1 Understand the central dogma of molecular biology and some exceptions to the original Lecture 7 model Central Dogma DNA RNA protein Replication transcription translation Exceptions Retrovirus reverse transcription SARS Cov 2 RNA replication Prions infectious proteins Reverse Transcription RNA dependent DNA polymerase Makes DNA strand from existing RNA template RNase H Degrades RNA strand leaving DNA DNA dependent DNA polymerase Uses single stranded DNA for template to make double stranded cDNA 2 Know the essential features of DNA structures including the concept of base pairing antiparallel nature of strands in a double helix covalent bonds and hydrogen bonds in the structure of DNA Nucleotide Nitrogen base carbon sugar deoxyribose phosphate group Written 5 to 3 carbon of phosphoryl group to hydroxyl group of sugar Structure Double Helix Stabilized by hydrogen bonds hydrophobic stacking Edges of base pairs make hydrogen bond contacts Planar surfaces are hydrophobic base stacking Also includes van der waal interactions 10 ish base pairs per turn Complementary two strands in opposite directions Adenine forms 2 H bonds with thymine Guanine forms 3 H bonds with cytosine Chargaff s rule of A of T of G of C of purines of pyrimidines Phosphodiester Bond Covalent bond Between 3 hydroxyl of sugar and 5 carbon of other sugar near phosphate Glycosidic Bond Melting temp ssDNA bases In DNA is the linkage between 9 nitrogen purine or 1 nitrogen pyrimidine and the 1 carbon of the sugar group Covalent bond responsible for binding nitrogen base to backbone Temp where half of the DNA strands are in random coil or single stranded state DNA in B form have major and minor grooves Presence of grooves allows access for hydrogen binding capabilities of exposed Allows for sequence specific interactions in replication and transcription 3 Understand how intercalating agents inhibit DNA replication Intercalating Agents Distort double helix Several hydrophobic molecules containing aromatic rings that insert between stacked base pairs of DNA Cancer inducing reagents Ethidium bromide acridine orange actinomycin D Mostly in minor grooves of DNA 4 What are the basic structural components of nucleosomes Nucleosomes DNA is packaged with protein forming chromatin The eight histones of core particle are arranged as octamer DNA wraps outside octamer Nucleosome is the structure of histones DNA Are fundamental units of chromatin Linked in 30 nm fibers further folding generates the chromosome 5 Describe the enzymes involved in DNA replication and understand their roles Understand the differences between leading and lagging strand synthesis DNA Helicase Bind to single strand DNA and unwind the DNA double helix ATP replication fork Single Strand DNA binding protein SSB Bind to single strand DNA Keep single strand DNA separated Protect DNA from cleavage Primase Primes the replication using RNA primase DNA polymerase III Recognizes RNA primer and synthesizes DNA 5 to 3 Leading vs Lagging Strand Leading synthesized 5 to 3 Continuous and is replicated towards replication fork Lagging synthesized 5 to 3 but away from replication fork There are okazaki fragments Discontinuous as more DNA needs to be exposed to replicate away from replication fork multiple primases DNA polymerase I DNA Ligase Topoisomerases Removes RNA primers and fills the gaps in lagging strand Can also go 3 to 5 and proofread lagging strand Nicks are sealed Phosphodiester bonds between backbones are repaired Bond covalently to DNA phosphate and break the phosphodiester linkage Removes supercoils in DNA strand Type 1 creates temporary single strand nick to relieve stress Type 2 creates temporary double strand nick to relieve stress 6 Understand the key features of the reactions catalyzed by DNA polymerases Define template and primer as they relate to DNA polymerases 7 Relate the 3 5 nuclease activity of DNA polymerases to the fidelity of DNA replication by proofreading DNA polymerase III Can also go 3 to 5 and do exonuclease activity Proofreads and removes mismatched nucleotide 8 Understand how topoisomerase gyrase inhibitors can act as antibacterial agents and anticancer agents Know the topoisomerase targets of these drugs Topoisomerase gyrase Bacterial Ciprofloxacin gyrase and Quinolones both topoisomerase II trap these enzymes on the DNA strand not allowing them to re fix nicks Causes DNA to break and makes cell go under apoptosis Anti cancer Etoposide human topoisomerase II Camptothecin Indenoisoquinoline both topoisomerase I trap these enzymes not allowing them to fix nicks Nucleoside Analog Rnase H Modification on hydroxyl group at the 3 carbon of deoxyribose Removes RNA primers usually on lagging strand 9 Understand the DNA or RNA synthesis inhibitors as drugs e g in cancer and infection and the potential side effects from some of the drugs Chemotherapy Targets DNA replication aims to prevent replication and transcription by modifying DNA Cisplatin reacts with DNA guanine and disrupts DNA structure cross linking 2 purines and causing cell death Side effects occur in bone marrow gut epithelium skin and hair follicles 10 Understand how telomerase makes DNA of defined sequence in the absence of a DNA template Telomerase chromosome Elongated chromosomes by adding TTAGGG sequences to the end of existing AAUCCC is actually added from telomerase RNA sequence via reverse transcriptase in the 3 overhang Telomerase activity is enhanced in cancer cells as cells divide rapidly and need to preserve their telomeres 11 Understand the DNA synthesis catalyzed by reverse transcriptase 1 Understand the difference between transition and transversion mutation Lecture 8 Transition Mutation When one purine turns into a different purine Or when one pyrimidine turns into another pyrimidine Transversion Mutation When one purine becomes a pyrimidine Or when one pyrimidine becomes a purine 2 Know the different types of DNA damages Endogenous Damage from inside body Replication errors mismatches spontaneous loss of nucleosides base alteration transition of transversion C G pair to T A pair oxidative deamination transition Most common point mutation in cancer cells G C pair to T A oxidation transversion Alkylation chemical reaction that entails a transfer of an alkyl group Chemical exposure base alteration Exogenous Damage from outside body Chemical exposure base alteration Transversion G C to T A UV light exposure thymine dimer Ionizing radiation double strand break Chromosomal