replikations-unabhängige DNA Synthese
und „by- pass” Polymerasen, werden vorgestellt und Krebsprädispositions-Syndrome,
die mit mangelhafter DNA Reparatur verbunden sind, besprochen.
In einem ersten Schritt wird der Klinische Fall geschildert und die Diagnose erstellt. Die
Studierenden werden dann aufgefordert über die Mechanismen der DNA Reparatur und
Replikation zu diskutieren. Die folgenden Themen werden in Form eines Seminars
wiederholt oder vorgestellt:
a) DNA Reparaturmechanismen (Repetition)
b) DNA Replikationsmechanismen (Repetition)
c) DNA Replikations-unabhängige DNA Synthese
d) By-pass Polymerasen (neu)
e) Krebsprädispositions-Syndrome, welche auf mangelhafte DNA Reparatur
zurückzuführen sind (neu − Diskussion)
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DNA replication, DNA repair and cancer
Although most people believe that the DNA of our cells is primarily damaged by
exogenous agents such as ionizing radiation and noxious chemicals in the environment,
most of the mutations in our genomes arise through spontaneous (endogenous) damage.
DNA bases are oxidized (e.g. guanine to 8-oxoguanine) and hydrolytically deaminated
(e.g. cytosine to uracil), or spontaneously lost to give rise to abasic sites. Mutations can
also arise during DNA replication, through errors introduced into the newly-synthesized
strand by DNA polymerases. All these types of damage are potentially mutagenic and
must be repaired if the genetic information is to be preserved. If they are left unrepaired,
50% of progeny cells will inherit mutations that may give rise to inactive or
malfunctioning proteins, and thus also to disease.
In the following pages, I shall briefly review the mechanisms of DNA replication and
repair, with particular emphasis on their link to human malignancy.
DNA replication is semi-conservative; this means that each replicated DNA molecule
consists of an old (template) strand and a newly-synthesized (daughter) strand.
To replicate the 6.6 x 109 base pairs of human DNA without errors is a major
challenge for the cellular machinery. Because DNA polymerases are not sufficiently
precise to accomplish this task, other functions are recruited to help. These include the
proofreading exonucleases and DNA repair mechanisms such as mismatch repair.
During DNA replication, the leading DNA strand is synthesized continuously by
DNA-polymerase-δ/ε, but the lagging strand requires the involvement of primase,
polymerase-α and polymerase δ/ε. It is not hard to imagine that this complex handover is
error-prone, especially as the primase and polymerase-α do not possess a proofreading
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activity. The importance of proofreading was first noted in E. coli, where the mutD strain,
which has a mutation in the proofreading subunit of pol III, could be shown to have an
extremely strong mutator phenotype. More recently, experiments with human cells
showed similar results. The current estimates tell us that proofreading contributes about
two orders of magnitude towards replication fidelity.
Mismatch repair (MMR). Other E.coli strains with a strong mutator phenotype are
mutS, mutL and mutH. These genes encode proteins that participate in the repair of
mismatches that arise during DNA replication and recombination. The mutS and mutL
genes are highly conserved through evolution and MutS homologues (MSH) and MutL
homologues (MLH) are found in every organism with the exception of some Archaea. In
humans, mutations in the hMSH2 and hMLH1 genes segregate with more than 60% of
families with Hereditary Non-Polyposis Colon Cancer (HNPCC), an autosomal dominant
syndrome that accounts for ~4% of all colorectal cancers (CRCs). In addition, the hMLH1
gene is transcriptionally silenced in ~10% of CRCs.
Why does a mismatch repair defect make epithelial cells of the colon (and also of the
endometrium and ovary) prone to transformation? Because these cells divide very rapidly
(the colonic epithelium turns over on average every 5-7days) and cells that acquire
mutations in genes that control cell proliferation/migration/differentiation are selected for
and can grow into tumours. Which genes are these?
Sporadic CRC and FAP. In normal colon, the stem cells near the bottom of the colonic
crypts divide in a very controlled manner by the Wnt signaling pathway. A key protein
that is required for replication and migration is β-catenin, but this protein is prevented
from fulfilling its role in unstimulated cells by phosphorylation. This posttranslational
modification is mediated by the GSK3β kinase, which resides in a large complex
consisting of APC/Axin/PP2Ab/PP2Ac. Phosphorylated β-catenin is first ubiquitylated
and then degraded in the proteasome. Upon receipt of an external signal, which arrives in
the form of a Wnt (wingless) ligand binding to a cell surface receptor of the frizzled
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family, the kinase activity of GSK3β is inhibited and β-catenin can now travel to the
nucleus, where it can activate the transcription of genes that are required for S-phase
initiation. In addition, it can travel to the cell membrane, where it can control cell
migration by interacting with E-cadherin. In colon cancer, this equilibrium is disturbed
most frequently through the loss of function of the APC (adenomatous polyposis coli)
gene. This gene is believed to be mutated in most sporadic CRCs, which account for
~95% of all colon cancers. Individuals who inherited one mutated APC allele from one of
the parents suffer from Familial Adenomatous Polyposis (FAP), a cancer-predisposing
syndrome with ~100% penetrance. The affected individuals acquire hundreds to
thousands of adenomatous polyps already in their teens (through the loss of the
remaining, wild type APC allele), which transform to CRC thereafter. The only remedy is
total colectomy.
