-1-Chromosome StructureBiochemistry 201Advanced Molecular BiologyApril 5, 1999Doug BrutlagIntroductionThe supercoiling of prokaryotic DNA is induced and maintained by the action ofthe enzyme DNA gyrase. The supercoiling of eukaryotic DNAs results primarily fromthe folding of the DNA about histone proteins to form chromatin.The formation of chromatin is the first step in a series of folding events thatreduce the length of DNA 10,000 fold from its extended form into a compact mitoticchromosome. An average human mitotic chromosome 5 microns in length contains 5cm of DNA.In the electron microscope, chromosomes appear to be composed of fibers 250 to300 Å in diameter.When chromatin is spread under more dissociating conditions (low ionicstrength) these fibers extend into 100 Å diameter threads.The 100 Å fibers are about 6 to 10 times longer than the 300 Å fibers from whichthey are derived.The final level of unfolding of the chromosome into DNA requires the removalof the histone proteins themselves.Chromosome Structure-2-Histones and Chromatin StructureHistones can be resolved into five major classes by biochemical fractionationprocedures and by several physical properties.1. Histone H1, the most unusual histone, is larger than all the others (20,000daltons) is very rich in the amino acid lysine, and has the lowest affinity for DNA.2. Histone H1 can be dissociated from chromatin at ionic strengths as low as 0.5M NaCl, while 2.0 M salt is needed to remove the other four classes of histone.3. Histone HI has an unusual distribution of amino acids. Both the N-terminusand the C-terminus are rich in basic residues while the center of the molecule is richerin hydrophobic residues and has an equal number of acidic and basic amino acids.4. Physical studies indicate that the center of the molecule folds into a beta-pleated sheet structure while the ends of the molecule remain extended in a randomcoil conformation.There are four other histones called H2A, H2B, H3, and H4 for historical reasons.These histones are among the most highly conserved protein sequences known. Thesequence of histone H4 from cows and pea plants differs by only 3 out of 104 residuesand these are very conservative amino acid substitutions. This conservation speaks toChromosome Structure-3-the important nature of the structure of the histones and suggests that an alteration ofeven a single amino acid side chain is either lethal or strongly selected against.Histone-histone associationsPhysical studies indicate that the predominant structural feature of these fourhistones is the presence of alpha-helical segments in the hydrophobic regions. Thesehistones also tend to associate with each other in a very specific pattern of interactions.The interactions were first investigated by studying physical parameters, sincethe amounts of helical structure in each histone increased upon associating withanother histone molecule. The pattern of associations is:H3=======H4   H2A=====H2BHistones H3 and H4, the most arginine rich histones, interact very strongly witheach other and form a very specific complex, a tetramer. Histones H2A and H2B alsointeract with each other strongly and can form primarily dimers and some higheroligomers.The first direct evidencethat such associations existedwithin chromatin came fromthe work of Roger Kornberg andJean Thomas. By gentlyextracting histones fromchromatin at very high proteinconcentration it was possible toisolate specific complexes ofhistones of even larger size.Octamers are the largest specifichistone complex and theyconsist of 2 molecules each ofhistones H2A, H2B, H3, and H4.An octamer of histone interactswith an equal weight of DNA(160-200 base pairs) inchromatin.NucleosomesChromatin is composedof repeating subunits that wereChromosome Structure-4-based on an octamer of histones and 200 base pairs of DNA, referred to as a nucleosome.Evidence for a repeating nucleosome structure includes:- Low angle X-ray diffraction indicating a 110 Å repeating unit.- Nuclease treatment of chromatin cleaves DNA at 200 base pair intervals.- Nucleosomes can be visualized in the electron microscope as beads on a string.Chromosome Structure-5-- The DNA ofthe virus SV40replicates in thenucleus and iscoated withhistones in amanner quitesimilar to the hostcell chromatin.When SV40chromatin is spreadat low ionic strength, one can see the beaded appearance and separation of the adjacentnucleosomes.- The DNA between the nucleosomes is very sensitive to micrococcal nucleaseand is referred to as linker DNA.- The resistant DNA with its associated histone octamer is referred to as thenucleosome core particle or sometimes just the core particle.- Folding of SV40 DNA into chromatin results in the compaction of thecircumference of these two circles by 7 fold. About 200 base pairs of DNA which by itselfis 680 Å in length is converted into 100 Å diameter nucleosome structure.Evidence that the DNA is wrapped on the outside of the core histone came fromexperiments in which either chromatin or isolated core particles are cleaved withanother endonuclease, called pancreatic DNAse. Pancreatic DNAse, unlike themicrococcal nuclease, does not cleave preferentially in the linker region betweennucleosomes. Instead it nicks only one strand of the DNA helix. The lengths of thesefragments differ by about 10 bases, each fragment being about 10 bases longer than thefragment before it.Chromosome Structure-6-Finch, Klug and Lutter proposed the wrapping of DNA around the histoneoctamer based on these patterns of sensitivity to DNAse. Core particles contain about140 base pairs of DNA that they proposed was wrapped in two turns about the octamers.Chromosome Structure-7-The isolation of core particles has also allowedthe crystallization of the nucleosome and a lowresolution 3-dimensional picture has been obtained.The histone octamer is in the core. The 140base pairs of DNA associated with octamer is knownas the core DNA. Together they are known as the coreparticle. The linker DNA joins one nucleosome tothe other.Histone H1 is associated with the linker DNA.Nucleosomes and Supercoiling of Eukaryotic DNAReconstitution of core histones with circular SV40 DNA in the presence of aDNA topoisomerase shows the formation of beaded nucleosome structure and theinduction of 1.25 supertwists per nucleosome core particle.The winding of DNA twice about the histone core in a left-handed way shouldcause a writhe of two negative twists for 200 base