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Purdue BIOL 10200 - DNA and Replication Notes

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Nucleic Acids and Bases:In 1869, Swiss chemist Friedrich Miescher discovered a white substance from the nuclei thatwas slightly acidic, so he called it nucleic acid.DNA contains 3 main parts1. A 5-carbon sugar2. A phosphate group3. A nitrogenous (nitrogen-containing) base.The base can be a large purine (A / G) or a small pyrimidine (T / C).C and T form two hydrogen bonds (and are therefore easier to melt)G and A form three (and are therefore harder to melt)We refer to the five carbons on a 5-carbon sugar with the prime (‘) symbol. In DNA, thephosphate connects to the 5’ carbon atom and the base connects to the 1’. A free hydroxyl(-OH) is connected to the 3’ carbon atom.The phosphodiester bond links the hydroxyl of the 3’ atom to the phosphate of the 5’ atom. Itis called that because the phosphate is now linked by way of ester bonds. This forms long acidpolymersErwin Chargaff discovered that DNA isn’t constant. It’s complex, and it varies a lot, but italways has the same amount of A and T, and it always has the same amount of C and G. Soeven though one species might have more C than the other, there will always be G to match itUsing x-ray diffraction (where crystals of a molecule are bombarded with X-rays), we provedthat DNA is a helix shape.The different structural forms of bases (called tautomers) are due to whether they have keto(C=O) versus enol (C-OH) and whether they have amino (-NH2) versus imino (C=NH)The two strands of the helix are made of repeating sugar and phosphate units, which we call thephosphodiester backboneA single phosphodiester strand has inherent polarity because the 3’ side ends with OH and the5’ side ends with PO4. Thus, they are referred to as having 3-to-5 polarity or 5-to-3 polarity.Strands are antiparallel meaning one side runs 3-to-5 and another runs 5-to-3DNA Replication Models:There are three main models of DNA replication, but they all begin with a two-strand parent andthey all end in a pair of two-strand daughters (four strands total)1. A conservative model where the original strand is preserved alongside the copy2. A semiconservative model is where each daughter has one strand of the copy and onestrand of the original3. A dispersive model is where bits and pieces of the original and copy are dispersed ineach of the four strandsThe Meselson-Stahl experiment supported the semiconservative model.They took a batch of bacteria, replicated it and replicated it again. Here’s the thing: after the firstreplication, a strand of the bacteria (15N) turns into a strand of the much lighter 14N.So basically, after the first replication, their two-strand, heavy 15N helix now had a copy thatwas one heavy strand and one light strand.When they replicated it again during the second round: the heavy strand once again became alight strand and a heavy strand; and the light strand became two light strands.DNA replication requires something to copy (DNA), something to do the copying (enzymes) andbuilding blocks to make the copy (nucleoside triphosphates)The three stages are:1. Initiation process begins2. Elongation majority of building blocks are added3. Termination process endsProkaryotic ReplicationProkaryotic replication starts at a single origin; this origin + a chromosome = the replicon.This is not only the origin of replication but where the replication is controlled. The textbook usesE Coli as an example, and its origin is called oriC.Prokaryotes have one replicon, eukaryotes have multipleDNA polymerase are a class of enzymes that use DNA template to assemble a newcomplimentary strand, but they only assemble in the 5-to-3 direction and they require a primerOther times, polymerases can act as a nuclease and cleave the nucleotide chain into smalleracids. Either they are an endonuclease (and can do it from inside) or an exonuclease (whichcan only do it at the end on the outside).The textbook covers three main DNA polymerases:● DNA POL III is the main replication enzyme● DNA POL I removes primers and replaces them with DNA● DNA POL II is involved in repairA helicase is an enzyme that unwinds the DNA to replicate itWhen unwound, the helix undergoes a form of strain called torsional strain and it supercoilsTopology studies how forms twist and coil so this supercoiled state of DNA is called theTopological StateTopoisomerases are enzymes that relieve the torsional strain and prevent supercoiling. DNAgyrase is the topoisomerase involved in DNA replicationBecause they are antiparallel, DNA strands must be synthesized in opposite directions. Onestrand (the leading strand) is synthesized continuously from a single primer, and the otherstrand (the lagging strand) is synthesized discontinuously (in short bursts) from lots of primers.Each fragment of the lagging strand is called an Okazaki fragment. The Okazaki fragments arethen joined by a DNA ligaseDNA having to be opened up slightly to be replicated creates a shape called replication forkThe enzyme DNA primase synthesizes primers, which attracts DNA polymerases (whichcreates a new strand to go with one of the split-off template strands)All of these many enzymes involved in DNA replication come together as a macromoleculecalled the replisomeEukaryotic ReplicationEukaryotic replication is complicated by linear chromosomes and large amounts of DNA.The process and the replisome is different for a few reasons:● There are multiple origins and multiple replicons, but each origin can only be used onceper cycle.● The replication fork is more complex● The Okazaki fragments are shorter● The ends of eukaryotic chromosomes have special structures called telomeres● Eukaryotic replications proteins are rooted in evolution● There is a gradual shortening of chromosomes with each round of cell divisionTelomeres are made by an enzyme called telomerase. This activity is high but it goes downduring old age, causing the ends of chromosomes to get

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