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RNA and DNA Consist of Polymerized Nucleotides 5’-end O O O O 5 O 4 3 O O O P O 5’–PO 4 O P O O O O Base Base O O O Base O O O O O P O Base 1 2 P O O P O O O O O 3’–OH O P O O Base 2’–OH O Base O 3’-end Ribonucleic acid (RNA) Deoxyribonucleic acid (DNA) G. Magnusson 2003 Components of RNA and DNA O O O O P O N O O O N O O N P O N O O O P O O O P O O O P O O N O N N O O N N N N N dCMP O O O P O O N O O N O O N GMP N O O N N O O N O O CMP O O P O O dAMP O O O N O O AMP N N N O O N O N N O O O P O O N N dGMP O UMP N N O O dTMP G. Magnusson 2003 RNA Is More Labile Than DNA RNA is labile in a water solution because the phosphate groups can form phosphodiester bonds with both the 2’ and 3’ –OH groups of ribose. O O O O O P O O O P O O Base Base O O P O O O O P O O O O Base + O O O O P O O O O Base O Base O O P O O O O Base O O O G. Magnusson 2003 1 The Strands of DNA are Anti-Parallel 5’-end 3’-end O O O O P O O A T O O O O P P O O O G C O O O O O O O O O P P O O O O O 3’-end A T O O P O O O 5’-end G. Magnusson 2003 Structure of Double-Stranded DNA B-form of DNA Base-pairing of B DNA 10 base pairs 3.4 nm G. Magnusson 2003 The Size of Cellular DNA Escherichia coli (a bacterium) has one DNA molecule (chromosome) with a size of ca. 106 nucleotides Homo sapiens cells have 46 chromosomes, each consisting of one DNA molecule, with a total size of ca. 109 nucleotides G. Magnusson 2003 2 Nuclear DNA Forms Chromatin Histones are among the most conserved proteins in eukaryotes H2B H4 H4 H3 H3 H2A The nucleosome core is composed of an octamer of histone molecules. Two each of the histones H2A, H2B, H3 and H4. H1 is a linker histone. G. Magnusson 2003 Synthesis of DNA Enzymes catalysing the synthesis of DNA: DNA polymerases. DNA polymerases have some general properties q DNA molecules are extended at the 3’-end. DNA is synthesised in the 5’ to 3’ direction q Deoxynucleoside triphosphates (dATP, dCTP, dGTP, dTTP) are used as building blocks q The polymerisation reaction is directed by A–T, G–C base pairing to a template strand Primer 5’ 3’ Template strand G. Magnusson 2003 Polymerisation of deoxynucleotides O O P O O Growing strand ’Primer’ N O N O N N O O P O O P O O Mg2+ P G N N C N N O N O O O O Template strand T O N O O O N N N Active site of DNA polymerase O O N O P O O O N A N O N T N O N N A N N G. Magnusson 2003 3 Fidelity of DNA synthesis In mammals, the error frequency of DNA synthesis must be <10-9 The base pairing mechanism leads to one error in ~104 polymerised nucleotides G. Magnusson 2003 Reasons for Incorrect Base-pairing H H H N N Correct base-pairing N N N N N N H N N Tautomer of A can base pair with C instead of T G. Magnusson 2003 Correction of Replication Errors Proofreading activity of DNA polymerases 5’ Correct synthesis 3’ Template strand 5’ Misincorporated nucleotide 3’ Template strand 3’ to 5’ exonuclease activity of DNA polymerase catalyses the removal by hydrolysis of 3’-terminal nucleotides that are not base-paired to the template strand. 5’ 3’ Template strand With this mechanism the error frequency of DNA synthesis is decreased to ~10-6. G. Magnusson 2003 4 Correction of Replication Errors DNA polymerase I from E. coli has two catalytic activities, although the enzyme consists of a single polypeptide chain. Pol Pol Exo Exo View of a DNA polymerase bound to a primer-template junction in its synthesis (left) and proofreading (right) modes Pol denotes the polymerase active site; Exo, the 3’ to 5’exonuclease active site. From Baker & Bell (1998) Cell, 92:295 G. Magnusson 2003 Correction of Replication Errors Postreplication mismatch repair 5’ 3’ Template strand Methylated base In DNA, some of the C (and A) bases are methylated. Methylation is a slow process. Newly replicated DNA is undermethylated. Mismatch repair enzymes recognise mismatched nucleotides and remove the nucleotide in the undermethylated strand. 5’ 3’ Template strand The mismatch repair system increases the fidelity of DNA replication to ~10-9. G. Magnusson 2003 Replication of Chromosomal DNA Semiconservative DNA replication + A half-replicated DNA molecule What happens at the replication fork? G. Magnusson 2003 5 The Replication Fork, a Simplified View 5’ 3’ Template strand Newly synthesised DNA Lagging strand Discontinuous DNA synthesis DNA polymeras a/Primase DNA chain ~100 nucleotides RNA primers ~10 nucleotides DNA helicase Topoisomerase I RF-A, single-strand DNA binding protein Leading strand Continuous DNA synthesis DNA polymerase d with sliding clamp (PCNA) 5’ 3’ G. Magnusson 2003 Reaction Catalysed by DNA Topoisomerase I Tyr Topoisomerase I DNA OH Tyr H2N O O Rotation around intact DNA strand 5’ O P O O Tyrosine Tyr HO 3’ G. Magnusson 2003 The Replication Fork, a Simplified View 5’ 3’ Lagging strand Discontinuous DNA synthesis DNA polymeras a/Primase RNA primers ~10 nucleotides Template strand Newly synthesised DNA DNA chain ~100 nucleotides DNA helicase Topoisomerase I Leading strand Continuous DNA synthesis DNA polymerase d with sliding clamp (PCNA) RF-A, single-strand DNA binding protein 5’ 3’ G. Magnusson 2003 6 Lagging Strand Synthesis 5’ 3’ 5’ 3’ 5’ 3’ 4, 5 3 1, 2 DNA polymerase RNAase H DNA ligase 1. 