HNPCC. As mentioned above, this syndrome is linked to inherited mutations in the
mismatch repair (MMR) genes. As HNPCC individuals do not acquire a large number of
polyps, the APC gene is not the primary target for mutation. A MMR defect increases the
number of point mutations, but it also destabilizes repeated sequences such as AAAAAA,
CACACACACA etc., the so-called microsatellites. Microsatellite instability (MSI) is a
hallmark of HNPCC and of sporadic MMR-deficient CRCs. The genes mutated in most
MMR-deficient tumours have microsatellites in their coding region. One such gene is the
tumour growth factor beta type II receptor (TGFβIIR), which has an A10 repeat that is
frequently mutated to A9 or A8 in the tumours. This causes frameshift mutations that give
rise to truncated, non-functional protein. [The picture shows MSI in HNPCC tumors (T)
of patients 2-4 as compared to normal tissue (N) from the same patients.]
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Damage reversal. Some repair enzymes are able to recognize damaged DNA and
catalyse its conversion back to undamaged DNA. In humans, the enzymes discovered to
date reverse methylation damage. Methylguanine methyltransferase (MGMT or AGT)
converts O6-methyguanine back to guanine.
This enzyme is of great importance in the chemotherapy of cancer, as many cytotoxic
drugs (dacarbazine, procarbazine, streptozotocin, temozolomide) are DNA methylating
agents. Because MGMT levels vary widely among patients and among tumours, it is
necessary to establish the level of expression of this protein before commencement of
therapy. Tumours expressing high levels of MGMT are resistant to therapy with these
drugs and thus the side effects of chemotherapy will most likely outweigh the efficacy of
treatment. Best indications are for patients expressing high MGMT levels in blood
lymphocytes and presenting with tumours that express low MGMT levels. This is not an
infrequent scenario, as the MGMT gene is often transcriptionally silenced by cytosine
methylation in tumour cells.
O
N
O
N
O
O
O
N
NH
N
NH2
O
N
Me+
MGMT
O
CH3
N
N
NH2
O
Base excision repair (BER). Damaged or modified bases, such as uracil (product of
cytosine deamination), 8-oxoguanine (product of guanine oxidation) and 3-methyladenine
and 7-methylguanine (products of purine methylation) are removed from DNA by BER.
In this pathway, enzymes named DNA-glycosylases recognize the aberrant base and
remove it from the sugar-phosphate backbone of the DNA. The baseless sugar-phosphate
is then removed by AP-endonuclease and polymerase-β, the single nucleotide gap is
filled in by polymerase-β and the remaining nick is sealed by DNA ligase III/XRCC1.
Eight DNA-glycosylases with different substrate specificities have been identified to
date. In this section I shall discuss only the processing of 8-oxoguanine (GO). This
aberrant base is excised from DNA by hOGG1 to leave an abasic site opposite cytosine.
Polymerase-β can then insert an unmodified G and thus correct the damage. However, if
GO is not removed prior to DNA replication, it may base-pair with A and thus give rise
to GO/A mispairs. Left unrepaired, these mispairs would bring about G to T transversion
mutations in the progeny DNA. The human MutY homologue (hMYH) DNA-glycosylase
removes adenines from GO/A mispairs. Because polymerase-β inserts C opposite GO,
hMYH helps to prevent mutagenesis by giving hOGG1 another chance at repair.
Several patients with multiple adenomatous polyps were shown to have G to T
mutations in the APC gene. Because these mutations are a hallmark of oxidative damage,
the hOGG1 and hMYH genes were sequenced. About 35% of these patients were shown
to carry germline mutations in both alleles of the hMYH gene. As both alleles of hMYH
have to be mutated in the germline, this (autosomal recessive) syndrome is rare.
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Nucleotide excision repair (NER). This is probably the only DNA repair pathway that
has evolved to deal with damage brought about by an exogenous agent: ultraviolet
radiation (UV). UV generates pyrimidine dimers in DNA, which cause large distortions
in the DNA helix. These are recognised and processed by a complex consisting of as
many as 30 polypeptides. The complex possesses two DNA endonucleases, which incise
the damaged strand on either side of the photodimer and remove a stretch of ~30
nucleotides. This gap is subsequently filled-in by polymerase-δ/ε.
Defects in NER were linked to Xeroderma pigmentosum (XP), a rare, autosomal
recessive syndrome characterized by extreme sensitivity to sunlight and predisposition to
skin cancer. Experiments carried out in the late sixties showed that cells of XP patients
were NER-deficient, but that extracts of NER-deficient cells of one patient could be made
NER-proficient by the addition of extracts of NER-deficient cells from another patient. In
this way, XP was divided into seven complementation groups, XP-(A-G). Subsequent
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work showed that the XP-(A-G) genes encoded polypeptides involved in the repair of UV
damage.
