DNA Organization Jan. 2025 PDF

HUBI 2004: Fundamentals of Modern Molecular Biology DNA Organization Pavan K. Kakumani Jan. 21, 2025 1  Learning Objectives How are bacterial chromosomes compacted/orga...

HUBI 2004: Fundamentals of Modern Molecular Biology DNA Organization Pavan K. Kakumani Jan. 21, 2025 1  Learning Objectives How are bacterial chromosomes compacted/organized? What are the proteins associated with chromosomes in bacteria and their functions in DNA organization? What are histone proteins, and how are they involved in the assembly of nucleosomes? What is the linker DNA? What is the difference between a chromatosome and a nucleosome? What is a heterochromatin and a euchromatin, and what are the differences between these two states of chromatin? 2 What is, and Why Organize (or Package) DNA? Packaging of DNA (or Organization) means condensing the DNA in a very orderly way…!!! It allows you to access your genes in a controlled way…!!! 3 Books in a library vs Books in a pile 4 Human genomic DNA Adapted from Genomics Education Program 5 https://www.youtube.com/@GenomicsEducation Dimensions of Human sperm cell = 5.1 µm x 3.1 µm Volume of HeLa cell = 2,000 µm3 End-to-end Length of Human DNA = 3 x 109 bp x 0.34 nm = 1.02 m 6  DNA Packaging (or Organization) Involves several levels 1) Supercoiling 2) Nucleosomes 3) Chromatosomes All mechanisms to efficiently and effectively organize the 4) Solenoids DNA into a usable form 5) looping onto matrix Required to produce a mitotic 6) higher order coiling chromosome 7 Viruses are Infectious Particles Containing Nucleic Acid Genomes Virus - a non-cellular infectious particle containing a small nucleic acid genome with a limited number of genes Bacteriophage - a virus that infects (“eats”) bacteria The nucleic acid can be single-stranded or double-stranded The nucleic acid can be DNA or RNA Physical size and Genomic size of viruses varies (a lot!) 8 Packaging of viral genomes Enveloped and non-enveloped viruses Capsid - protein coat that contains viral genetic material. A non-enveloped virus contains genetic material in only a protein shell. An enveloped virus has an envelope of host cell cytoplasmic membrane surrounding the capsid. 9  Bacterial Chromosomes Are Organized by Proteins Bacterial genomes are haploid They most often have a single dsDNA chromosome. dsDNA: double stranded DNA. Some bacteria have more than one chromosome 10  Bacterial Chromosome Compaction Bacterial chromosomes are densely packed to form a small The nucleoid of E. coli. region called the nucleoid. The chromosome is organized into a series of tight loops. These allow for efficient packaging of relatively long DNA molecules into very small spaces. 11  Bacterial Chromosome Compaction Bacterial chromosomes are compacted in two ways: 1. Proteins help organize the DNA into loops that pack the chromosome into the nucleoid 2. The circular DNA undergoes supercoiling 12  Proteins Associated with Chromosomes Bacterial chromosome Small nucleoid-associated condensation by proteins proteins participate in the DNA bending that contributes to folding and condensation of bacterial chromosomes Structural maintenance of chromosome (SMC) proteins holds DNA in coils or V shapes Other proteins also interact in the nucleoid to compact DNA 13  Supercoiling Covalently closed circular chromosomes exist in various (super)coiled forms The relaxed circle is the least twisted Supercoiling compacts DNA as a result of over or under rotations of helical twisting 14 Eukaryotic Chromosomes are Organized into Chromatin The DNA and associated proteins of a eukaryotic chromosome are called chromatin Proteins that organize chromosomes are essential, and they provide a mechanism for condensation, segregation, and organization of chromosomes Eukaryotes have multiple chromosomes that undergo cyclic condensation for cell division 15  Histone Proteins and Nucleosomes Each chromosome is approximately half DNA and half protein About half of the proteins are histone proteins, small basic proteins that tightly bind DNA There are five types of histone proteins in chromatin: H1, H2A, H2B, H3, and H4; they are highly conserved among eukaryotes The remaining proteins, the non-histone proteins, are very diverse and perform a variety of tasks in the nucleus 16 Histone Protein Characteristics 17 There are just 2 amino acid differences in Histone 4 (H4) between cows and peas 18 DNA associates with histone proteins to form nucleosomes ▪ 8 histone proteins found in core nucleosome H2A H3 2 each of H2A, H2B, H3 and H4 H4 H2B ▪ DNA is wound around protein octamer 1.65 times (~146 bp) ▪ In humans, there is ~50 bp space between core nucleosomes (= linker region) This wrapping of DNA introduces 19 supercoils into the molecule  Nucleosome core particles—fundamental units of histone protein organization with two molecules each of histones H2A, H2B, H3, and H4 (octamer) A span of DNA 146 bp long (core DNA) wraps around each octamer to form a nucleosome Condensing the Nuclear Material 20  Nucleosome Assembly Histones H2A and H2B assemble into dimers; H3 and H4 also form dimers Two (H3-H4) dimers form a tetramer, then two (H2A-H2B) dimers associate with it to form the octamer The wrapping of DNA around the octamer is the first level of DNA condensation and compacts the DNA about sevenfold. 