2. 3. 4. 5. RNA primer synthesis: DNA pol a/Primase DNA chain synthesis: DNA pol a/Primase Removal or RNA primer: RNAase H Extension of DNA chain to close the gap: DNA polymerase Joining of DNA chains: DNA ligase 5’ 3’ G. Magnusson 2003 Reactions Catalysed by DNA Ligase O 1 E–lys–N+– P E–lys–NH2 + ATP N O O O O N O + O P O P O N O O N N O OH 2 O O P O O O O E–lys–N+– P N O O O O N N O O OH P O O P O O N N N N N O N O N O O O G. Magnusson 2003 Reactions Catalysed by DNA Ligase O 3 E–lys–NH3+ O O OH P O O O P O O O N N O P O O N N + O N O O O P O N O O N N N N O O G. Magnusson 2003 7 Replication of Chromosomal Ends (Telomers) RNA primer New DNA 5’ 3’ 5’ 3’ Template strand Degradation of RNA primer Loss of genetic information at chromosomal ends is prevented by the addition of nontemplated nucleotides by telomerase New RNA primer 5’ 3’ 5’ 3’ Telomerase product G. Magnusson 2003 Mechanism of Telomerase Activity Telomerase is a DNA polymerase that uses an RNA template. This template is a subunit of the enzyme and consists of an RNA molecule with a AAUCCC sequence repeat. Telomerase product 3’ GGGTTAGGGTTAGGGTTA AAUCCCAAUCCCAAU 5’ Telomerase RNA 5’ 3’ In the new-born, the telomeres have a length ~10~4 nucleotides with ca. 102 unpaired nucleotides at the 3’-end. G. Magnusson 2003 Initiation of DNA-Synthesis at an Origin From Bogan et al. (2000) J. Cell. Physiol. Vol. 184, 139 G. Magnusson 2003 8 Frequent Spontaneous DNA Damage O P O O O Frequency of individual lesions of nucleotides is suggested by the size of the arrows. N O NH2 N A N N O O P O O Depurination is the most frequent lesion ~104/day/cell. NH2 O Deamination of C to form U occurs ~103 times per day in a cell. N C N O O O P O O N O O N G N N O N O P O O O O N T N O O G. Magnusson 2003 UV-Induced Damage to DNA Absorption of UV-radiation (200–320 nm) by DNA induces two major types of potentially mutagenic lesions. Skin cells not protected by pigmentation accumulate ~104 lesions per hour when exposed to strong sun-light. O O N N N O PO4 N O OH N N N O PO4 O DNA backbone PO4 PO4 Cyclobutane thymine dimer kinks DNA 7°–9° N O PO4 PO4 Thymine (6–4) pyrimidone photoproduct kinks DNA 40° G. Magnusson 2003 Mechanisms of DNA Repair In the cell, DNA damage is recognised by specific proteins either as damage to individual bases or as distortion of the double helix. G. Magnusson 2003 9 Base Excision Repair of DNA DNA-glycosylase 5’ 3’ Damaged base e.g. uracil resulting from deamination of a C e.g. uracil-DNAglycosylase Apurinic/apyrimidinic endonuclease Excision endonuclease DNA polymerase b DNA ligase G. Magnusson 2003 Repair of Lesions Distorting the DNA Helix Global genome repair 5’ 3’ The XP factors were identified as a result of defective function in cells of individuals with the inheritable disease Xeroderma Pigmentosum DDB1 XPE XPC hHR23B Unwinding Transcription coupled repair TFIIH RNA pol XPF TFIIH XPA XPA XPG ERCC1 II RPA RPA Stabilised high specificity complex Incision 3’ and 5’ to lesion Excision of 27–29 nucl. Repair of patch DNA pol DNA ligase G. Magnusson 2003 Ames’ Mutagen Test • Salmonella bacteria requiring histidine for growth • Use strains with several different types of mutations in the his genes • Treat bacteria with test chemical, directly or after metabolic activation in liver cell extract • Plate bacteria on agar in histidine deficient medium • Count colonies and calculate mutation frequency G. Magnusson 2003 10 Mouse Carcinogen Test • Treat animals with test substance. For substances with low potency, use high doses or many animals • TD50 (mg/kg bodyweight/day during lifetime) calculated as the dose of chemical that would decrease the number of tumour-free animals by 50% • Tumours have to be analysed by a pathologist • TD50 for various substances has a range of >107 G. Magnusson 2003 Risk Assessment • All environmental risks cannot be avoided • There is no inherent difference in the toxicity of natural and synthetic substances • Human exposure has to be considered in the assessment of risk • HERP (human exposure rodent potency). Percentage of the rodent potency (TD50) received by a human during lifetime exposure G. Magnusson 2003 HERP HERP (%)a • 3.6 Beer, 500 ml (etyl alcohol, 23 ml/day) • 0.4 Typical indoor air (formaldehyde, 600 µg/day) • 1 x 1 0- 6 Lindaneb (32 ng/day) aA HERP value of 100 means that the exposure is identical to the TD50 in rodent tests. bLindane is a mutagenic insecticide that is banned from use in Swedish agriculture. G. 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