Nucleosome Packing

Nucleosome Packing

Main driving force for DNA bending around histones is:

Bending rigid DNA duplex around small histone octamer radius about 4.2 nanometers overcomes electrostatic repulsion and mechanical stiffness. Strong driving force arises from extensive attractive interactions between positively charged lysine and arginine side chains lining histone octamer superhelical ramp and negatively charged phosphate groups, plus multitude hydrogen bonds between histone main chain amides and DNA backbone phosphates and numerous water-mediated contacts. Charge neutralization lowers energetic barrier permitting sharp curvature. Hydrophobic interactions stabilize octamer co

Ref: Luger et al., Nature 1997 Structure; Lodish et al., Chapter 8: Driving Force DNA Bending Charge Neutralization

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Nucleosome Packing

Diameter of metaphase chromosome is approximately:

Chromosome morphometry quantifies hierarchical folding ratios. Starting from 2 nanometer DNA helix, beads-on-string 11 nanometer, 30 nanometer helix, 300 nanometer looped domain scaffold attached, finally fully condensed metaphase chromatid about 700 nanometer wide, complete metaphase chromosome with two sister chromatids about 1400 nanometer or 1.4 micrometer diameter. Dimensions illustrate approximately ten thousand-fold linear compaction enabling 2 meters human diploid DNA fit nucleus. Electron microscopy of isolated metaphase chromosomes shows width near 1400 nanometer. Values 300 nanomete

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 4 and 19: Diameter of Metaphase Chromosome Approximately 1400 nm

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Nucleosome Packing

Diameter of DNA double helix is:

B-form DNA double helix diameter measured by Franklin Wilkins X-ray diffraction patterns approximates 2.0 nanometers, corresponding to distance across base pair plus phosphate backbone van der Waals radii. This baseline dimension explains how 147 base pairs wrapped around 11 nanometer octamer achieves first level compaction. Subsequent levels: nucleosome 11 nanometer, solenoid 30 nanometer, looped scaffold 300 nanometer, metaphase chromatid 700 nanometer represent stepwise coiling. Recognizing 2 nanometer starting point underscores approximately seven-fold linear compaction achieved at nucleos

Ref: Watson and Crick 1953 Nature; Alberts et al., Molecular Biology of the Cell, Chapter 4: DNA Double Helix 2 nm Diameter

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Nucleosome Packing

11 nm fiber is best observed when chromatin is isolated in:

Isolation ionic conditions profoundly affect visualization of chromatin hierarchical levels. In low salt buffer like 1 millimolar EDTA or 10 millimolar Tris-HCl, electrostatic repulsion between linker DNA segments prevents folding and H1-mediated compaction weak, leaving extended 11 nanometer beads-on-string fibers easily observed by electron microscopy with clear spacing. Adding NaCl to 100 millimolar or Mg2+ to 2 millimolar plus stoichiometric H1 induces coiling into 30 nanometer dense fiber. High salt beyond 0.6 molar strips histones, detergent solubilizes membranes, heat denatures proteins

Ref: Olins and Olins 1974 J Cell Biol EM; Alberts et al., Chapter 4: 11 nm Fiber Observed in Low Salt Buffer

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Nucleosome Packing

Micrococcal nuclease preferentially digests:

Micrococcal nuclease from Staphylococcus aureus, calcium dependent endo-exonuclease, preferentially cleaves accessible linker DNA between nucleosomes due to steric exposure, showing mild sequence bias toward AT-rich regions. Limited digestion time course generates characteristic ladder pattern on agarose gel with fragments in multiples of nucleosome repeat length, enabling mapping of positioning and repeat determination. Core DNA tightly wrapped around octamer protected early, centromeric heterochromatin more resistant due to compaction, GC-rich naked DNA not preferentially digested. Assay rem

Ref: Noll M 1974 Nature; Lodish et al., Molecular Cell Biology, Chapter 8: Micrococcal Nuclease Digests Linker DNA

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Nucleosome Packing

Histone octamer conservation during replication occurs for:

During semi-conservative DNA replication parental histone octamer distribution must conserve epigenetic information. Pulse-chase labeling combined with density gradient shows H3-H4 tetramer (H3-H4)2 remains intact as unit, does not split into dimers, segregating randomly to one of two daughter duplexes behind fork. This allows recycling of parental marks like H3K9 methylation to reestablish heterochromatin on both daughters via reader-writer mechanisms. In contrast H2A-H2B dimers exchange rapidly, mixing old and new, H1 readily dissociates. Therefore H3-H4 tetramer conservation underlies epige

Ref: Jackson V 1990 Biochemistry; Alberts et al., Molecular Biology of the Cell, Chapter 5: H3-H4 Tetramer Conservation During Replication

