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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) 1 Mantelstudium/Jiricny Sept. 2009 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 2 Mantelstudium/Jiricny Sept. 2009 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 3 Mantelstudium/Jiricny Sept. 2009 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.] 4 Mantelstudium/Jiricny Sept. 2009 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. 5 Mantelstudium/Jiricny Sept. 2009 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 6 Mantelstudium/Jiricny Sept. 2009 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. 7 Mantelstudium/Jiricny Sept. 2009 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. 8 Mantelstudium/Jiricny Sept. 2009 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 (?) 9