Chemical reactivity of nucleic acids Chemical methods in DNA studies DNA damage Miroslav Fojta Olsztyn-Lańsk, September 19th, 2007 Institute of Biophysics Department of Biophysical Chemistry and Molecular Oncology Centre of Biophysical Chemistry, Bioelectrochemistry and Bioanalysis Chemical reactivity of DNA •DNA chemistry is derived from chemistry of its costituents •phosphodiester bonds •N-glycosidic bonds •deoxyribose •nitrogenous bases • 2-deoxyribose phosphate •Chemical modification of DNA: • •damage to the genetic material • •analytical use •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) • DNA hydrolysis •two main sites susceptible to oxidation attacks: • – –C8 of purines (ROS) – –C5-C6 of pyrimidines Oxidation + 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 5mC • reactions with electrophiles •attacking N and/or O atoms •nitrous acid (HNO2) 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 ones 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 -NH2 of guanine, then G-N7, G-O6 and A-N6) • •similar pathway of • aflatoxin activation – bay region anticancer drugs •some types of antineoplastic agents act via formation of DNA adducts •metallodrugs: mainly platinum complexes (ineffective) cisplatin: reaction with DNA in certain sequence motifs some adduct types preferred (and/or more stable than others) 1,2-GG and 1,2-AG IACs = the main cytotoxic lesions 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 pyrimidine products purine products (mainly C8 hydroxylation or opening of the imidazole ring) chemical nucleases species containing redox active metal ions mediating production of hydroxyl radicals (or othe reactive oxygen species) via Fenton and/or Haber-Weiss processes bleomycine Fe or Cu Men + H2O2 → Men+1 + •OH + OH- iron/EDTA complex Cu(phen)2 complex Chemical approaches in DNA studies (several examples) Maxam and Gilbert method of DNA sequencing HCOOH (acid depurination) DMS (preferential methylation of G at N7; spontaneous depurination) G A T C hydrazine (modification &breakdown of the pyrimidine ring) hydrazine+NaCl (selective modification of cytosine) at sites of base modification (removal) the sugar-phosphate backobone is labile towards alkali treatment with hot piperidine → cleavage at such sites •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 HCOOH G A T C G A T C A T C G A T C G T C G A T C G A T C A T C G A T C G T C or or or only the „subfragment“ bearing the label is „visible“ in the following detection step G A T C A T C piperidine G A T C A T C G A T C G A T C A T C G A T C T C G A T C A T C T C G A T C G A T C Sangerovo („dideoxy“) sekvenování značení a sekvenování DNA F. Sanger 2‘,3‘-dideoxynukleotidy: terminátory syntézy DNA (není kam napojit další nukleotid) značené dideoxy: navázání značky podle komplementární báze napojování nukleotidů přes 5‘-OH a 3‘-OH templát primer syntetizovaný úsek Syntéza DNA in vitro („primer extension“) Deoxynukleotid trifosfáty (dNTP) DNA polymeráza templát primer ZNAČENÉ dNTP DNA polymeráza vložení značek do DNA pomocí DNA polymeráz template primer ZNAČENÝ ddNTP +normální dNTP DNA polymeráza vložení značek do DNA pomocí DNA polymeráz značení a sekvenování DNA F. Sanger -směs normálních deoxy a značených dideoxy -pro každou bázi jiná barva -různě dlouhé produkty, dělení elektroforézou -značka (barva) odpovídá koncové bázi templát primer Detekce a identifikace mutací a polymorfismů -důležité pro diagnostiku (určité mutace v určitých pozicích jsou spojeny s určitým onemocněním) -zjišťuje se, jaká báze je v konkrétní pozici G G C C T A T A DNA „footprinting“: determination of binding sites of other molecules (e.g. proteins) at the DNA sequence level complex free DNA DMS (guanine) piperidine DNaseI UO2 UV Cu(phen)2 OH radicals single strand-selective chemical probes Open local structures in negatively supercoiled DNA relaxed circular DNA negatively supercoiled DNA (linking deficit) stress related to the negative superhelicity (the linking deficit) can be absorbed in local open structures DNA segments of specific sequence can adopt „alternative“ local structures cruciform DNA (inverted repeat) Open local structures in negatively supercoiled DNA unpaired bases Triplex DNA (homopurine·homopyrimidine stretch with mirror symmetry) e.g. TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT (intermolecular triplex) Open local structures in negatively supercoiled DNA Otevřené lokální struktury v negativně nadšroubovicové (sc) DNA Intramolecular triplex (homoPu•homoPy segment within negatively supercoiled DNA) T T T T T T A A A A A A A Chemicals selectively reacting with unpaired bases: osmium tetroxide complexes (Os,L) (T, more slowly C) chloroacetaldehyde (CAA) (A, C) diethyl pyrocarbonate (DEPC) (A, G) footprinting of CGC+ triplex by DMS Using the Maxam-Gilbert technique, it is possible to determine with a high preciseness which nucleotides are forming the local structure Ø modification of supercoiled DNA Ø restriction cleavage, radiactive labeling Ø hot piperidine Ø sequencing PAGE the structure can be deduced from the modification pattern TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 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