2

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 Organism

Cell Structure & Function

Disease Proteins
Mutation DNA Replication
Variety Perpetuation
of Life of Life
Genetic Engineering
Genetic Testing
Engineered
Biopharmaceuticals
Life Forms
• What characteristics make
something a “living thing”?

• Where do these
characteristics come from?
 GENES
• Mendelian Genetics
– HEREDITY: Traits or characteristics are
passed on from one generation to the next as
discrete entities called GENES
– Two copies of each gene (one from each parent)
– What is the physical nature of the gene?
– Less than 100 years ago showed these “genes”
are on chromosomes
 Molecule of Life,
in the Chromosomes

85% protein - chemically complex

15% DNA - chemically simple

 What is the Molecule of Life?

We now know because of the work by…

1944 Avery and co-workers

1952 Hershey & Chase

1952 Watson & Crick

Oswald Avery, Colin MacLeod and Maclyn McCarty
built on work done previously done by Frederick Griffith:

Griffith (1929)
Pneumococci
Smooth Form
Rough form
Transformation

Virulent
Avirulent
• Nonvirulent cells change to virulent cells

• Change in the characteristics of the cells

• Changed by the heat killed cells (or a

component of the cells)

• Is this transforming factor the “Molecule

of Life”?
 Avery and Co-workers

Pneumococci

Rough form Smooth Form

Cell extract from

Smooth form
Virulent
(Transforming factor)
Purification of Transforming Factor

Could you test this further?

The rough cells are transformed with the gene
encoding the enzyme to make the polysaccharide
coat.

Rough bug
Smooth bug DNA
cell wall

+ chromosomal DNA
gene encoding
polysaccharide
coat enzyme
transformation

polysaccharide coat

smooth bug
• Alfred Hershey and Martha Chase

• Different experiment

• Also supported the hypothesis that DNA

was the “Molecule of Life”

• Used T2 bacteriophage

• Radioactively labeled the DNA and Protein

Bacteriophage (just DNA & protein)
• Bacteriophage changes the characteristics of
the bacteria. Sounds like the Molecule of
life…

• Track the Bacteriophage DNA and Protein

using radioactivity

• Is 35S (Protein label) passed on?

• Is 32P (DNA label) passed on?

• Is the molecule that is passed on the

“Molecule of Life”?
• Is this enough data to convince
scientists that chemically simple
DNA could be the “Molecule of
Life” and carry all that genetic
information?

But wait……
There’s more!
 Watson & Crick Paper
Nature 171, 737-738 (1953) WATSON, J. D. & CRICK, F. H. C.
Medical Research Council Unit for the Study of Molecular Structure of Biological Systems, Cavendish Laboratory,
Cambridge.

A Structure for Deoxyribose Nucleic Acid

We wish to suggest a structure for the salt of deoxyribose nucleic acid (D.N.A.). This structure has
novel features which are of considerable biological interest.

Figure 1

This figure is purely diagrammatic. The two ribbons symbolize the

two phophate-sugar chains, and the horizonal rods the pairs of
bases holding the chains together. The vertical line marks the
fibre axis.
 Watson & Crick

Cellular DNA exists as a double helix:

• Two anti-parallel polynucleotide strands

• Held together by complementary base pairs

Deduced from molecular modelling studies using:

• X ray diffraction data

• Chargaff’s rule
 X-Ray Picture Looking Down the
End of a DNA Fibre

X-ray diffraction pattern. (Rosalin Franklin/Science Source/Photo Researchers.)

 Chargaff’s Rules
DNA source A T G C

Human 30.3 30.3 19.5 19.9

Rat 28.6 28.5 21.4 21.5
Sea urchin 32.8 32.1 17.7 17.7
Wheat 27.3 27.2 22.7 22.8
Yeast 31.7 32.6 18.3 17.4
E. coli 26.0 23.9 24.9 25.2
Mycobacterium 15.1 14.6 34.9 35.4

The amount of Adenine in a cell equals

the amount of Thymine.

