DNA damage and repair nDNA: the genetic material ensuring n npreservation of the genetic information nits transfer to progeny nits transcription and translation into proteins n nDamage to DNA may n nlead to change of the genetic information (mutation) naffect gene expression nhave severe health impacts Why is it important to study „DNA damage“? DNA damage, mutation, lesion, mismatch…? nmutation may arise from (among others) DNA damage which is not repaired prior to DNA replication, e.g.. n AGCCGATTAACTTAGCTTAGT TCGGCTAATTGAATCGAATCT AGCCGATTAGCTTAGCTTAGT TCGGCTAATCGAATCGAATCT semi-conservative replication (two cycles without repair) AGCCGATTAGCTTAGCTTAGT TCGGCTAATCGAATCGAATCT „wild type“ (homo)duplex DNA „wild type“ homoduplex mutated homoduplex AGCCGATTAGCTTAGCTTAGT TCGGCTAATUGAATCGAATCT cytosine deamination heteroduplex containing a single base mismatch DNA damage lesion/mismatch mutation (base pair substituted) nmutations arise from unrepaired DNA damage (or from replication errors) ndamaged DNA is not mutated yet! (damage is usually repaired in time i.e. before replication – lesions and/or mismatches are recognized by the reparation systems) nDNA with mutated nucleotide sequence does not behave as damaged! All base pairs in such DNA are „OK“ (no business for the DNA repair machinery) but the genetic information is (hereditably) altered. DNA damage, mutation, lesion, mismatch…? DNA in the cells is permanently exposed to various chemical or physical agents Ø endogenous - products and intermediates of metabolism Ø exogenous - environmental (radiation, pollutants) Scharer, O. D. (2003) Chemistry and biology of DNA repair, Angew. Chem. Int. Ed. 42, 2946-74. single-strand break double-strand break interruptions of DNA sugar-phosphate backbone abasic sites interruption of the N-glykosidic linkage Øreactive oxygen species Øaction of nucleases Øconsequence of base damage Ø Øspontaneous hydrolysis (depurination) Øconsequence of base damage Ø Most frequent products of DNA damage („lesions“) guanin adenin cytosin thymin base damage: chemical modifications Øalkylation Øoxidative damage Ødeamination Ødamage by UV radiation (sunlight) Ømetabilically activated carcinogens Øanticancer drugs Ø Ø Ø Most frequent products of DNA damage („lesions“) •estimated number of DNA-damage events in a single human cell: 104-106 per day!! •only a small number of base pairs alterations in the genome are in principle sufficient for the induction of cancer •DNA-repair systems must effectively counteract this threat •in an adult human (1012 cells) about 1016–1018 repair events per day • Importance of DNA repair p53 and others DNA damage cell cycle arrest DNA replication postponed until DNA repair apoptosis only then DNA replication followed by cell division damaged cell eliminated genomic instability mutations cancer… if unrepairable? if everything fails DNA repair pathways •direct reversal of damage •base excision repair •nucleotide excision repair •mismatch repair •repair of double strand breaks Direct reversal of DNA damage •photolyases: repair of cyclobutane dimers • • • • •O6-alkylguanine transferase: reverses O6-alkylguanine to guanine by transferring the alkyl group from DNA to a reactive cysteine group of the protein • UV photolyase Base excision repair •repair of damage by deamination (U, I), oxidation (8-oxoG), and alkylation •initiated by DNA glycosylases, which recognize damaged bases and excise them from DNA by hydrolyzing the N-glycosidic bond •substrate specificity of the glycosylases: developed to repair expectable „errors“? •second enzyme is AP-lyase introducing single strand break next to the abasic site •replacement of the abasic sugar by proper nucleotide •sealing the break • • • Nucleotide excision repair •removes bulky base adducts (such as those formed by UV light, various environmental mutagens, and certain chemotherapeutic agents) from DNA •broad substrate specificity: dealing with unexpected environmental DNA damaging agents •excision of the damaged oligonucleotide •then filling the gap & the sealing break • • • Mismatch repair •dealing with replication errors •polymerases introduce about one erroneous nucleotide per 105 nucleotide; their 3’→5’- exonuclease activity decreases incidence of the errors to 1:107 •the MMR contributes to replication fidelity by a factor of 103 by removal of base-base mismatches, insertions and deletions (hence the resulting incidence of mutations due to erroneous replication is only 1:1010) •the system must be able discrimitate between parental and daughter DNA strand! •MutS binds to mismatches and insertion/deletion loops •„repairosome“ formation, removal of a part of the daughter strand by 5’→3’- exonuclease •new DNA synthesis and ligation Repair of double strand breaks •consequences of DSBs can be very severe (chromosome aberrations) •two repair pathways: •homologous recombination: an intrinsically accurate repair pathway that uses regions of DNA homology (such as sister chromatids) as coding information. • • Repair of double strand breaks •consequences of DSBs can be very severe (chromosome aberrations) •two repair pathways: •non-homologous end joining: conceptually simple pathway that involves the religation of broken ends (without using a homologous template •less accurate: may loss of a few nucleotides at the damaged DNA ends • • Examples of techniques used to detect DNA damage 1.Techniques involving complete DNA hydrolysis followed by determination of damaged entities by chromatography or mass spectrometry HPLC: 8-oxo guanine determination fig1 fig3 32P-postlabeling: analysis of base adducts 1.Techniques involving complete DNA hydrolysis followed by determination of damaged entities by chromatography or mass spectrometry 2. 2.Monitoring of changes in whole (unhydrolyzed) DNA molecules: electrophoretic and immunochemical techniques detection of strand breaks: relaxation (and/or linearization) of plasmid supercoiled DNA scDNA (intact) ocDNA (ssb) linDNA (dsb) (damaged) flcomets 3dna „comet assay“ (dsb) „alkaline elution assay“ (ssb + alkali-labile sites) imunochemical techniques when antibodies against the adducts available ØELISA Ø ØIn situ techniques 8-oxo guanine detection in situ in kidney tissue