Chromosome Organization September 20, 2017

Chromosome Organization Prokaryotic and Eukaryotic cells have a DNA packaging problem The DNA in one human diploid cell is about 2 meters long (6.5ft!). It needs to squeeze into 15μm nucleus reducing its length as much as 10,000 fold How does it do this? DNA in prokaryotic and eukaryotic cells are organized into chromosomes. Chromosomes are defined as the DNA and its associated proteins

Chromosome Organization Why is chromosome organization important? Chromosomes are compact forms of DNA—allows DNA to fit inside a cell Chromosomes stabilize DNA and protect it from damage Allows for equal partitioning of DNA into daughter cells during cell division Organizes the accessibility of other proteins to DNA. Critical for the regulation of DNA replication and transcription Prokaryotic and Eukaryotic cells have different mechanisms for compacting DNA

DNA in cells Prokaryotic OR Eukaryotic? Do not have membrane bound organelles; no nucleus Linear chromosome Have membrane bound organelles; have a nucleus Circular chromosomes Plasmid DNA is a small circular piece of DNA, often present in multiple copies. Typically not essential for growth. Most cells are diploid (two copies of each chromosome), but cells can be haploid (one copy; gametes) or polyploid (many copies of each chromosome).

DNA in cells Prokaryotic Cells Do not have membrane bound organelles; no nucleus Circular chromosomes Plasmid DNA is a small circular piece of DNA, often present in multiple copies. Typically not essential for growth. Eukaryotic Cells Have membrane bound organelles; have a nucleus Linear chromosome Most cells are diploid (two copies of each chromosome), but cells can be haploid (one copy; gametes) or polyploid (many copies of each chromosome).

Prokaryotic Chromosome Organization Prokaryotic Chromosomes: Prokaryotic circular DNA molecules are much larger than the bacteria cell (x1500) Bacterial DNA is compacted and organized into a nucleoid. A nucleoid contains DNA, proteins and RNA

Prokaryotic Chromosome Organization Prokaryotic Chromosomes: Prokaryotic circular DNA molecules are much larger than the bacteria cell (x1500) Bacterial DNA is compacted and organized into a nucleoid. A nucleoid contains DNA, proteins, and RNA DNA is looped around anchoring proteins and loops are supercoiled

Eukaryotic Chromosomal Organization Half the molecular mass of a eukaryotic chromosome is protein. What are the majority of proteins? Histones, Chromatin Histones!

DNA Packaging

The majority of DNA is packaged into nucleosomes Nucleosomes: Composed of a histone core and the core DNA. Histones: Small positively charged proteins that associate with DNA. Form the protein “core” of a nucleosomes Histone core: 2 copies of histones H2A, H2B, H3 and H4 (8 total!) Core DNA: DNA that is most tightly associated with the nucleosome. Average length is 147bp Linker DNA: DNA between each nucleosome. The size of linker DNA is highly variable. Chromatin: The term given to describe DNA with its associated proteins. Histones, Chromatin https://www.dnalc.org/resources/3d/08-how-dna-is-packaged-advanced.html

The core DNA sequence is 147 bp? Perform MNase Assay MNase = Micrococcal Nuclease Assay MNase cleaves DNA non-specifically MNase cleaves protein- free DNA. Isolate DNA from the protein DNA band was 147bp

DNA Fragmentation in Apoptosis

DNA Fragmentation in Apoptosis

How is DNA packaged into nucleosomes? Core Histones Positively charged amino acids! Linker Histones

Histone Protein Structure Positive The histone-fold domains of histones are conserved Each core histone has an unstructured N-terminal tail

Nucleosomes Assembly The histone core is composed of 2 copies of each core histone H3 and H4 form dimers. H3-H4 dimers interact to form tetramers H2A and H2B form dimers. H2A –H2B dimers interact to form tetramers.

Nucleosomes Assembly Nucleosomes are assembled in an ordered fashion! Step 1: Two H3-H4 dimers form a tetramer Step 2: H32:H42 tetramer binds to double stranded DNA Step 3: Two H2A- H2B dimers bind are recruited to the DNA molecule forming a complete nucleosome Step 1 Step 2 Step 3

How histones interact with DNA There is a high affinity of histones for DNA The DNA becomes distorted when bound to histones DNA sequence specificity?

How histones interact with DNA There is lack of DNA sequence specificity. Histones form about 140 hydrogen bonds with the sugar-phosphate backbone and with the minor groove of DNA. Why does binding to the sugar-phosphate backbone suggest a lack of DNA sequence specificity? Answer: Histones form hydrogen bonds with the minor groove

Major and Minor Groove In a double helix, one edge of the base pair is exposed in the minor groove and other edge is exposed in the major groove. Therefore: The edges of each base pair are exposed in the major and minor grooves! Minor and Major Groove: https://www.youtube.com/watch?v=QD1TjeszTHQ Readings: Chapter 4 84-86

If a protein was making chemical bonds with the major groove, could a G-C pair be distinguished from a C-G pair? Same question but with minor groove?

Nucleosomal Organization How could you use the MNase assay to determine if DNA sequences that wrapped around a histones was similar?

Eukaryotic Chromosomal Organization Nucleosomes and DNA Compaction DNA wrapping around the histones (10nm) results in 6 fold compaction Needs to be about 10,000 fold—depending on genome size Nucleosomes link together to build a 30-nm fiber with help of Histone H1 https://www.youtube.com/watch?v=9kQpYdCnU14

Histone H1 binds to the linker DNA 10nm 30nm Yes– It increase it from 147 to about 167.

Histone Tails are required to generate the 30nm fiber Yes– It increase it from 147 to about 167.

True nature of the chromosome scaffold remains a mystery. Further compaction Histones alone are not the answer. The histone compaction (with Histone H1) results in about a 40 fold reduction. What does the rest? True nature of the chromosome scaffold remains a mystery.