The amount of Cytosine in a cell equals

the amount of Guanine.
 Structure - base
• 4 bases in DNA: A, G, C, T
• 4 bases in RNA: A, G, C, U
DNA bases can form hydrogen bonds to “pair up”:
 base + sugar = nucleoside
base + sugar + phosphate = nucleotide

Monomer of DNA =nucleotide

(Deoxynucleotide)

Base

PO4
5’

4’ 1’

3’ 2’
OH
Note:
Specific base pairing,
Hydrogen bonds,
Antiparallel stands,
Positions of Sugars, Phosphates and Bases
• DNA consists of 2 polynucleotide chains wound around each
other in a clockwise manner to form a double-helix
• The 2 chains are anti-parallel
• The sugar-phosphates are on the outside of the helix, and
the bases on the inside
• Base pairing is specific: A to T, and C to G
• The 2 chains are held together by hydrogen bonds between
bases: 2 between A and T, 3 between C and G
• One turn of the helix contains 10 bases
• The helix is uneven, forming major and minor grooves
• A nitrogenous base is linked to a single ribose sugar which is
linked to a phosphate group (base + sugar + phosphate =
nucleotide)
• Learn the structure of DNA!
 DNA = very long, thin molecule.
How does this long molecule fit into the nucleus of a eukaryote?
Histone proteins and multiple levels of packaging.
 Central Dogma of
Molecular Biology

Explore this central dogma in the next lectures…

Molecular Genetics
GENE 2000

DNA Replication
Week 1 – Part 2

But first let’s discuss a

potential exam question…
ELECTRONIC WARNING NOTICE FOR
COPYRIGHT STATUTORY LICENCES

WARNING

This material has been reproduced and communicated to you by

or on behalf of Curtin University in accordance with section 113P
of the Copyright Act 1968 (the Act)

The material in this communication may be subject to copyright

under the Act. Any further reproduction or communication of this
material by you may be the subject of copyright protection under
the Act.

Do not remove this notice.

Good uni students and scientists read widely.
Reasons to do this include:
• Reading about areas which you don’t fully understand
from the lectures/labs.
• Expanding on unit content that you find interesting.
• General reading to broaden your knowledge.
• Learning latest developments.

How would you answer this question?

Show your broad knowledge of Molecular Genetics
by briefly discussing an area that you have read
about (in any scientific textbook, or scientific
journal). It should be an area that has not been
covered in the lectures. Include citation.
Central Dogma of Molecular
Biology
 DNA Replication
If DNA was the “molecule of heredity” it must be able to be
passed on from one generation to the next

ie, must be capable of being replicated

Watson & Crick Model of DNA Structure

• proposed how DNA could be replicated

• predicted that DNA replication would be semiconservative

 Semiconservative Model
of DNA Replication
5’-ATGGCTACAAAGTCACAT-3’
3’-TACCGATGTTTCAGTGTA-5’

5’-ATGGCTACAAAGTCACAT-3’
+
3’-TACCGATGTTTCAGTGTA-5’
Semiconservative
Replication

Old 5’-ATGGCTACAAAGTCACAT-3’
New 3’-TACCGATGTTTCAGTGTA-5’
+
New 5’-ATGGCTACAAAGTCACAT-3’
Old 3’-TACCGATGTTTCAGTGTA-5’
Matthew Meselson and Franklin
Stahl experiment in 1958

Demonstrated that DNA replication is semiconservative

Support for Watson & Crick model of DNA structure

 Meselson-Stahl Experiment
Hi Guys

E. coli

• Grew bugs in media containing heavy N15 as a nitrogen

source.
– All DNA contains N15 in its bases

• Transfer bugs to fresh media containing N14 as a nitrogen

source
– Newly synthesised DNA will contain N14 (“light”).
– Old DNA N15 (“heavy”).
 Ultra Centrifugation
CsCl Gradients
Centrifuge at ~ 50,000 rpm for 2-3 days

DNA in CsCl solution

in centrifuge tube

Centrifugal force
Bugger !
Low CsCl High CsCl
concentration concentration

Less dense DNA Dense DNA

 Predicted Results
• If DNA replication was semiconservative
N15
Parents

Heavy
N14
1st generation
Intermediate
2nd generation

Intermediate Light Intermediate

Hypothesis accepted! DNA Replication is Semiconservative
 DNA Polymerase
DNA Polymerase incorporates complementary bases.
Requirements include:
- Single stranded DNA template
- deoxy nucleotide triphosphates (dATP, dCTP, dGTP, dTTP)
- Primer
DNA is synthesised in a 5' to 3' direction

Mg++
 •DNA is double stranded
•Replication occurs at a Replication Fork

Replication Fork

Direction of Replication
Paradox ?!
• Replication Fork migrates in one direction only

• DNA polymerase synthesises DNA in 5’ to 3’ direction only

• Two DNA strands used as templates are antiparallel

 Solution:
• One new strand of DNA is synthesised in a continuous
manner
= LEADING STRAND

• The other new strand is synthesised in a discontinuous

manner
= LAGGING STRAND

DNA replication semi-discontinuous

Party Time at the Replication Fork!

