UCL DNA Repair Pathways PDF

UCL Cancer Institute Faculty of Medical Sciences DNA Repair Pathways Prof John Hartley [email protected] MSc Cancer 2 Part 1 Direct reversal, mismatch repair and base excision repair Learning Outcomes Following this topic, students should be able to: • Discuss the main cellular response...

UCL Cancer Institute Faculty of Medical Sciences DNA Repair Pathways Prof John Hartley [email protected] MSc Cancer 2 Part 1 Direct reversal, mismatch repair and base excision repair Learning Outcomes Following this topic, students should be able to: • Discuss the main cellular responses to DNA damage • Describe the principles and steps involved in direct reversal of DNA damage, DNA mismatch repair and DNA base excision repair 3 Cellular responses to DNA damage - 1 Cells activate complex signaling networks which decide cell fate • DNA repair – Direct reversal of damage – Excision of damage – Double strand break repair • Damage tolerance For cells that are not as expendable - need to survive – Translesion synthesis • Cell cycle arrest • Cell death if the cell is expendable or the DNA damage is too great – Apoptosis, autophagy, necrosis, senescence 4 Cellular responses to DNA damage - 2 • Pathways that dictate cell fate have key roles in cancer initiation and progression. • Outcome is determined by threshold of pro-survival factors versus pro-death factors. • The threshold can differ greatly in different cell types. • Many DNA repair mechanisms are conserved from bacteria and yeast through to humans. 5 DNA repair – Nobel Prize 2015 6 Direct reversal of DNA damage - 1 Removal of pyrimidine dimers by photoreactivation Found through experiments in bacteria • Photolyases are enzymes that repair damage caused by UV. • They require light for their own activation and for repair. • They are present from bacteria to fungi to plants to animals. • This mechanism is not used in humans. photolyase is able to recognise the dimer in the DNA and then absorbs a higher absorption of light to transfers the dimer off the DNA to restore the DNA back to normal 7 Direct reversal of DNA damage - 2 Repair of O6-methylguanine by O6methylguanine methyltransferase • The methyl group is transferred from guanine to a cysteine group in the active site of the enzyme. • It is a ‘suicide’ enzyme as it can only catalyse a single reaction. • The gene is silenced in some tumours e.g. 40% of gliomas. • • • evolved an enzyme just to reverse the damage of the O6-methylguanine to transfer the methyl from the G to the enzyme in the active site contains an SH group which is where this reversal takes place the enzyme isn’t able to do the catalysis again - which is why it is called a suicide enzyme 8 * in humans there is a whole list of components to be able to recognise the different errors Excision of damage – mismatch repair - 1 • Repairs mispaired bases formed: – During DNA replication – During genetic recombination – As a result of DNA damage • * Can repair: – – – – Base-base mismatches e.g. G:T One base insertions/deletions > 1 base insertion/deletion loops Recombination intermediates • MutSa and MutLa collaborate to initiate repair of mismatched DNA MutS is the recognition protein - which recruits the MutL protein MutL finds the first break adjacent to the error - it coils the DNA up to that point and a new protein comes and removes all the newly synthesised DNA up to this point - and the synthesis resumes 9 Excision of damage – mismatch repair - 2 10 Pairing up the different proteins - results in different specificity Up to about 12 nucleotides • MutS and MutL are heterodimers • MutS components include MSH2, MSH3, MSH4, MSH5 and MSH6 • MutL components include MLH1, PMS2, PMS1 and MLH3 • Different combinations determine the type of mismatch repair e.g. MSH2/6 and MLH1/PMS2 repairs a base-base mismatch Excision of damage – mismatch repair - 3 generates mutation 11 • Defects in mismatch repair are responsible for the accumulation of a variety of mutations in the genome including microsatellite instability. • Genetic defects in mismatch repair genes play an important role in cancer susceptibility syndromes (e.g. hereditary nonpolyposis colon cancer) and sporadic cancers. • Approximately 90% of mismatch repair mutations occur in MSH2 and MLH1. They are involved in a lot of the combinations of the MutS MutL complexes • Hereditary nonpolyposis