DNA REPAIR molecular mechanisms and biological consequences of DNA damage and repair Mgr.Lumír Krejčí, PhD. Department of Biology, Faculty of Medicine, Masaryk University NCBR, Faculty of Science, Masaryk University International Clinical Research Center Brno Take care of your DNA as you do not know how long it will last To do … DNA damage - sources, thymine dimer; single strand breaks; double strand breaks; Mutations – definition and types of gene mutations DNA repair - mechanisms of DNA repair; base excision repair; nucleotide excision repair; mismatch repair; DSB repair Human disease linked with DNA repair - Xeroderma Pigmentosum, Cockayne, Bloom and Werner syndromes, Fanconi Aneamia Mutations and epigenetic aberrations in cancer Common Types of DNA Damage and Spontaneous Alterations Radiotherapy Ionizing Radiation X-rays Chemotherapy (Alkylating agents) Cisplatin Mitomycin C Cyclophosphamide Psoralen Melphalan UV (sunlight) Pollution (hydrocarbons) Exogenous Sources Endogenous Sources Oxidative damage by free radicals (oxygen metabolism) Replicative errors Spontaneous alterations in DNA Alkylating agents Smoking Foodstuffs • loss of base –26,000 • deamination of cytosin – 1 000 • alkylation of base – x 10 000 • dimerization of pyrimidins – 50 000 • ssDNA breaks – 100,000 Total ~ 500 000 damage/day DNA damage in human cell per day: UV-induced damage Covalent linkage between neiboring thymins – thymin dimers (pirimidin dimers). Loss of base and deamination Biological consequences of deamination and depurination Deamination – base exchnage Depurination – deletion Intercalating mutagens fit between adjacent base pairs • cause base insertions, leading to translational frameshifts Same size as a base pair Mutations Genomic (numeric aberration of chromosomes) Chromosomal (structural aberration of chromosomes) Gene (point) mutations Somatic versus germline mutations Somatic mutations manifested in 1 cell – clone – not transmitted but can lead to cell death or cancer transformation – 1 individual is affected Mutations of germinal cells – carrying to offsprings – abortion or inborn deffects – Mutations affecting the germ cells are passed on to future generations. DNA Damage Metabolism Exogenous Endogenous DNA Replication Permanent Genetic Alteration Disease Cell Cycle Arrest DNA Repair Apoptosis DNA damage and repair 1. Direct reversals 2. Excision repair - Base Excision Repair (BER) - Nucleotide Excision Repair (NER) 3. Mismatch repair (MMR) - replication errors 4. Recombination repair (HR and NHEJ) - multiple pathways - double strand breaks and interstrand cross-links 5. Tolerance mechanisms - lesion bypass (TLS) - recombination DNA Repair Pathways Damage Recognized: Thymine dimers 6-4 photoproduct Gene Products Required: Photolyase Related disease: Photolyase not yet found in placental mammals T T Visible light T T Repair by Direct reversal: photoreactivation 1) Recognition and removal of lession 2) Gap filling 3) Joning DNA strands Steps 2 and 3 are same in other DNA repair pathways. Base Excision Repair (BER) Repair of deamination, oxidative damage and other small base lesions • Two pathways of global genomic repair • Transcription-coupled pathway • Many components of the global pathways are essential • Defective TCR causes Cockayne’s syndrome • Repairs wide variety of base damage - oxidative damage - alkylation damage - ionizing radiation damage - incorrect base (deamination of cytosine to uracil) - abasic sites - some types of UV damage Further reading: Fortini et al. Biochimie 85, 1053 (2003) Summary BER Nucleotide Excision Repair 1. Recognition by protein factors 2. Incision on each site from the lession (nuclease). 3. Unwinding of DNA (DNA- helicase) 4. Synthesis from the complementary strand (DNA polymerase δ a ε) 5. Joining DNA ends by DNA ligase Repair of dimmers, crosslinks and other helix distorting damage XPB & XPD - DNA helicases XPC - damage recognition XPA & RPA - damage validation & complex stabilization XPG - 3’ incision ERCC1-XPF - 5’ incision CSA & CSB - role in processing RNAP II? XPC not required Mammalian NER Pathways 5’ incision is 22 nuc. from lesion 3’ incision is 6 nuc. from lesion Cockayne’s Syndrome • Occurrence: 1 per million population • Genetic: autosomal recessive, genes (XPA, B, D & G) • Disorder: arrested development, mental retardation, dwarfism, deafness, optic atrophy, intracranial calcifications; (no increased risk of cancer) Genetics of NER Trichothiodystrophy • Occurrence: 1-2 per million population • Genetic: autosomal recessive, (TTDA, XPB