Chapter 11

DNA Organization and Chromosomes

• There is a higher order structure of DNA within cells:
The chromosome
The genetic material of chromosomes can be:
– linear or circular molecules of nucleic acid
– single-stranded and double stranded
– RNA or DNA
• Whatever the nature of the genetic material, generally there has to be some level of
packaging in order to fit within the cell

Bacterial Chromosome (E. coli)

• Double-stranded DNA (dsDNA)
• 4.6 X 106 bps (4.6 Mb)
• Single circular chromosome,…no 5’ and 3’ ends
• 90% DNA, some protein
• Centrally located in the nucleoid, not membrane encased
• ~1,600 m in length [~1000 X E.coli (1 m)]
• How was this length calculated?
• How does it Fit??
Condensation of DNA by Supercoiling
• The nucleic acid within a cell complexes with a variety of different proteins that assist in
forming higher order, tertiary structures of the DNA, that results in tight packaging of
the material
• One type of higher order structure seen in bacteria and eukaryotes is supercoiling, or
coiling of a coil
Bacterial Chromosome
• Bacterial chromosome exists as a “covalently closed circle” or CCC
• A nick in one of the DNA strands forms a “nicked open circle”
• A nick in both of the DNA strands forms a linear DNA
• Supercoiling can only exist with CCC molecules
• Consider a telephone cord (before the days of cordless telephones)

Supercoiling

Covalently Closed Circle (CCC)  Supercoiled (SC)

• Negative supercoiling, counter-clockwise twist, untwisting, underwound

• Positive supercoiling, clockwise twist, overwound
• In order to introduce supercoiling, one end of the CCC molecule has to be anchored and
either one or both the the DNA strands has to be nicked
 Supercoiling by Topoisomerases
• Most bacterial DNA is negatively supercoiled
• The degree of supercoiling is usually mediated by special enzymes called
topoisomerases
• Supercoiling is a topological isomer of its relaxed double helical form
• Topoisomerases are therefore enzymes that alter the topology or shape of a DNA
molecule creating a spatial or topological DNA isomer
Relaxed versus supercoiled DNA

Topoisomerase Activity
• There are two major types of topoisomerases
– Topoisomerase I: introduces a transient single-stranded nick in DNA molecules
and relax negatively supercoiled DNA by removing one supercoil at a time
(adding a positive supercoil)
– Topoisomerase II: introduces transient double-stranded nicks in DNA molecules
and introduces negative supercoils, 2 supercoils at a time
– DNA gyrase is a special type of class II topoisomerases that is essential for DNA
replication. It reduces torsional stress at the DNA fork by introducing negative
supercoils (unwinds)
Bacterial chromosomes are organized into
supercoiled loops
Topoisomerase II can untangle DNA
• Each loop can be independently relaxed or
molecules condensed.

Schematic drawing of the

folded supercoiled E. coli
chromosome, showing DNA
loops (blue) attached to a
protein core (orange) and
The opening of loops by
nicks

Topoisomerase II untangles a pair of DNA molecules by cleaving one DNA duplex and
passing the other duplex through the gap
Eukaryotic Chromosome
• The eukaryotic genome consists of linear, dsDNA divided into multiple units: Each unit
represents a single chromosome
– Human haploid genome is 3 X 109 bp
• How much DNA is in each somatic cell?
– 23 chromosomes
• Complex structure made up of chromatin
– Stains intensely with dyes used to visualized chromosomes
– Compact complex of DNA and protein (~50:50)

Histone proteins
– Small basic proteins with a net positive charge allowing binding to DNA which
has negatively charged phosphate backbone
– Highly conserved
– Five histones: H1, H2A, H2B, H3, H4
Histone 1 : is used as clamp
• Non-histone proteins
– Most are acidic proteins, negatively charged, thus binding to the positively
charged histones
– Variable among cell types and among organisms and even vary with cell stage

Eukaryotic
DNA is
organized in
nucleosomes

Organization of nucleosomes b) Effect of treatment with micrococcal nuclease

Nucleosomes compact into

chromatin fibers

A model of the chromatin fiber in which the

DNA (blue-gray) is wound around each
nucleosome
Electron micrograph of the 30-nm
chromatin fiber in mouse chromosomes
 Stages in DNA and
chromatin
condensation in
eukaryotes

Stages in the condensation of DNA

Eukaryotic Chromosome Structure

• The metaphase chromosomes are the most compact form of eukaryotic chromosomes
• Characteristic structure of two sister chromatids held together at a constriction point
called the centromere
• The centromere is a functional unit of the chromosome that is involved in the
segregation of chromosomes during mitosis and meiosis mediated through attachment
of spindle fibers
• The structural ends of the chromsomes are called telomeres

