Chemical reactivity of DNA • DNA chemistry is derived from chemistry of its costituents • phosphodiester bonds • N-glycosidic bonds • deoxyribose • nitrogenous bases Chemical modification of DNA: • damage to the genetic material • analytical use DNA hydrolysis • both phosphodiester and N-glycosidic bonds susceptible to acid hydrolysis • N-glycosidic bond more stable toward hydrolysis in pyrimidine than in purine nucleosides (and more in ribo- than in deoxynucleosides) • stable in alkali (unlike RNA) • alkali-labile sites: upon DNA damage • enzymatic hydrolysis (N-glycosylases, nucleases, phosphodiesterases) Oxidation • two main sites susceptible to oxidation attacks: – C8 of purines (ROS) – C5-C6 of pyrimidines reactions with nucleophiles • C4 and C6 are centres of electron deficit in pyrimidine moieties • reaction with hydrazine: pyrazole derivative and urea residue bound to the sugar • with T the reaction is disfavored in high salt: Maxam-Gilbert sequencing technique reactions with nucleophiles • hydroxylamine: cytosine modification • the products‘ preferred tautomer pairs with adenine →mutagenic • bisulphite: cytosine modification inducing its deamination to uracil →mutagenic • 5-methyl cytosine does not give this reaction: genomic sequencing of 5^mC reactions with electrophiles • attacking N and/or O atoms • nitrous acid (HNO[2]) causes base deamination (C→U, A→I) – affecting base pairing, mutagenic • aldehydes: reactions with primary amino groups • formaldehyde: two step reaction DNA alkylation • hard or soft alkylating agents • hard attack both N and O atoms, soft only N • dimethyl sulfate: typical soft alkylating agent • N-alkyl-N-nitroso urea: typical hard alkylating agent • modifies all N + O in bases as well as phosphate groups (forming phosphotriesters) • analytical use (sequencing, foorprinting) Biological consequences of base alkylation • N-alkylation: the primary site = N7 of guanine (accessible in both ss and dsDNA) – does not change base pairing; easily repairable • N3 of adenine or guanine: located in minor groove – cytotoxic modification (DNA/RNA polymerization blocked) • N1 of guanine: interferes with base pairing • O-alkylation (G-O6, T-O6) the bases „locked“ in enol forms → improper base pairing → mutagenic • chloro- (bromo-) acetaldehyde: two reactive centres (aldehyde and alkylhalogenide) • reaction with C or A • chemical probes (react only with unpaired bases) • diethyl pyrocarbonate: acylation of purines (primarily A) at N7 • modification leads to opening of the imidazole ring • chemical DNA probing Metabolically activated carcinogens • some substances became toxic after their metabolic conversion • detoxifying machinery of the organism acts here as a bad fellow • microsomal hydroxylase complex, cytochrome P450 • the role of this system is to introduce suitable reactive groups into xenobiotics enabling their conjugation with other molecules followed by removal from the organism • but…. Metabolically activated carcinogens • aromatic nitrogenous compounds (amines, nitro- or azo- compounds): • aromatic amines are converted into either (safe) phenols, or (dangerous) hydroxylamine derivatives • azo- compounds: „cleaved“ into amines • nitro- compounds: reduced into hydroxylamines Metabolically activated carcinogens • polycyclic aromatic hydrocarbons like benzo[a]pyrene: three-step activation – P450 introduces epoxy group – epoxide hydrolase opens the epoxide circle – P450 introduces second epoxy group • DNA adduct formation (primarily -NH[2] of guanine, then G-N7, G-O6 and A-N6) • similar pathway of aflatoxin activation anticancer drugs • some types of antineoplastic agents act via formation of DNA adducts • metallodrugs: mainly platinum complexes cisplatin: reaction with DNA in certain sequence motifs other platinum complexes tested as cytostatics mitomycin C • reactive aziridine group, quinone group • reductive activation • bifunctional adducts Photochemical DNA modifications • mainly pyrimidines • excitation at 240-280 nm: reactive singlet state • water addition at C5-C6 • excitation at 260-280 nm: photodimerization of pyrimidines • photoproducts of C can deaminate to U (mutagenic effects) effects of ionizing radiation • mostly indirect – through water radiolysis • each 1,000 eV produces ~27 •OH radicals that attack DNA • sugar damage:abstraction of hydrogen atoms from C-H bonds • a series of steps resulting in strand breakage effects of ionizing radiation • base damage: hydroxylation and/or (under aerobic conditions) peroxylation chemical nucleases Chemical approaches in DNA studies (several examples) Maxam and Gilbert method of DNA sequencing • DNA fragment is end-labeled (radionuclide, fluorophore) • the sample is divided into four reactions (HCOOH, DMS, hydrazine, hydrazine + NaCl) • the conditions are chosen to reach only one modification event per DNA molecule single strand-selective chemical probes Open local structures in negatively supercoiled DNA Open local structures in negatively supercoiled DNA Open local structures in negatively supercoiled DNA Otevřené lokální struktury v negativně nadšroubovicové (sc) DNA Chemicals selectively reacting with unpaired bases: Using the Maxam-Gilbert technique, it is possible to determine with a high preciseness which nucleotides are forming the local structure DNA damage and repair Why is it important to study „DNA damage“? DNA: the genetic material ensuring • preservation of the genetic information • its transfer to progeny • its transcription and translation into proteins Damage to DNA may • lead to change of the genetic information (mutation) • affect gene expression • have severe health impacts DNA damage, mutation, lesion, mismatch…? • mutation may arise from (among others) DNA damage which is not repaired prior to DNA replication, e.g.. DNA damage, mutation, lesion, mismatch…? • mutations arise from unrepaired DNA damage (or from replication errors) • damaged 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) • DNA 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. Importance of DNA repair • estimated number of DNA-damage events in a single human cell: 10^4-10^6 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 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 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 10^5 nucleotide; their 3’→5’- exonuclease activity decreases incidence of the errors to 1:10^7 • the MMR contributes to replication fidelity by a factor of 10^3 by removal of base-base mismatches, insertions and deletions (hence the resulting incidence of mutations due to erroneous replication is only 1:10^10) • 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 • Techniques involving complete DNA hydrolysis followed by determination of damaged entities by chromatography or mass spectrometry • Techniques involving complete DNA hydrolysis followed by determination of damaged entities by chromatography or mass spectrometry • Monitoring of changes in whole (unhydrolyzed) DNA molecules: electrophoretic and immunochemical techniques to be continued: Electrochemical sensing of DNA damage tomorrow 11:00