More than just DNA Polymerase

 Lagging Strand Synthesis

DNA Polymerase I has a 5’-3’ polymerase activity and a 5’-3’ exonuclease activity
DNA Replication In vivo
• Origin of replication
• Replication bubble forms
• Replication fork forms
• Strand separation (helicases + SSB proteins)
• RNA primers (DNA Pol α /primase in primosome)
• Strand elongation (DNA polymerase ε, δ/ III)
• Strands kept untangled (DNA topoisomerases)
• RNA primers removed (eg DNA polymerase I)
• Single stand breaks sealed (DNA ligases)
Start at Replication Origin / RO/ Ori Site
Replication Bubble > two Replication Forks
Primers form and strand elongation
 Prokarytoes Eukaryotes

RNA primer primase Polymerase a

/ Primase Complex
ssBP 1 subunit 3 subunits

Lagging strand DNA polymerase III Polymerase d

synthesis

Leading strand DNA polymerase III Polymerase d/ pol e

synthesis
Okazaki fragments 1000- 2000 nt 100-200nt

Speed of synthesis 1 1/10

Are other DNA Polymerases. 5 DNA Polymerases in bacteria,

5 families (up to 15 different polymerases) in eukaryotic cells.
 Similar in eukaryotes
but more complicated
Consider
nEukaryotes - unwind chromatin (DNA packaging was
discussed at the end of the last lecture)

Modes of replication
nProkaryote – circular chromosome

nEukaryotes – linear chromosomes

Circular DNA Replication Mode:
Circular DNA Replication Mode:
Linear DNA Replication:
 DNA replication tightly regulated
Eukaryotic Cell Cycle
Chromosomes
visible
M

G2

G1

S
DNA
Replication
DNA replication occurs at a specific time in the cell cycle - S phase
M = mitosis or meiosis
G1 = growth phase 1
S = DNA synthesis phase
G2 = growth phase 2

G1 + S + G2 = interphase
• Chromosomes
- Long linear DNA molecules
• Multiple origins of replication
• Replicons expand in bidirectional manner
- Adjacent replicons fuse
 Telomeres
Getting to the End
Telomere Centromere Telomere

Ori’s

TTAGGG TTAGGG TTAGGG TTAGGG TTAGGG TTAGGG TTAGGG T TAGGG

AATCCC AATCCC AATCCC AATCCC AATCCC AATCCC
5’-P 3’-OH

(TTAGGG)n where n > 1000 The End

Telomere act as a buffer protecting the genes

near the end of the chromosome
 End Replication Problem
• Each time cell replicates telomere shortens
– 50 to 100 bp of DNA lost per division

• Due to inability of DNA polymerase to replicate 3’ end of

chromosome
– lagging strand synthesis

Lagging Strand RNA primer removed last RNA primer

DNA pol cannot

Next Generation fill in gap

RNA primer removed

Next Generation
 Telomeres & Mortality

• Normal cells have finite replicative capacity

– Hayflick limit
– approx. 70 divisions then cells become senescent
(arrested in G1)
– fibroblasts taken from old people undergo fewer
generations in culture than cells taken from young people
• Replicometer counting mechanism that measures telomere
shortening
– After 70 shortenings cell division is stopped
 Telomerase & Immortality

Normal Cells Cancer Cells

• Cancer cells are immortal

– grow indefinitely in culture
• Telomeres of cancer cells do not shorten
• Produce enzyme called Telomerase
– ribonucleoprotein
• Telomerase uses its RNA as a template to fill in the gap left
at the end of the telomere
 Telomerase - End Filling

Telomerase carries a short RNA molecule that acts as a template for the addition of
the complementary DNA sequence at the 3’ end of the double helix. In the ciliate
Tetrahymena, the DNA sequence added is TTGGGG.

Telomerase - active in cancer cells and during

early embryonic development
 Telomeres & Longevity

• Correlation between no. of generations that cells

can under go in culture and longevity of the
species
– Humans live longer than other mammals and human cells
can under go more generations compared to other
mammalian cells

• Genetically engineered mice that have a mutated

telomerase gene
– age more rapidly and die earlier than normal mice
 Dolly
from adult cell Baa

Mum!

• Cloned from adult cells

– preshortened telomeres
– premature aging?
• Evidence that nuclear transfer resets telomere clock?
• Other issues and problems
• Potential for Immortality ?