Slide 1Slide 2Relaxing DNATwo Types of TopoisomerasesSlide 5DNA Gyrase is a special Type II TopoisomeraseSlide 7Two Types of TopoisomerasesType I TopoisomeraseSlide 10Slide 11Supercoiling Affects Electrophoretic MobilitySlide 13Clicker QuestionSlide 15All Cells Require TopoisomerasesSlide 17Slide 18The Bacterial ChromosomeThe Compaction ProblemDNA in eukaryotic cells exists as chromatinForms of Chromatin (Electron Micrographs)Slide 23Slide 24Eukaryotic Chromosomes are Packaged Around HistonesNucleosomeSlide 27Slide 28Nucleosome StructureH1 Histone and Histone Tails are Required to Form 30 nm FibersH1 and the 30 nm FiberSlide 32Slide 33Slide 34Slide 35Slide 36Are Eukaryotic Chromosomes Supercoiled?Wrapping of DNA Around Histones Introduces Negative SupercoilsSlide 39Slide 40Scale of the ProblemDNA Replication is SemiconservativeSlide 43Replication proceeds 5’ to 3’The substrates for DNA replication are dNTPsChemistry of DNA ReplicationSlide 47So, can Igo to thelake?Remind him that I could really use some relaxation!Let me see what your Dad thinks.If he relaxes any more, his DNA will unwind.MCB 250 Lecture 10 Chromosome StructureDNA Replication - 1Relaxing DNAFig 4-19Two Types of Topoisomerases •Type II Topoisomerase–Example: E. coli TopoII, TopoIV (note even number)–ATP-Dependent – energy is required –Passes BOTH strands through another DNA double strand–Changes Linking number by 2–Covalent intermediate 5’phosphate to tyrosine–Except for Gyrase these enzymes act to relax supercoiled DNA (i.e., to remove supercoils)Fig 4-21Type II TopoisomeraseDNA Gyrase is a special Type II Topoisomerase•Adds negative supercoils •Changes linking number (Lk) by -2 each time it acts•Found only in bacteria•Negative supercoils introduced by gyrase are important for facilitating opening of the double helix for replication and transcription.•Target of quinolone antibiotics, such as ciprofloxacinGyraseTwo Types of Topoisomerases •Type I Topoisomerases–Example: E. coli TopoI and TopoIII (note odd numbers)–Relax DNA – fewer supercoils–ATP-independent –Pass ONE strand through the other–Change linking number by 1–Covalent intermediate: 5’phosphotyrosine linkage to the enzymeType I TopoisomeraseFig 4-22Mechanism of Type I TopoisomeraseMechanism of Type I TopoisomeraseLike Fig 4-24Supercoiling Affects Electrophoretic MobilityNicked CircleLinearSupercoiledRemember: Movement is dependent on charge and SHAPEA solution of DNA containing highly supercoiled DNA and relaxed DNA is treated with a type I topoisomerase.Time 0 The Lk of each band differs from the next band by 1.Clicker Question1 2 3 4You start with sample of plasmid isolated from E. coli. Lane 1 shows this sample run on a gel and stained with ethidium bromide. You treat one aliquot with Topo I and another aliquot with Gyrase (and ATP) and runs these samples on a gel. Which lanes best represent the expected results? Topo I GyraseA Lane 1 4B 2 3C 3 1D 4 2A. Relax supercoiled DNA (or, for gyrase only, add negative supercoils)Functions of TopoisomerasesB. Untangle knotsC. Resolve (decatenate) catenanesAll Cells Require TopoisomerasesE. coli has 4: 2 type I’s and 2 type II’sMammals have 6 and 5 of them are essential.Not all topos do all of the reactions on the previous slide equally well, some are specialized. Example: E. coli TopoI specifically removes negative supercoils, TopoIV is good at resolving catenanes.Like Table 8-1E. coli ChromosomeSize of Intact CellDNA is 1 mmCell is 1 mmThe Bacterial Chromosome•The E. coli chromosome is a closed circle (a few bacteria have linear chromosomes).•Bacteria have histone-like proteins but there is not the repeating higher order structure seen in eukaryotes.•There is a higher order structure or scaffolding that is apparent in electron micrographs – “Nucleoid”.•Nobody understands the nature of this scaffolding–Unknown proteins?–Gyrase/TopoI?The structure is not static.–There is no evidence that the same piece of DNA is always associated with the scaffold–All regions of the chromosome can apparently find any other regionThe Compaction Problem•The compaction problem is even greater for eukaryotic cells than for prokaryotes.•A human cell contains 3 x 109 bps per haploid set of chromosomes. 3 x 109 bp x 3.4 Å/bp = 1010Å = 1m. In a diploid cell there will be 2 meters of DNA to accommodate in a nucleus of diameter 10 - 15 m.DNA in eukaryotic cells exists as chromatin•Chromatin: the complex of DNA and proteins found in the nuclei of eukaryotic cells•Histones are the major proteins found associated with eukaryotic DNA.•Many non-histone proteins are also present.•Half of the mass of a eukaryotic chromosome is protein.Forms of Chromatin (Electron Micrographs)Chromatin isolated at low saltChromatin isolated at physiological salt (0.15M)Fig 8-17Compaction States of Eukaryotic DNAThe structures at the left are well characterized. The higher order structures at the right are largely speculative.The “beads on a string” of the 10 nm fiber are nucleosomes.Eukaryotic Chromosomes are Packaged Around Histones•Histones are small (11 - 21 kDa), very basic (20 -25% lys + arg) proteins.•Four histone proteins (the “core histones”) form a disk that the DNA wraps around.–The disk is made up of 2 copies each of H2A, H2B, H3, and H4.•H1 is a “linker” protein between the “core” histones.•Histone proteins are nearly identical in all eukaryotes, i.e., they are highly conserved. –H4 from cows differs by 2 amino acids from H4 of peas so histones have not changed very much in the ~ 109 years since plants and animals diverged.Nucleosome•dsDNA wraps around histone core ~1.67 times = Nucleosome or bead on a string (10 nm)•146 bp of DNA plus 20–60 bp linker DNA10nmFig 8-18 and Box 8-1Figs 8-21 and 8-22Binding of the histones to the DNA backbone bends the DNA. Note that all of the histones share a similar fold - the “histone fold”. Like Fig 8-25Interactions are primarily with the phosphates and a few minor groove bases – NOT sequence specificNucleosome StructureH3 (blue) H4 (green)H2A (yellow) H2B (red)Fig 8-20Note N-terminal tails (NTDs, N-terminal domains). There are 8NTDs: 2 H3, 2H4, etc. About 25 - 30% of the total mass of the core histones is in the NTDs. NTDs extend out of the central disc and are unstructured in the crystal structure.H1 Histone and Histone Tails are Required to Form 30 nm