BIO 138 Chapter II.

Organization of the DNA into Chromosomes Page |1

June 21, 2012
II. ORGANIZATION OF THE DNA INTO CHROMOSOMES

PROPERTIES OF A GENETIC MATERIAL

1. Storage of genetic information

-organisms traits
-biological diversity (based on the information stored in the DNA)
*stores information through sequences (great number of combination of A, T, G, C)

2. Accurate transmission
-traits
-signals: methylation pattern
*specific base pairing (A-T, G-C)
*error repair/proofreading mechanism (it’s a process that the DNA undergoes, not a feature of the DNA
itself)

3. Stability
-low mutation
rate
-flexible
(adapt to the
environment
esp. change in
its secondary
structure)

4. Mutability
-capacity for
genetic
change
-->variation--
>evolution

A.1. THE PRIMARY STRUCTURE OF THE DNA: linear sequences

-the molecular configuration of the DNA showing
1. components of the nucleotide
(phosphate group, 2-deoxy-D-ribose, nitrogen base)
2. chemical bonds
phosphodiester bond-connects the phosphate and the sugar,
n-glycosidic bond- a purine is attached to the C1 of the sugar through its N9: C1-N9; a
pyrimidine is attached to the C1 of the sugar through its N1:C1-N1,
hydrogen bond-weak, but numerous strong
3. 2 antiparallel strands (strands runs in opposite direction)
4. acidic due to its phosphate group (highly negative)
5. helps stabilized by chemical bonds and hydrophobic interaction among the nitrogen bases
(hiding from water; it is inside the DNA)
6. covalent backbone- made up of phosphodiester bridges (because of the phosphate and the
sugar)
7. specific base pairing (--> accuracy in transmission)
~ phosphate group in 5'; hydroxyl group in 3' end
~ pairing analogies

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NITROGEN BASES
-heterocyclic (N,C atoms found in
the ring)
-N renders basic character
-2 groups: [see handout for
structures]
*purines : 2 rings
A (6-amino purine),
G (2-amino-6-oxypurine)

*pyrimidines : 1 ring
T (2,4-dioxy-5-methylpyrimidine or
5-methyluracil),
C (2-oxy-4-aminopyrimidine),
U (2,4-dioxy pyrimidine)

Tautomeric shifts-could endanger the base pairing of atoms;

however it occurs rarely
-O usually in keto (C=O)-more normal; rarely enol (COH)
-N atoms in rings usually amino (NH2); rarely imino (NH)
*hydrogen atoms within the ring have relatively stable locations
--> if not , A-C or G-T
--> mutation / error in replication

INFORMATION FROM
THE PRIMARY
STRUCTURE
1. Size/ length:
1Mb, 1000kb,
2. MW: 80,000
daltons
3. Configuration:
ds linear (human), ss
circular
4. Base
composition: 10% A,
50% G+C
5. Base sequence:
5' to 3' ( in letters)

DNA BASE COMPOSITION AND SIZE (1, 2 & 4)

-Erwin Chargaff and colleagues in Columbia University
-Chargaff's rule
1. *DNA base composition is a characteristic of a species
2. *different cells or tissues in an organism have identical or highly similar base composition
3. *base composition is not altered by age, development, nutrition, physiological, and
environmental factors
4. *closely related species have very similar base composition. This represents a taxonomic value
(% DNA homology)
5. *base composition among bacteria show greater variation than in eukaryotic species (divide
very fast, cell cycle, replication: more prone to errors/mutations)
6. *molar amounts of purine= molar amount of pyrimidines

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DNA CONTENT (5)
In haploids
-the total DNA content (C)

In diploids
-the DNA that contains one complete set of the genes (2C)

Observations on the DNA Content

a. The genomes of more complex organism require much more DNA.
-viruses and prokaryotes --> 1 DNA molecule
-eukaryotes --> several chromosomes; each chromosome has 1 big DNA molecule (46 in humans)
*human haploid cells > yeast cell > E. coli
*physical complexity vs. DNA content

~C value paradox
-exceptions: toad and South American lungfish > humans

b. The DNA content is also correlated with certain processes or properties of a cell.
-species with lower DNA content have faster cell division (fewer DNA to replicate faster cell cycle)
-in Asian rice species, wild species have higher DNA content than domesticated (cultivated) species
(man made the selection)

June 26, 2012

A.2. THE SECONDARY STRUCTURE OF THE DNA

SECONDARY STRUCTURE
1. -double helical form of the DNA
2. -two antiparallel strands
3. -right-handed helix rotation (counter clockwise)
4. -bases stacked in flat planes perpendicular to the axis
5. -~10 bp per turn (34 angstrom)
6. -major groove: 22 angstrom (for attachment of regulatory proteins)
7. -minor groove: 12 angstrom
8. -diameter is 20 angstrom (width of the base pairs /distance between sugar phosphate
backbone)
*plectonemic coil

