Lecture 1 Central Dogma detailed residue by residue transfer of sequential information Information cannot be transferred back Chargaff s rules amt of A T C G puring pyrimidine temperature of DNA strands are in random coil or single stranded ssDNA Intercalating agents Hydrophobic molecules containing flat aromatic and fused heterocyclic rings between heterocyclic rings Dina Pang DNA structure and DNA replication Structure Nitrogen base carbon sugar deoxyribose phosphate group Chains have directionality one end is phosphoryl group 5 carbon atom one end has free hydroxyl attached to 3 carbon Chains are written 5 to 3 Double Helix Stabilized by hydrogen bonds and hydrophobic stacking Edges of base pairs make hydrogen bond Hydrophobic effect base stacking van der waals A T forms 2 hydrogen bonds G C forms 3 hydrogen bonds Phosphodiester glycosidic bonds 9 nitrogen of purine bases or 1 nitrogen of pyrimidine bases 1 carbon of the sugar group Enzymes Glutamine phosphoribosyl pyrophosphate amidotransferase purine oroate phosphoribosyltransferase pyrimidine Major Minor Grooves are Lined by sequence specific Hydrogen bonding groups In B form has major minor groove Presence allows access to hydrogen bonding capabilities of the exposed bases Hydrogen bonding provides means of sequence specific interactions Intercalating Agents Distort the Double Helix Intercalating agents potential Cancer inducing reagents Intercalating agents that insert between DNA base pairs Ethidium bromide acridine orange actinomycin D Steps in prokaryotic DNA synthesis Separation of the complementary DNA strands Problem 1 where to open double helix Origin AT rich recognized by DnaA Replication is bidirectional and semi conservative Formation of replication fork Problem 2 how to open double helix DNA helicase Problem 3 how to maintain opened DNA Single strand DNA binding protein SSB Problem 4 how to start DNA replication Priming the replication Action of DNA Polymerase Needs a primer to add onto an existing nucleic acid Key is 3 OH group DNA polymerase will only synthesize DNA from 5 to 3 Elongation of the leading and lagging strands Problem 5 how to extend the polynucleotide chain Leading strand 5 3 Continuously Lagging strand 3 5 DIscontinuously Removal of RNA primer and filling of the gaps Problem 6 how to fill in the gap in the lagging strand Fill in the gap polymerase DNA polymerase I remove RNA primers 5 3 exonuclease fill gap Seal the nick ligase DNA polymerase III Problem 8 How to deal with incorrectly incorporated nucleotides Polymerase II has two roles 5 3 polymerase activity DNA synthesis 3 5 exonuclease activity proofreading Problem 9 how to take care of the supercoils DNA topoisomerase break the phosphodiester linkage Type I and Type II Antimicrobial Ciprofloxacin Quinolones Eukaryotic DNA Replication DIfferences between prokaryotic eukaryotic multiple origins vs single origin S phase in cell cycle Also differences by multiple enzymes Rnase H remove H primers Topoisomerase Anti cancer drugs Etoposide human topoisomerase II Camptothecin topoisomerase I Indenisoquinoline topoisomerase I Assembly of DNA into Chromosomes Eukaryotic DNA is found packaged with protein forming a substance called chromatin Eight histones in core of particle composed of octamer DNA wrap outside of octamer The Compaction of DNA into a eukaryotic chromosome 1 Naked DNA 2 Nucleosome beads 3 Nucleofilament 30 nm fiber 4 Nucleofilament is coiled and anchored to scaffold proteins Nucleosomes in 30 nm fibers arranged into 2 interwound left handed helical stacks which generate chromosome Problem 10 how to replicated the ned of DNA of the lagging strand Removal of RNA primer leads to the shorting of the chromosome after each round of replication Telomerase is an enzyme made of protein and RNA subunits Telomeric DNA TTAGGG AAUCCC Telomerase and Cancer Low telomerase activity is normal in somatic cells without telomerase chromosomes are shortened each time cell divides High telomerase activity in cancer cells DNA synthesis via reverse transcription a RNA dependent DNA polymerase RIbonuclease H DNA dependent DNA polymerase i High affinity for RT 100 fold higher no repair system to remove it Inhibition of DNA synthesis modified hydroxyl group at 3 carbon Cancer Chemotherapy target DNA replication Methotrexate 5 fluorouracil Cisplatin reacts w DNA nitrogens of adjacent purines guanine replaced with chloride atoms cross linking two purines and leads to cell death Highly proliferative cells bone marrow gut epithelium skin and hair follicles side effects of chemotherapy Know essential DNA structure how intercalating agents inhibit DNA replication DNA replication cycle and key mediators key features of reactions catalyzed by DNA polymerases know DNA replication inhibitors understand DNA synthesis catalyzed by reverse transcriptase Antiviral herpes complex vidarabine Anticancer leukemia cytarabine Summary Lecture 2 Dina Pang DNA Damage Repair and Cancer Chromosomal mutations segmental mutations involve changes to the entire chromosome or sections of it Point mutations changes to one or few base pairs in DNA in form of deletion substitution or insertion Mismatches errors in DNa replication Transition mutations replace a purine or a pyrimidine with a pyrimidine Mutations 20 000 DNA damaging events per cell per day Chromosomal mutation 1 Point mutations DNA replication transition between pyrimidines is a During mismatch DNA polymerase delta 3 5 exonuclease methylation deamination 2 Base alteration spontaneous G T A T transition mutation a Oxidative deamination Most common point mutation in cancer b Oxidation ROS such as hydroxyl radical Guanine oxidized into 8 oxoguanine which binds to A guanine has lowest oxidation potential cells i proofreading c ALkylation G C T A i ii cytochrome P450 into rxn with dna by a free radical Methylation is the most common type of alkylation transfer of methyl group CH3 3 methylcytosine Deamination oxidation methylation and depurination alter thousands of bases per cell genome each day by exogenous mutagenic agents d Depurination destabilize the covalent bond to deoxyribose resulting in the loss of the purine or pyrimidine base from DNA AP sites misread by DNA polymerase Thymine dimer and break 3 Thymine dimer UV radiation leads to cross linking of pyrimidines 4 Double strand break ionizing radiations and free radical products Cellular Responses to DNA Damage Cell cycle activation DNA repair Dna repair base excision