Lecture 7 DNA Structure and Replication 1 Understand the central dogma of molecular biology and some exceptions to the original model The Central Dogma of molecular biology was made by Francis Crick in 1958 While some of it remains true some parts have been proven wrong The central Dogma says two primary things The first is that information is transferred residue by residue which is always true I believe Second it says that information flows in a singular direction From DNA to RNA to protein This second part of the central dogma has many expectations Some of which include reverse transcription RNA DNA prions proteins proteins and RNA replication RNA RNA Covid 19 was an example of RNA replication 2 Know the essential features of DNA structure including the i concept of base pairing ii antiparallel nature of strands in a double helix iii covalent bonds and hydrogen bonds in the structure of DNA Some essential features of DNA include Base Pairing Base stacking is induced predominantly by the hydrophobic effect and also some Van der Waals interactions The bases themselves are also hydrophobic Adenine always bonds with Thymine and there are two hydrogen bonds between them And Guanine always bonds with Cytosine with 3 hydrogen bonds Note that this means that it is energetically easier to break A T bonds than C G Chargaff s rule states that the numbers of T and A are equal and the numbers of C and G are equal So if we are given the amount of one base in a sample of DNA we can easily calculate the amounts of the other three bases Antiparallel strands This means that DNA strands are complementary They run in opposite directions and have opposite bases This allows A to bond with T and G to bond with C The two helices also run in opposite directions One runs 5 to 3 and the other 3 to 5 Covalent and hydrogen bonds DNA has two covalent bonds that we discussed One is the phosphodiester bonds These run along the backbone of the DNA and connect the phosphate groups to two deoxyribose sugar groups each one of which is connected at the 3 position and the other is at the 5 placement The second are glycosidic bonds These are in the inside of the helix and connect the base to the sugar This is done by linking the base at the 9 Nitrogen position of purine bases or the 1 Nitrogen position of pyrimidine bases to the 1 carbon on the pentose sugars DNA also has hydrogen bonds between base pairs A T has two h bonds and the G C bonds have three hydrogen bonds These H bonds form major and minor grooves in DNA which allows access to the hydrogen bonding capabilities of the now exposed bases This provides the means for DNA s interactions with other molecules 3 Understand how intercalating agents inhibit DNA replication Intercalating agents distort the double helix Intercalating agents are non covalent aka hydrophobic molecules that contain things like flat aromatic and fused heterocyclic rings These rings insert themselves between the stacked base pairs of DNA This predominantly occurs in the minor grooves of DNA This can cause cancer as it inhibits DNA replication Some intercalating agents are ethidium bromide acridine orange and actinomycin D 4 What are the basic structural components of a nucleosome A Nucleosome aka beads on a string consists of histones wrapped in DNA that has been packaged with a protein DNA that is packaged with this protein is called chromatin Eight histones make up the octamer core of the nucleosome Something called nucleofilament is what the core of the histones are made of And this nucleofilament is coiled and anchored to scaffold proteins when it is not part of a nucleosome Nucleosomes are arranged in 30 nm fibers and wound into two interconnected left handed helical stacks When they are folded more they become chromosomes 5 Describe the enzymes involved in DNA replication and understand their roles Understand the differences between leading and lagging strand synthesis The leading strand is synthesized continuously as it has an available 3 to add to The lagging strand is synthesized discontinuously as it has an available 5 rather than 3 This causes some complications and the DNA must be made in small okazaki segments discontinuously These segments are then sealed with polymerase and then ligase DNA Helicase binds to single strands of DNA and functions to unwind the double helix like a zipper Single Strand Binding proteins Bind to single separated DNA strands They keep the DNA strands separate and also protect the DNA from cleavage DNA polymerase DNA Polymerase adds nucleotides down like a track to existing nucleic acid strands It can t add a nucleotide without an existing nucleic acid to add to It always attaches a new unit to the 3 OH group of the last sugar Another way to say that is that DNA polymerase only makes DNA 5 3 Primase synthesis RNA primer 6 Understand the key features of the reactions catalyzed by DNA polymerases Define template and primer as they relate to DNA polymerases The RNA sequence acts as a template for DNA DNA must lay a primer as in a first unit of a nucleotide down to attach the next one tp DNA Polymerase I removes RNA primers and fills the leftover gap DNA polymerase II does two things It has a polymerase activity of DNA synthesis which occurs 5 3 And it also has an exonuclease activity of proofreading in which it can remove units 3 5 This allows DNA polymerase II to remove incorrectly incorporated nucleotides 7 Relate the 3 5 nuclease activity of DNA polymerases to the fidelity of DNA replication by proofreading DNA nucleotide units can be removed from the strand 3 5 This is done to remove an incorrect base This can be due to base pair mismatching flipped bases etc When even one base is wrongly incorporated the DNA is mutated and its proliferating abilities can be lost DNA polymerase II proofreads the DNA and can repair wrongly incorporated bases 8 Understand how topoisomerase gyrase inhibitors can act as antibacterial agents and anticancer agents Know the topoisomerase targets of these drugs Topoisomerases bond covalently to DNA phosphate as they break the phosphodiester linkages between adjacent nucleotides Some antimicrobial agents that target topoisomerases are ciprofloxacin and quinolones These drugs stop topoisomerases from relaxing supercoils Ciprofloxacin DNA gyrase topoisomerase II Quinolones Topoisomerase II 9 Understand the DNA or RNA synthesis inhibitors as drugs e g in cancer and infection and the potential side effects for some of these