a D) • Disorder: sulfur deficient brittle hair, mental and growth retardation, peculiar face with receding chin, ichthyosis; (no increased cancer risk) Xeroderma Pigmentosum • Occurrence: 1-4 per million population • Genetic: autosomal recessive, seven genes (XPA-G) • Disorder: multiple skin disorders; malignancies of the skin; neurological and ocular abnormalities • The human genome is 109bp (~ 2m) per cell • During the course of our lives we synthesise a ‘light year’ (1016m) of DNA • During this talk, we will each synthesise ~ 20 billion metres of DNA. • Mutations in any of a large number of genes can cause cancer... • ... yet 2/3 of us will not get cancer! Replication errors Mechanisms for Insuring Replicative Fidelity 1. Base pairing 10-1 to 10-2 2. DNA polymerases 10-5 to 10-6 - base selection - proofreading 3. Accessory proteins 10-7 - single strand binding protein 4. Mismatch correction 10-10 Further reading: A. Bellacosa, Cell Death and Differentiation 8, 1076 (2001) M. J. Schofield & P. Hsieh, Ann. Rev. Microbiol. 57, 579 (2003) DNA Mismatch Repair 1) MutS recognizes and binds at the site of a base pair mismatch 2) MutH is activataed to create a nick in newly synthesized strand 3) „Marked“ strand is removed by exonuclease, resynthesized by DNA polymeraze a joined by DNA ligáse. Mismatch Repair Mismatch Repair Mutations in Hereditary Nonpolyposis Colon Cancer (HNPCC) • MMR mutations in 70% of families • MLH1 (50%), MSH2 (40%) • Minor role for MSH6, PMS1, PMS2 • Population prevalence 1:2851 (15-74 years) • 18% of colorectal cancers under 45 years • 28% of colorectal cancers under 30 years Translesion Bypass DNA Polymerases Pol eta - inserts adenosines opposite TT dimers - in general has low fidelity - low processivity - may be error-prone with other lesions - Pol eta is a product of the XPV gene Pol zeta and Rev 1 - Rev 1 inserts random bases opposite dimer - Pol zeta extends bypass by a few bases - Both polymerases have low fidelity and low processivity Generation of DSB: • Induced by radiation & chemicals • During replication of damaged DNA template • Initiation of meiotic recombination • Part of immune reponse Failure to repair DSB: • Cell death • Chromosomal aberation • Meiotic aneuploidy • Immuno-deficiency Double-strand breaks (DSB) Recombinational repair Adapted from Surralles et al., Genes Dev. (2004) Recombination Translesion bypass Model of ICL repair (Interstrand CrossLinks) NER + FA Core Complex FancD2-Ub Brca2 Rad51, etc. MMR NER/HR Congenital abnormalities - skeletal - skin pigmentation - short stature - male genital - mental retardation - cardiac abnormalities - hearing Cancer - myeloid leukemia - solid tumors Review: Tischkowitz & Hodgson, J. Med. Genet. 40, 1 (2003) 15 genes in FA BRCA2 is deficient in FANCD1 Fanconi aneamie Summary for DNA Repair Pathway Error-free Error-prone Direct reversal + NER + BER + Gene conversion + SSA + BIR + NHEJ + MMR + Lesion bypass + + Mutations and cancer • 1016 cell divisions in human body during the live • Environments withouth mutagens 10-6 chance of mutation/cell division = 1010 mutations each day Cancer incidence as a function of age © Espero Publishing, s.r.o. !!! 1 mutation is not sufficient; most of mutations are repaired !!! Epigenetic aberrations and cancer corelation between increased histone acetylation and augmented transcription Global DNA hypomethylation – striking feature of neoplasiachromosomal instability, transcription of genes that were silenced DNA hypermethylation and chromatin hypoacetylation of tumoursupressor genes (RB1) - cancer Hypomethylation of specific genes –oncogenes Chromatine alterations – post-translational histone modifications Loss of imprinting – activation of the normally silenced allele or silencing of the normally active allele Mutations and cancer Future perspectives Future perspectives Synthetic lethal approach (i.e. BRCA2-patients with PARP inhibitors, MLH1-patients with Pol γ and β inhibitors) Thank you for your attention and take care of your DNA !!!! Epigenetic DNA modifications Epigenetic: something that affects a cell, organ or individual without directly affecting its DNA. Non-sequence-based alterations that are inherited through cell division DNA methylation – a covalent modification that can occur at cytosines within CpG-rich regions of DNA and is catalysed by DNA methyltransferases. Lower DNA accesibility to transcriptional complex – no transcription. Histone modifications – post-translational modification, i.e. acetylation, methylation, phosphorylation…