Various Helical Forms of DNA

ABCDEFZ
ABZ-the most common conformations in nature
-differ in (see handout)

Significance of Base Sequence to the Secondary Structure of DNA

-not only does it carry genetic information
-I also determines the helical form of that region
~variation in the exposure of bases to DNA binding proteins (repressors or activators)
~affects gene expression

Other secondary structures

Cruciform
Hairpin
Stem and loop/lollipop
Cloverleaf

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Cruciform
-has inverted repeats or palindromes
-formed by ds

Hairpin
-formed by ss DNA or RNA
-palindromes
-

Stem and loop

-ss DNA or RNA
-bigger loop compared to hairpin
-palindromes in its stem

Cloverleaf
-tRNA molecule
-multiple palindromes
-3 stem and loops in one molecule (anticodon, d,tyc arms)
-acceptor arm

Importance elf these Secondary Structure

-formation of functional tRNA and rRNA
-stability of viral DNA (ESP for ss)
-landmarks for regulatory proteins
~replication of priming sites in the circular DNA of some viruses
~operator and terminator sites of bacterial genomes

SUPERCOILED DNA
-axis of the helix is curved
-occur in two forms: with scaffold - occurs in nucleosome; in circular DNA

~chromatin of higher organism

*dna wound around the histone core
~supercoiling of cccDNA (covalently closed circular DNA)
*no histone core
*twisted rings of DNA
--positive (when you over-wined the DNA) and negative supercoil
--eg:bacterial DNA, plasmids, DNA of tumor viruses, mitochondrial DNA, chloroplast DNA

Discovery of supercoiled DNA

1963: John Cairns observed ds rings in replicating bacterial DNA
1965: polyoma virus; ds DNA ---CsCl density gradient centrifugation (they move base on their density) -
--> 3 bands
:Jerome Vinograd's explanation--why 3 bands? (see handout); mw/ length identical; variation is due to
differences in molecular compactness of DNA (density)

Experimental detection of covalently closed circles

-sedimentation at alkaline pH
pH > or = 11.3
-DNA denatures
-no h bonds between the 2 strands
-the denatured DNA forms sediments differentially

Differences between 3 forms of DNA upon sedimentation at alkaline pH

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-equilibrium centrifugation in CsCl containing ethidium bromide (EtBr-a flat molecule that intercalates
DNA causing unwinding--> changes supercoiling of DNA depending on its concentration; direct
relationship)

Effect of increasing EtBr concentration in supercoiling of cccDNA

-gel electrophoresis
~smaller or more compact molecules willsmove faster; pass through the matrix easier and
migrate farther than large molecules
~in terms of rate of migration
Supercoiled>relaxed circular
~linear DNA migrate according to size

Properties of supercoiled DNA

Twist (Tw)
-the ideal number of helical turns in a relaxed DNA

Linking number (Lk)

-the number of times a strand of DNA winds in the right handed direction around the helix axis
when the axis is constrained to lie in a plane
-the actual number of helical turns in a DNA molecule

Writhe (Wr)
-a measure of the coiling of the axis of the double helix
-the number of turns of the axis about the superhelical axis
-the measure of the degree of coiling

Lk= Tw + Wr

Principle Behind Supercoiling

 Adding (or substracting) turns to a relaxed cccDNA imposes strain
 This strain causes cccDNA to contort into a new shape (e.g. simple figure – 8)
o  Supercoil

relaxed (+)
(-) supercoil
DNA supercoil
Lk 95 100 95
Wr -5 0 5
Tw 100 100 100
due to due to
underwiding overwinding
*e.g
extracted
DNA

Sample Problems
1. Given 3 cccDNA molecules of the same length and sequence but varying Lk. Predict their
expected configuration.

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# of
number pb expected SC turning
Lk Electrophoresis
of bp per configuration direction
turn
1000 100 10 relaxed

overwinding --
1000 200 5 clockwise
> (+) SC
faster than
relaxed
underwinding
1000 50 20 counterclockwise
--> (-) SC

2. The SV40 virus contains dsDNA with 5200 bp

a. Compute the theoretical number of forms of its DNA turns of a relaxed circular DNA
b. However, the isolated SV 40 DNA contained only 475 turns. Predict its configuration.