colon cancer accounts for approximately 3% of all colon cancers. Excision of damage – base excision repair - 1 highly conserved • Base excision repair tends to repair lesions that derive from endogenous sources Is what gives specificity to the process • It is initiated by a group of DNA glycosylases which recognise an abnormal base and cleave its bond to deoxyribose. • Each DNA glycosylase is specialised to recognise a unique abnormal base e.g. uracil DNA-glycosylase recognises uracil and then removes it. also have other glycosylases to recognise other toxic bases 12 Excision of damage – base excision repair - 2 13 Base excision repair of uracil in DNA • Uracil DNA-glycosylase recognises uracil and removes it. Cuts the glycosidic bond • The base-free site is excised by an apurinic/apyrimidinic endonuclease (APE). • The gap is filled by a DNA polymerase and sealed by a What is shown is ‘short patch’ base DNA ligase. excision repair. • Proofreading can occur to An alternative ‘long patch’ repair involves ensure correct incorporation. removal of several nucleotides Excision of damage – base excision repair - 3 14 Poly ADP-ribose polymerase 1 (PARP1) Poly-(ADP ribose)polymerase-1 (PARP1) • DNA repair enzymes involved in base excision repair are recruited to single strand breaks by the action of PARP1. • It binds to the breaks and attaches multiple ADP-ribose units to itself and to other proteins. • The ADP-ribose chains act as docking sites for the repair enzymes. • Zinc finger domain binds to ssb, cleaves NAD+ and attaches multiple ADP-ribose units to DNA, histones and repair enzymes Summary 15 • DNA repair mechanisms are crucial for survival and are highly conserved. • Repair of O6-methylguanine is one of the few examples of direct reversal. • Mismatch repair is a major mechanism for preventing mutations arising from DNA replication, recombination and as a result of DNA damage. • Base excision repair, involving specific DNA glycosylases, repairs many lesions that derive from endogenous sources. 16 Part 2 Nucleotide excision repair, double strand break repair, DNA repair genetic disorders, translesion synthesis and DNA damage response. Learning Outcomes Following this topic, students should be able to: • Discuss the main principles of nucleotide excision repair, double strand break repair repair and translesion synthesis • Describe genetic disorders resulting from defects in DNA repair • Understand the concept of DNA Damage Response (DDR) 17 Excision of damage – nucleotide excision repair - 1 18 • In contrast to base excision repair, nucleotide excision repair largely repairs lesions created by exogenous agents. • It repairs bulky, helix-distorting alterations. • Rather than removing a single base, it removes damagecontaining oligonucleotides from DNA. • It is highly conserved and has a broad specificity. • It involves the product of over thirty genes. • Transcription factor TFIIH is an essential component. doesn’t have the same specificity as the glycosylases - just recognises that something is wrong with the DNA through the distortions or bulky additions 19 Excision of damage – nucleotide excision repair - 2 Scans the DNA distortion recognition & partial opening • The main steps of nucleotide excision repair in humans 5' 3' TFIIH recruits other proteins (TFIIH) formation of open structure & recognition of damaged strand Function is to unwind the DNA as they are helices XPC-HR23B XPA 5' 3' XPB XPD RPA ERCC1-XPF dual incision by structurespecific endonucleases excision proteins ERCC1-XPF and XPG have different polarities - always cuts on specific sides excision & DNA repair synthesis 5' XPG XPB XPD 3' PCNA 5' 3' Around 28-30 bases are removed the other strand is then used as a template for DNA pol RFC DNA pol e or d DNA ligase RPA XP (xeroderma pigmentosum) - patients with defects in excision repair - but depends which protein which gives subtypes of the disease Excision of damage – nucleotide excision repair - 3 Unable to repair the DNA • A defect in nucleotide excision repair can lead to the genetic disorder xeroderma pigmentosum (XP). • Individuals with XP syndrome have a 2000-fold increased risk of skin cancer before the age of 20 and a 100,000 fold increased risk of squamous cell carcinoma of the tip of the tongue. Cancer incidence in XP versus non-XP populations 20 Excision of damage – nucleotide excision repair - 4 21 Xeroderma pigmentosum • Affected individuals have extreme sensitivity to UV light. • They have dry parchment-like skin (xeroderma) and many freckles (pigmentosum) Excision of damage – nucleotide excision repair - 5 22 Nucleotide excision repair has two subtypes: • Global Genomic Repair (GGR) • Transcription-Coupled Repair (TCR) – Repairs all regions of the – Repairs template strand during genome transcription – Repairs all types of bulky – Protein complex dislocates adducts regonition protein stalled transcription machinery – requires XPC and all other and provides access to repair nucleotide excision repair proteins proteins except CSA and CSB – requires CSA, CSB and all other – GGR is defective in p53nucleotide excision repair mutant cells proteins except XPC Excision of damage – nucleotide excision repair - 6 Cockayne syndrome (CS) 23 • CS is another inherited syndrome associated with a defect in nucleotide excision repair. Didn’t manifest in the same way as XP • Cells have increased sensitivity to UV light. not to the same extent as XP patients • Despite a defect in DNA repair, this disease is not associated with increased cancer risk. • Patients with CS have normal global genomic repair, but defective transcription-coupled repair. Repair of DNA double strand breaks - 1 • Homology-directed repair (HR) – Occurs during late S and G2 phases of cell cycle – Requires undamaged sister chromatid – Important components are proteins RAD51, BRCA1 and BRCA2 – The process is error free 24 Repair of DNA double strand breaks - 2 • Nonhomologous end joining (NHEJ) Main mechanism but there is more – Used when a sister chromatid is not available e.g. in G1 – Has a normal function in V,D,J gene rearrangement – Important components are proteins KU70, KU80 and DNA-PK – The process is error-prone 25 Tolerance of DNA damage • As a last resort cells have distinct DNA polymerases that can bypass some types of DNA damage in a process called translesion synthesis (TLS). as the cell needs to replicate to survive • This process is highly errorprone due to the high incidence of misincorporated bases. 26 DNA repair capacity • DNA repair capacity differs greatly between cell types – Human embryonic stem cells repair most types of DNA damage more effectively than differentiated cell types – Monocytes and muscle cells are defective in base excision repair – Some cancer cells show up-regulation of DNA repair – Others have defects in specific DNA repair pathways – DNA repair can be an important determinant of inherent sensitivity, or a mechanism of acquired resistance, to genotoxic drugs. 27 If DNA repair fails 28 • If DNA repair fails, DNA damage impedes replication and transcription. • The activated DNA Damage Response (DDR) can signal downstream death pathways. Surviving DNA damage 29 • Ability of a cell to survive DNA damage depends on the: – – – – – – – Amount of critical DNA damage Overall DNA repair capacity of the cell Effectiveness of activating DNA repair genes Proliferation state of the cell p53 status of the cell Status of key DNA damage response (DDR) proteins e.g. ATM/ATR Ability to execute downstream cell death pathways 30 The role of p53 • DNA damage can cause a rapid increase in p53 levels. • P53 protein undergoes posttranslational modifications and induces a number of responses. • This can include cell cycle arrest which can allow time for DNA repair, and mobilisation of DNA repair proteins. • In certain circumstances, it can also trigger apoptosis. DNA damage response proteins ATM and ATR 31 • DNA double strand breaks activate ATM • Stalled or damaged replication forks recruit and activate ATR. • ATR and ATM phosphorylate substrates such as histone H2AX, and CHK1 and CHK2, respectively. • A deficiency of ATM leads to the disease ataxia telangiectasia • Leads to cell cycle arrest, and and to hypersensitivity of cells activation of pathways including to ionising radiation. DNA repair and apoptosis. Defectiveness of DNA damage RESPONSE not repair Summary 32 • Nucleotide excision repair repairs bulky helix-distorting lesions produced by exogenous agents. • Some inherited syndromes are associated with defects in nucleotide excision repair. • DNA double strand breaks are repaired either by homology-directed repair or nonhomologous end joining. • DNA damage response can result in DNA repair, cell cycle arrest and cell death.