DNA damage by-pass. One group of Xeroderma pigmentosum patients puzzled scientists
until 1998. These patients had classical XP, yet their cells were proficient in NER. They
were referred to as XP-V (variant). Only recently could it be shown that the XP-V gene
encodes a DNA polymerase-η, which is capable of by-passing UV-photodimers during
DNA replication. This is an important function, as photodimers are DNA polymerase
blocking lesions and cause arrest of the replication fork. Because NER cannot remove
damage from single-stranded DNA, DNA polymerase-η extends the arrested primer
strand by a few nucleotides and then hands over to polymerase-δ again. The damage
remains, but is now in double-stranded DNA and can be repaired. Interestingly, this bypass process is largely error-free. If polymerase-η is mutated, the by-pass reaction is
catalysed by another enzyme of the by-pass polymerase family (there are more than 10
known), but in an error-prone way. This gives rise to mutations, which lead in turn to
photosensitivity and skin cancer in a similar way as unrepaired UV lesions do.
Double-strand break (DSB) repair mechanisms are not completely characterised at the
moment. We know that DSBs caused by e.g. ionizing radiation or arising during
replication fork arrest are efficiently repaired during a cell cycle arrest that is activated by
several DNA damage signaling pathways at the G1, S and G2 checkpoints. These arrest
periods are triggered by strand-break sensitive protein kinases and their downstream
targets.
Patients with germline mutations in the ATM gene have ataxia telangiectasia (AT), a
progressive neurodegenerative disorder that also predisposes to acute lymphocytic
leukemia (ALL) and lymphomas. The ATM protein kinase is not activated directly by
strand breaks. These have to be recessed by an exonuclease complex consisting of the
MRE11/RAD50/NBS1 proteins. Germline defects in MRE11 cause ATLD (AT-like
disorder), and NBS1 mutations are linked with Nijmegen breakage syndrome, another
neurodegenerative syndrome associated with genomic instability.
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Following resection of the DSB to generate long 3’-overhangs, the ends are repaired
by one of two mechanisms: homologous recombination (HR) that uses the intact sister
chromatid to copy missing genetic information, or non-homologous end-joining (NHEJ),
which links the ends together at regions of microhomologies, but results in a loss of
genetic information. Both mechanisms deploy DNA helicases in the search for
homologies and in the resolution of the recombination intermediates. Defects in the
helicase function are also deleterious: genetic defects in BLM cause Bloom’s syndrome,
characterized by erythema and telangiectasia, dwarfism and cancer predisposition.
Mutations in the WRN gene cause Werner’s syndrome. Patients with this disease show
many symptoms of premature ageing, including hair greying and loss, cataracts,
atherosclerosis and osteoporosis. They also display some characteristics not directly
associated with ageing, including reduced fertility and a predisposition to sarcomas.
There are also other genes that predispose to cancer when mutated. Thus, germline
mutations in the BRCA1 and BRCA2 genes predispose to breast cancer. They encode very
large proteins that are believed to function in recombination, but their precise roles are
not well understood at the moment. Fanconi anaemia (FA), a familial syndrome
consisting of pancytopenia associated with short stature, small skull, characteristic faces,
hypogonadism, patchy melanotic pigmentation of the skin, as well as non-specific
chromosomal changes is also believed to be linked with a defect in strand break repair
and DNA damage signalling, but no detailed information about the molecular roles of the
FANC proteins is available at this time, other that they act in post-translational
modification (ubiquitylation) of several replication and repair proteins. Interestingly,
BRCA2 has recently been shown to be FANC-D1, which links these two break repair
pathways together in an unknown manner.
DNA-dependent protein kinase (DNA-PK) is activated by DSBs arising during VDJ
and class switch recombination. Patients carrying germline mutations in the genes
encoding the DNA-PK complex are afflicted with SCID (severe combined immune
deficiency), because they do not produce a full range of antibodies. Interestingly, they are
not predisposed to cancer. This is most likely
linked to the finding that DSBs arising
spontaneously are processed differently from
those generated by the RAG1/2 endonucleases
that initiate VDJ recombination. The DNA
damage signalling kinase that triggers cell
cycle arrest upon e.g. ionizing radiation
treatment, which leads to the generation of
DSBs, is ATM (see above) and not DNA-PK.
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Lernziele
1. Know different types of DNA polymerases
replicating (pol-α, pol-δ/ε)
base excision repair (pol-β)
by-pass (pol-η, pol-ι, pol−µ, pol-κ, pol-λ)
2. Know different types of DNA repair
direct reversal
BER
NER
MMR
Strand break repair (HR, NHEJ)
3. Know which syndrome is linked to which DNA repair defect
Multiple adenomatous polyposis – BER (MYH)
XP – NER
HNPCC – MMR
AT, ATLD, NBS, SCID, hereditary breast cancer – DSB repair
Bloom’s, Werner’s, Rothmund-Thomson syndromes – DNA helicases (HR)
Fanconi anaemia – interstrand cross-link repair, DSB repair (?)
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