Biological Significance of Supercoiling

1. Removal of supercoil (strain) is energetically favored
2. Promote binding of proteins
3. Specific examples
a. Replication of ᶲX174
 Initiated only when it is SC
b. Transcription: RNA polymerase binding and activity
 (+) SC inhibits promoter recognition
 (-) SC enhance strand separation
c. Recombination
 SC needed in integration of λ DNA in E. coli DNA
4. Packing of DNA
a. Viral DNA, bacterial DNA
b. Increase in levels of coiling in eukaryotic DNA
5. Gene recognition
a. SC loop formation in DNA  wrap around proteins
 Same principle with hetero/euchromatin
b. SC  Z-DNA formation  recognition sites for proteins

TOPOISOMERS
 DNA molecules with the same length but different Lk
 Can be interconnected by connecting one or both strands and then rejoining them
o By topoisomerases
 Relieve torsional strain
 e.g. gyrase
1. Toposisomerase I
a. Monomeric protein; 100,000 MW
b. Break only one strand
c. Involved in the formation of “catenates”
d. E. coli Topoisomerase I relaxes (-) SC DNA without using external energy
e. Introduces a change of increments [n+1 or n-1] of 1 in Wr

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2. Topoisomerase II (DNA gyrase)
a. Two subunits
i. Gyr A
 105 kDa
 Inhibited by nalidixix acid and oxolinic acid
ii. Gyr B
 95 kDa
 Inhibited by coumermycin and novobiocin
b. Supercoils DNA at the expense of ATP hydrolysis
c. Relaxes both (-) and (+) SC DNA
d. Introduces a change in increments [n+2 or n-2] of 2 in Wr

Significance of Toposiomerase
1. Facilitate strand separation
 Replication, transcription, recombination
2. Assist structural transition in DNA
 Gene accessibility  expression
 E.g.
o BZ
o Cruciform formation

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July 5, 2012

THE PROKARYOTIC CHROMOSOME STRUCTURE

The Prokaryotic Model: E. coli chromosome

 A single cccDNA molecule
 Length: 4.6 Mb
 Replicated continuously
 Packaged into the nucleoid
o Nucleoid= DNA + proteins
 DNA
 Very high in [ ]
 30-50 mg/mL
 Proteins
 Polymerases
 Topoisomerases
 Packaging proteins

The E. coli Chromosome

 Negatively supercoiled DNA (not relaxed one packaging)
 The ccc DNA winds around itself
o More compact structure
o An ideal way of packaging
 Consist of 50-100 DNA domains or loops
 Each domain 50-100kB long
 The ends of each domain are constrained by binding to a membrane protein complex or
scaffold
 Individual domains are supercoiled independently
o Unwinding of one domain will not cause the other domain to unwind

Experimental Proof for DNA Domains

-electron microscopy
 DNA was carefully isolated free of attached proteins
 Introduction of a break in the DNA
 Rotation at the break site
 Loss of supercoiling in only a small segment of the molecule

Packaging Proteins in the Prokaryotic Chromosome

 Histone-like proteins (makes the DNA not naked)
 Essential for
o Packaging of the DNA into the nucleoid
o Stabilizing and constraining the supercoiling
 Protein Hu and H-NS
o Hu protein
 The most abundant
 Small basic [(+) charged attract (-) DNA] dimeric protein
 Binds DNA non-specifically
 Forms a tetramer, around which ~60bp of DNA becomes wound a form of
writhing or supercoiling
 ~60,000 Hu proteins per E.coli cell
 Evenly spaced along the DNA?
 Restricted to the core region?

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o H-NS protein
 Formerly “protein H1”
 A monomeric neural protein
 Binds DNA nonspecifically
 But seems to prefer regions of DNA which are intrinsically bent
PLASMID
 Extrachromosomal
 Small piece of circular DNA
 Coexist with the main chromosome
 Can integrate into the main genome
o Via integrons [(+) in all plasmids]
 Carry non-essential genes
o Resistance to antibiotics
o Fertility
o Degradative enzymes
o Virulence
But recently, these features of the E. coli chromosome were found to be absent in some prokaryotes…

Some Complications on the E. coli theme

1. The discovery of linear chromosme in some prokaryotes
e.g. Streptomyces
Borrelia burgdorferi (Lyme disease)
2. Discovery of “megaplasmids”
a. Vibrio cholera has 2 circular DNA molecules
i. 2.96 Mb (71% of the genome)
Genes for
 Central cellular activities
 Genome expression
 Pathogenicity
ii. 1.07 Mb
Genes for
 Central cellular activities
 Integrons (makes it a plasmid)
A megaplasmid
b. Borrelia burgdorferi B31
i. has 1 linear chromosome plus
ii. 17 or 18 linear ( a unique feature) and circular plasmids
iii. Essential genes are distributed in these
~some prokaryotes may have highly multipartite genomes

