1DNA packaging summary1. Problem is packaging2. Levels of chromatin structure (nucleosomes, 30-nmfiber, loops, bands)3. Histone code marks active and inactive sequences4. DNA elements for chromosome structure include (ARS),TEL and CEN.5. CEN promotes the assembly of the kinetochore, a giantprotein complex that attaches the chromosome to thespindle at division.High resolution scanning immunogoldelectron micrograph.Yellow = phosphorylated H3 in the pericentromeric region.A severe problem of packaging1. Largest human chromosome: ~3 x 108 bp2A severe problem of packaging1. Largest human chromosome: ~3 x 108 bpHow long is it?A severe problem of packaging1. Largest human chromosome: ~3 x 108 bpHow long is it?3 x 108 bp x 3.4 Å/bp x 1 m/1010 Å = 10 x 10-2 m = 10 cm!3A severe problem of packaging1. Largest human chromosome: ~3 x 108 bpHow long is it?3 x 108 bp x 3.4 Å/bp x 1 m/1010 Å = 10 x 10-2 m = 10 cm!2. A typical cell = 10 µm = 10 x 10-6 mA severe problem of packaging1. Largest human chromosome: ~3 x 108 bpHow long is it?3 x 108 bp x 3.4 Å/bp x 1 m/1010 Å = 10 x 10-2 m = 10 cm!2. A typical cell = 10 µm = 10 x 10-6 m3. Therefore the DNA must be compacted ~104-foldThis is like fitting an 11-mile-long string into a 6-foot box4Visualizing chromatin breakdown products1. Beads-on-a-string (low salt) 2. 30-nm fiber (0.15 M KCl)Electron micrographs of “chromatin” preparationsBeads contained “histones”: H1, H2A, H2B, H3, and H4 Packaging proteins discovered by DNase Idigests of nuclei1. Isolate nuclei2. Partial digest with DNase I (nonspecific endonuclease)3. Run agarose gel.4. Result: Ladder of multiples of 150-200 bp5Packaging proteins discovered by DNase Idigests of nuclei1. Isolate nuclei2. Partial digest with DNase I (nonspecific endonuclease)3. Run agarose gel.4. Result: Ladder of multiples of 150-200 bpConclusion: Something is protecting the DNA!Proteins = H1, H2A, H2B, H3 & H4Packaging proteins discovered by DNase Idigests of nuclei1. Isolate nuclei2. Partial digest with DNase I (nonspecific endonuclease)3. Run agarose gel.4. Result: Ladder of multiples of 150-200 bpConclusion: Something is protecting the DNA!Proteins = H1, H2A, H2B, H3 & H45. Treat with more DNase I.6. Run acrylamide gel.7. Result: Ladder of multiples of ~10 bp.6Packaging proteins discovered by DNase Idigests of nuclei1. Isolate nuclei2. Partial digest with DNase I (nonspecific endonuclease)3. Run agarose gel.4. Result: Ladder of multiples of 150-200 bpConclusion: Something is protecting the DNA!Proteins = H1, H2A, H2B, H3 & H45. Treat with more DNase I.6. Run acrylamide gel.7. Result: Ladder of multiples of ~10 bp.Conclusion: DNA is wrapped around theoutside of the nucleosome.Crystal structure of the nucleosomeRibbon drawings“Top” view“Side” view of 1/2nucleosome147 bp of DNA wrappedalmost twice around 8 corehistones: (H2A, H2B, H3, H4)2Carolyn Luger and Tim RichmondSpace-fillingdrawing7The histone foldRibbon drawingSimple.Conserved.Adopted by all 4 “core” histones (H2A, H2B, H3 and H4). Sequence schematicH3-H4 tetramer binds two H2A-H2B dimersto form the histone octamerMonomersDimersTetramerOctamer8The DNA is not smoothly bent Ribbon drawingof a single gyreH3/H4H2A/H2BWater mediates many histone:PO4 contacts“Stick” drawingProteinDNA Dots = Visible watersWaters on the DNA9Histone “tails” stick out between the gyres of DNAPost-translational chemical modification of the tails controls function.Modification patterns comprise the “histone code”.Examples of the “histone code” for H3/H4Ac - acetyl (lysine), Me - methyl (lysine), P - phosphoryl (Ser or Thr)10Hypo-acetylated histones in heterochromatinSir proteins (Sir 1, 3, 4) bind cooperatively to de-acetylated chromatin, tightly packaging the 30-nm filament. Sir2 is a histone deacetylase. The combined actions of the packaging proteins and the deacetylasespread heterochromatin (and silence packaged genes).Replication distributes nucleosomes to the daughtersExperiment: replicate mixed chromatin and nucleosome-free templates in vitroResults: Replisome copied through nucleosomes!Nucleosomes were found only on the daughters of the chromatin template.No nucleosomes moved to the daughters of the nucleosome-free template.11Levels of chromatin structureLarger DNA structures mediatemore compaction.Euchromatin: transcribed and lesscondensed“Loops” of 30-nm fibers seen atinterphaseHeterochromatin: more condensed,genes silenced, replicatedlater in S phase.Loops and scaffoldElectron micrograph of “histone-depleted” chromosome12DNA amplification produces visible “bands” instaining pattern of fly “polytene” chromosomesFunctional DNA sites -- Telomere & CentromereSchematic and electron micrograph of X chromosome.• Telomeres protect the ends.• Centromere is at the primary constriction. Itmediates chromosome cohesion, spindle attachmentand chromosome segregation.13Eukaryotic Cell DivisionKinetochores mediate chromosome-microtubule attachmentsTed SalmonKinetochore mediates attachment to the spindleSchematic drawing.Centromere (DNA segment) isat the primaryconstriction. Thekinetochore is a huge,complicated proteincomplex with severallayers. The outer layerprovides attachment sitesfor microtubules.14Centromeres contain special DNA sequences thatassemble kinetochores Does transcription promote replacement of centromeric histone H3?A model for the yeast kinetochoreYeast kinetochore assembly and architecture. Speculative modelfor the organization of known components. Protein complexes aredrawn approximately to scale.MicrotubuleDNA15Kinetochore complexity & structural challengeDam1 complexThe Kinetochore PuzzleThe Yeast Complex Dam1 SolutionWestermann et al. (2005) Mol. CellWestermann et al. (2006) NatureConly Rieder16Dam1: Mechanism and structureModel: Dam1 rings slide along shrinking microtubules!YACS = yeast artificial chromosomes50-1000 KbpNeeded to sequence the human genome17DNA packaging summary1. Problem is packaging2. Levels of chromatin structure (nucleosomes, 30-nmfiber, loops, bands)3. Histone code marks active and inactive sequences.4. DNA elements for chromosome structure include (ARS),TEL and CEN.5. CEN promotes the assembly of the kinetochore, a giantprotein complex that attaches the chromosome to thespindle at division. YACs = CEN, TEL, ARS + >50 Kb.High resolution scanning immunogoldelectron micrograph.Yellow = phosphorylated H3 in the pericentromeric