Not free to rotate turns added Unwinding of DNA during DNA replication E. coli Topoisomerase 1 Circular DNAs that differ in linking number can be Separated by gel electrophoresis Topoisomerase 2 (gyrase) Relaxed DNA substrate Supercoiled DNA + topoisomerase 1 Relaxed DNA + gyrase: 10 seconds Relaxed DNA + gyrase: 20 seconds Relaxed DNA + gyrase: 40 seconds 1 2 3 4 5 0 -1 -2 -3 -4 -5 -6 >-7 Relaxes negative supercoil DNA + topo 1 Introduces negative supercoils into DNA, requires ATP Chromatin Nucleosomes Histones Effect of condensins on supercoiling Problems: Ch. 24: 1, 5, 7-8 (4th ed 1, 5, 8, 9) Study guide: p.303 # 2-4 See animation of the packing of a duplex DNA into a chromosome Eukaryote chromosome structure Topoisomerases that introduce positive supercoils have never been found in nature. Most naturally occurring DNA molecules are negatively supercoiled due to underwinding. Negatively supercoiled DNA is poised to be unwound, preparing DNA for processes that require the separation of strands, such as replication, recombination and transcription. In contrast, positive supercoils make separation of DNA more difficult. The superhelical density of bacterial DNA is balanced by regulation of the net activity of topoisomerases I and II. Underwound DNA facilitates the formation of cruciform structures in palindromic DNA DNA interwinds and wraps about itself Supercoils in long, linear DNA arranged into loops whose ends are restrained A model for chromosomal DNA Plasmid or phage DNA Chromosomal DNA cannot freely rotate along its length but are organized into loops whose ends are restrained very much like a circular DNA molecule. Chromosome Structure Human DNA?s total length is ~2 meters! This must be packaged into a nucleus that is about 5-10 micrometers in diameter This represents a compaction of more than 10,000 fold! It is made possible by wrapping the DNA around protein spools called nucleosomes and then packing these in helical filaments Changes in chromosome structure during the eukaryotic cell cycle Negatively supercolied Positively supercoiled Different topoisomerases are involved in DNA replication, transcription and mitosis. The eukaryote topoisomerases, unlike the bacterial gyrase, cannot introduce negative supercoils, but they can relax positive or negative supercoils, depending on the enzyme. How do eukaryotes get underwound DNA when they lack enzymes that underwind DNA? Because of the protein spool (histone core) used for packing in eukaryotes, negative supercoils are introduced by the relaxation of positive supercoils. Bending of AT dinucleotide clusters facilitates the assembly of nucleosomes (nucleosome positioning). Nucleosome contacts minor groove which is compressed in A=T clusters. May play a regulatory role in transcription and DNA replication by changing the accessibility of regulatory sequences such as promoters or replication origins. A type I topoisomerase in animal cells introduces negative supercoils in steps of 1 Self-assembly of nucleosomes Chromatin, the chromosomal material, consists of fibers containing protein and DNA in approximately equal masses, along with a small amount of RNA. 30 nm 146 bp ~200 bp A nucleosome is the unit of organization of chromatin = one bead plus adjoining DNA that leads to the next bead or 200 bp DNA plus 2(H2A,H2B, H3, H4) See problem #9 of Ch 24 10 nm fiber [ The DNA in the chromatin is tightly associated with proteins called histones, which package and order the DNA into structural units called nucleosomes. Also found in the chromatin are many nonhistone proteins, some of which regulate the expression of specific genes. ] 10 nm 10 nm Electron micrograph of chromatin - ?bead-on-a-string? (susceptible to nuclease digestion) Histones undergo multiple forms of modifications such as phosphorylation, acetylation, methylation, ubiquitination and ADP-ribosylation. These modifications affect the net charge, shape and other properties of histones as well as the structural and functional properties of chromatin. They may also play a direct role in the recruitment of regulatory proteins in transcription and DNA replication. 100 Å DNA wrapped around a nucleosome core (histone octamer) A model for the higher order structure of eukaryotic chromosomes 10 nm Fiber 2 nm Nucleosome = ~200 bp DNA +2(H2A+H2B+H3+H4) ] Structure of SMC (structural maintenance of chromosomes) proteins Model for the effect of condensins on DNA supercoiling In contrast to the underwinding of DNA by nucleosome binding Condensation (over-winding) of chromosomes by condensins A partially unraveled human chromosome, revealing numerous loops of DNA attached to a scaffold-like structure. Loops of chromosomal DNA attached to a nuclear scaffold. H1 histone Topoisomerase II Related genes in the same loop may facilitate co-regulation 60,000 1 6-7 ~40 680 1.2x104 1.2x104 1,200 200 10 ~1.1x106 18 loops/ miniband scaffold There are 6 nucleosomes per turn of coil. A nucleosome is 10nm in diameter. 1 nucleosome = 200 bp of DNA 6 x 200 x 0.34 nm/10 nm = ~40 [ 10nm
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