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Nucleosome Packing

CENP-A mainly localizes to:

CENP-A containing nucleosomes localize almost exclusively to functional centromere, not dispersed throughout chromosome arms. High resolution immunofluorescence and chromatin immunoprecipitation sequencing show discrete foci at primary constriction colocalizing with inner kinetochore markers CENP-C and CENP-T during interphase and mitosis. At centromere CENP-A directly recruits CCAN network initiating kinetochore assembly ensuring spindle microtubule attachment and checkpoint signaling. It does not accumulate at telomeres containing TTAGGG repeats protected by shelterin, nor at nucleolar organ

Ref: Earnshaw and Migeon CENP-A Localization; Lodish et al., Chapter 19: CENP-A Mainly Localizes to Centromere

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Nucleosome Packing

CENP-A is a variant of histone:

Centromeric chromatin contains distinctive histone variant CENP-A that epigenetically specifies centromere location independent of underlying DNA sequence in most eukaryotes. CENP-A resembles canonical histone H3 in histone fold domain sharing about sixty percent similarity, but possesses divergent N-terminus and loop1 centromere targeting domain CATD directing specific deposition via HJURP chaperone during G1. It replaces H3 in subset of nucleosomes at active centromere creating specialized octamer recruiting inner kinetochore. H1 is linker histone, H2A variants include H2A.Z, H4 lacks centro

Ref: Palmer et al., 1987 PNAS; Alberts et al., Molecular Biology of the Cell, Chapter 4: CENP-A is Variant of H3

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Nucleosome Packing

SMC proteins are involved in:

Structural Maintenance of Chromosomes proteins represent large ATPase machines forming elongated coiled-coil dimers with hinge domain closing ring-like structures embracing DNA duplexes. Condensin complexes SMC2/SMC4 compact mitotic chromosomes via loop extrusion, cohesin SMC1/SMC3 holds sister chromatids together from S phase until anaphase cleavage by separase, SMC5/SMC6 aids homologous recombination repair at collapsed forks. Mutations produce chromosome missegregation, aneuploidy and repair defects. Proteins localize to scaffold attachment regions and utilize ATP hydrolysis to extrude loop

Ref: Hirano T 2016 Nat Rev Mol Cell Biol SMC; Lodish et al., Chapter 19: SMC Proteins Chromosome Maintenance

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Nucleosome Packing

Major scaffold protein involved in loop formation is:

Chromosome condensation during prophase requires scaffold proteins shaping 30 nanometer fiber loops into cylindrical chromatids 700 nanometers wide. Topoisomerase II alpha, type II enzyme cleaving both DNA strands, passing duplex through break and resealing, resolves sister chromatid catenations, adjusts supercoiling and forms axial scaffold along chromatid center overlapping condensin complex. Immunofluorescence shows topoisomerase II colocalizes with condensin along axis. DNA polymerase synthesizes DNA, RNA polymerase transcribes, ligase joins Okazaki fragments, but scaffold architecture and

Ref: Earnshaw and Laemmli 1983 Scaffold; Alberts et al., Molecular Biology of the Cell, Chapter 19: Scaffold Protein Topoisomerase II

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Nucleosome Packing

Scaffold attachment regions (SARs) are rich in:

Scaffold Attachment Regions also termed Matrix Attachment Regions designate DNA elements anchoring loop domains to nuclear scaffold. Sequence analysis from multiple loci reveals enrichment for AT-rich stretches with topoisomerase II cleavage consensus, origin recognition elements and base unpairing regions possessing high untwisting potential under superhelical stress. AT-richness favors minor groove interaction with scaffold proteins and propensity to become single stranded, facilitating protein binding. GC-rich islands associate with active promoters, CpG islands regulate imprinting, rRNA ge

Ref: Mirkovitch et al., Cell 1984; Lewin, Genes XII, Chapter: SARs Rich in AT Sequences

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Nucleosome Packing

Loop domains in chromosomes are anchored to:

Beyond 30 nanometer fiber, chromatin organizes into large looped domains estimated 20 to 100 kilobases each, anchored at bases to nonhistone chromosomal scaffold or nuclear matrix. Scaffold comprises topoisomerase II alpha resolving catenations, condensin SMC2/SMC4, KIF4 motor protein and lamins forming chromosome axis. Loop anchorage compacts fiber additional 40-fold achieving 700 nanometer chromatid. Centromeres provide constriction points, telomeres protect ends, nucleolus organizes rDNA repeats, but loop bases specifically attach to scaffold proteins revealed by histone-depleted metaphase

Ref: Laemmli UK 1978 Cold Spring Harbor; Alberts et al., Chapter 4: Loop Domains Anchored to Scaffold Proteins

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