THE EUKARYOTIC CHROMOSOME STRUCTURE

Importance of folded DNA structure

1. Storage in the limited size of the nucleus
2. neat segregation into newly formed cells during cell division
3. Manner of folding determines the activity of the genes in a cell ( gene expression)
 tightly packed DNA  unexpressed Chromatin
low ionic strength solution
1879 Walter Flemming (<5mM)
 “banded structures” in eukaryotic nuclei “beads on a string” under EM
 “chromatin”
 “chromosome” Bead: DNA-histone complex
= nucleosome (basic unit of
the eukaryotic cell)
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Nuclease Protection Analysis of Human Chromatin

Goal: to determin the length of the DNA directly associated with the histone core

Principle: the DNA associated with the histone core is less susceptible to nuclease digestion

Conclusions:
1. In human chromatin, protein complexes are space along the DNA at regular intervals, one for
each 200bp
2. 146pb are closely attached to each protein complex
a. Nucleosome
i. H2A,
ii. H2B,
iii. H3,
iv. H4
b. H1: attached to the linker DNA

The Chromatosome = nucleosome + H1

Chromosomal Proteins
Histones
a well defined class of structural proteins
primarily for packing DNA into chromosome
high in Arg and Lys [basic aa:(+) charged -->
binds to (-) DNA]
enormous quantity per cell
highly conserved gene sequences
5 types: H2A, H3B, H3, H4,H1 and related
proteins (H5 may replaced H1 in some
organisms)
NHCP (Non-histone Chromosomal
Proteins)
hundreds of different proteins
affect packing of DNA to control gene
expression
variable aa composition
variable amounts; <<< histones
variable
very many types (RNA polymerases, regulatory
proteins, HMG 14 and HMG 17, other enzymes
and protein factors)

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THE AMINO ACID COMPOSITION OF HISTONES
H2A, H2B, H3, H4
 Exceptionally constant in aa composition (across species the same aa composition)
 100 out of 140 aa of H4 from cow to pea
 Crucial role (important)
o Almost no aa substation was tolerated
H1
 Much less conserved aa composition
 Vary except in the central region
 In certain tissues, H1 is replaced by special histones
o E.g. in the nucleated RBCs of birds, H5 is present in place of H1

Histone Genes
 In clusters (gene families, ~like in operons)
 Gene clusters in a given species are located at specific chromosomal regions
o E.g. human: long arm of chromosome 7
 Each gene is transcribed separately (unlike operons)
 Vary in different species in terms of:
1. number of clusters per genome
 Drosophila: 100 copies
 Mammals: 20
 Chicken: 10
2. Size of the spacers (not an intron, i.e., it is found in the gene itself) that separate
genes in a cluster
3. Gene order in a cluster
4. Direction and template strand for transcription

*mRNA sequence are conserved

Next: variability within the genome

SOLENOID
 A chromatin fiber: 30 nm wide
 Formed from the folding and twisting further of the 10nm chromatin fiber (beads on a string) into
a supercoil
o Adjacent H1 proteins are in contact with each other
o H1 not only linked nucleosomes but also responsible for solenoid formation
 A left handed helix
 6 nucleosomes per turn

Interwound Type of Supercoiling in the Eukaryotic Chromosome

 Even though eukaryotic DNA is linear
 Due to “folded loops” (what makes the linear eukaryotic DNA circular)
o Chromatin fiber is looped and held fast at the base of each loop by the nuclear matrix
 Topologically independent
 Each loop: ~100kb of DNA
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The Role of NHCP and Nuclear Matrix in the Formation of Folded Loops

Nuclear Matrix
 A protein scaffold
 Metaphase scaffold
 Filamentous structure in interphase cells found in the interior of the nuclear membrane where
chromatin often appears to be attached

Chromatin Packing and Gene Expression

 Active chromatin/ euchromatin ( transcriptionally active)
 DNAse hypersensitive (proteins are removed ~experiment in nuclease)
 With altered packing

Hypothetical Events on Chromatin Uncoiling

Possible Mechanism for Alteration in Packing

1. Reduced binding of H1
 Chromatin decondenses  activation of genes
2. Covalent modification of core histones  change the charge
 Alter DNA(acidic)-histone (basic) interaction
a. Acetylation (acidic) of lysines (basic) near NH2 end of H3 and H4
o A (+) is neutralized
o Chromatin decondenses activation of genes
b. Methylation of lysines
o Prevents acetylation
o Chromatin condenses inactivation of genes
c. Phosphorylation of selected serines and threonines
o Histones becomes more (-)
o Lesser attraction for DNA
o Chromatin decondenses  activation of genes
3. Linkage of ubiquitin H2A
 May lead to degradation or loss of H2A
 Ubiquitin
o Highly conserved protein with 76 aa
 Ubiquitous(+) in all eukaryotic cells
o Ligated to the lysine residues of a protein that will be degraded
o Ubiquitinated: will be acted upon by proteases
activation
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