Eukaryotic Chromatin
– Euchromatin: Genetically active, condensed during cell division and uncoiled
during interphase, lightly stained
– Heterochromatin: Genetically inactive, more highly condensed, darkly stained
• Constitutive: Always condensed; repetitive sequences of centromeres
and telomeres
• Facultative: Has the potential to become condensed; inactivated X
chromosomes in females forming Barr bodies
Heterochromatin is highly repetitive and
low complexity
• Heterochromatin is located near the
centromeres and telomeres

Interpretive drawing of the metaphase chromosomes

Sequence Complexity of Eukaryotic Genomes
• Prokaryotic genomes consist mostly of unique sequence DNA
• Eukaryotic genomes are much more complex and contain both unique and repetitive
DNA sequence
• Denaturation and renaturation of DNA is a technique that is used to determine the
sequence complexity of any genome

• The DNA is denatured by heating which melts the H-bonds and renders the DNA single-
stranded
• The DNA is allowed to cool slowly,and sequences that are complementary will find each
other and eventually base pair again

Heating of DNA leads to denaturation of the two

strands
• The rate of denaturation depends on the G+C
content of the DNA

Mechanism of denaturation of DNA by heat

• Single copy sequences

– Found once or a few times in the genome
– Human genome is ~ 64% unique sequence
• 1-3% protein coding
– Many of the sequences which encode functional genes fall into this class
• Repetitive Sequences
– Moderately repetitive
– Highly repetitive
– Tandemly repeated DNA
– Dispersed repeated DNA
Eukaryotic DNA consists of three major components
• Unique sequences
– 30-75%
• Middle repetitive sequences
– 1-30%
– 10-1000 copies per genome
• Highly repetitive sequences
– 5-45%
– as many as 105 copies per genome

Highly Repetitive DNA

• The most abundant sequences in regards to copy number
• ~10% of human genome
• These sequences are found from 100,000 to a million times in the genome
• Range in size from a few to several hundred bases in length
• These sequences are found in heterochromatin, in particular centromeric and telomeric
DNA regions
• Centromeric DNA is also called Satellite DNAs
• Tend to be arranged as a tandem repeats

Eukaryotic Centromere
• Function to assure precise segregation of chromosomes in meiosis and mitosis
– Hold sister chromatids together
– Site of microtubule attachment (kinetochore)
• Alpha satellite DNA repeat units in higher eukaryotic organisms
– 170 bp tandem repeat (5,000-15,000 times) X 23 chromosomes (115,000-
345,000/genome)
– Structurally important? in centromere function?

Structure of a yeast
centromere

• Centromere DNA has

four centromere
determining elements
(CDEs)

A Centromere from the yeast Saccharomyces cerevisiae

Eukaryotic Telomere
• Consist of simple sequence tandem repeat units
– 5’-TTAGGG-3’ (humans)
– 5’-TTGGGG-3’ (protozoan Tetrahymena)
• Required for replication and stability of linear chromosomes
• Repeat sequences added to ends of linear chromosomes by telomerase
• Relationship of telomere length and age of cell (Hayflick Limit)
Cancerous cells remain young and continuously dividing
Replication of telomere DNA repeats by
telomerase

function of telomerase

Telomerase contains an internal RNA

template
• The RNA molecule serves as the template for
telomere DNA synthesis

Telomere formation in Tetrahymena

 Transposon structure

The transposon can exited and move somewhere and cause a mutation.
 Organelle DNA Has Unique
Characteristics

• Mitochondria and chloroplasts contain

DNA
• Encodes some polypeptides used by the
organelle, rRNA, and some tRNAs

Organelle DNA Has Unique Characteristics

• Endosymbiotic theory: proposes that mitochondria and chloroplasts were once free-
living bacteria
• Both organelles are similar to eubacteria, and DNA sequences found within them are
also similar to eubacteria

Traits Encoded by mtDNA

• erility

The Mitochondrial Genome

• High mutation rate in vertebrate mtDNA
• Number of genes and organization remains relatively constant
• Most copies of mtDNA identical

Damage to Mitochondrial DNA is Associated with Aging

• Many human genetic diseases associated with mtDNA appear in middle age or later
• Oxidative phosphorylation capacity declines with age; those with mutations in mtDNA
start life with decreased oxidative phosphorylation capacity
• Mechanism of age-related mtDNA damage unknown

Concepts
1.Transcription: DNA to RNA
2.Reverse transcription: RNA to DNA (reverse transcriptase is the enzyme)
3. Nuclease: Enzyme that degrade nucleic acid
4. Exonuclease:
5 Endonuclease:

-Telomerase is a reverse transcriptase (it makes DNA using RNA template)