21  Nucleosome structure The nucleosome model structure has been confirmed by X-ray crystallography 22  But nucleosomes are not just packaging material ▪ The ends of the histone proteins lie outside the nucleosome ▪ They can be chemically modified. ▪ Examples of chemical modifications include methylation, acetylation, phosphorylation. ▪ This is associated with (regulating) gene expression 23  The nucleosome is only the most basic unit of DNA packaging There are several more levels before you get to the state the DNA is, in a eukaryotic cell 24  Chromatin Structure Electron micrographs of chromatin in its least condensed state show a 10-nm fiber, or “ beads-on-a-string ” morphology—the “ beads ” are the nucleosomes Kornberg proposed the nucleosome- Condensing the Nuclear Material based model of chromatin in 1974 The variable-length “ string” between nucleosomes is linker DNA H1 may associate with linker DNA H1 with a nucleosome may sometimes be called a chromatosome 25  Association of nucleosomes with H1 histones (sometimes called chromatosomes) histone H1 binds to DNA at edges of histone particle covers another ~ 20 bp of DNA (total now ~166 bp) H1 DNA now wrapped about 2X around the histones Linker DNA 26  Solenoid Structure The 10-nm fiber is not observed under normal cellular conditions Instead, a 30-nm fiber (six times more condensed) is observed The 30-nm fiber forms when the 10-nm fiber coils into a solenoid structure, with six to eight nucleosomes per turn and histone H1 stabilizing the solenoid 27  Higher-Order Chromatin Organization and Chromosome Structure Solenoidal chromatin is looped and attached periodically to a nonhistone protein chromosome scaffold The loops on the scaffold form the 300-nm fiber This is the state of the chromatin in a functioning cell (interphase) 28  Higher Order Chromatin Condensation The radial loop - scaffold model of chromatin condensation. Chromatin loops of 20 to 100 kb are anchored to the chromosome scaffold by non-histone proteins at sites called MARs (matrix attachment regions) The radial loop-scaffold model suggests that the loops gather into “rosettes” and are further compressed by non-histone proteins Metaphase chromatin is compacted 250- fold compared to the 300-nm fiber 29  Heterochromatin and Euchromatin Chromosome condensation varies from one part of a chromosome to another Regions that contain actively expressed genes and are less condensed during interphase are called euchromatin Regions that remain condensed in interphase and contain many fewer expressed genes are called heterochromatin Heterochromatin is often associated with the methylation of histones 30 euchromatin = open structure, DNA is accessible to enzymes (= solenoidal form of chromatin) i.e., expressed genes are found here heterochromatin = compact, inactive DNA common at centromeres and telomeres (no genes)  Heterochromatin Actually, it comes in two forms Facultative heterochromatin not always heterochromatin. e.g. the sex chromosomes exhibits variable levels of condensation, related to levels of transcription of resident genes Constitutive heterochromatin permanently condensed, found prominently in centromeres and telomeres composed primarily of repetitive DNA sequences 32  Dynamic Chromatin Structure Changes in level of compaction regulate access to DNA by proteins for replication, transcription, recombination, or repair For example, chromosome centromeres consist of constitutive heterochromatin However, when chromosomes are being replicated in S phase of the cell cycle, the heterochromatin must dissipate Nucleosome core particles dissociate from DNA ahead of the replication fork and re-form centromeric heterochromatin after the fork passes The borders for reestablishing centromeres are variable and typically have no impact on gene expression 33  Dynamic Chromatin Structure Gene expression can be controlled by the state of chromatin in which a gene is located. Gene expression or gene silencing can be dictated by chromatin structure, which is transmissible from one cell generation to the next. 34  Dynamic Chromatin Structure Normally, the eyes of the fruit-fly Drosophila are red, thanks to expression of the w locus The w locus is usually located in a region of euchromatin near the telomere of the X chromosome  Dynamic Chromatin Structure Hermann Müller used X-rays and isolated mutants in which a segment of the X chromosome had inverted thereby placing the w locus near the centromere the result was that, in some eye cells, the gene was not expressed - due to the spread of heterochromatin to the centromere; while in other cells it was expressed giving a variegated appearance to the eyes.