CEL101:

REPLICATING AND REPRODUCING CELLS

Dr Ethan Newman – Botany Department, Room 212

e.newman@ru.ac.za
So far we have looked at the changes that occur in whole chromosomes throughout the cell cycle,
leading up to mitosis, as well as during cell division.

In this lecture we will examine chromosomes in more detail by deconstructing them down to the
basic units that they are made of – monomers called nucleotides – and then look at how these
monomers join up to form polymers called nucleic acids. This is DNA, and nucleic acids is the
overall name for DNA and RNA
S-phase

Most of this section will deal with the processes

that occur in the S-phase represented by the cell in
the middle, (above) in which each chromosome has
been replicated and now consists of two sister
chromatids.

The condensing of the chromosomes (above right)

marks the beginning of the mitotic phase, which we
covered in the last few lectures.
NEXT up we ask: What is the structure of DNA (and also RNA), and how is it
replicated with such accuracy?
THE STRUCTURE OF NUCLEIC ACIDS:
Deoxyribose Nucleic Acid & Ribose Nucleic Acid

Page 105 to
117
THE STRUCTURE OF NUCLEIC ACIDS:
Monomer is a
Nucleic acids are polymers made up of molecule forming a
monomers called nucleotides. single subunit
within a larger
monomer
A DNA double helix (left),
and a single ‘unit’ or monomer
called a nucleotide (below).
WHAT ARE THE COMPONENTS OF A NUCLEOTIDE?

A nucleotide is a monomer
made up of three main
components:
a phosphate group bound to
a 5-carbon sugar, which in
turn is bound to a
nitrogenous base.

Pg 106 of
textbook
 THE 3 COMPONENTS OF A NUCLEOTIDE: 1. THE SUGAR

Do not worry about the level of detail here, just focus on the differences between the two
sugars with respect to carbon 2 (C2).

• The ribose in ribonucleotides has a hydroxyl group (–OH) at C2, while the deoxyribose
has lost the oxygen and only has hydrogen (H) in this position. But on C3, both ribose and
deoxyribose sugars have an hydroxyl group (-OH)
 THE 3 COMPONENTS OF A NUCLEOTIDE: 1. THE SUGAR

Ribose, found in RNA, is a "normal" sugar, with one oxygen atom attached to each carbon
atom. Deoxyribose, found in DNA, is a modified sugar, lacking one oxygen atom (hence
the name "deoxy").
 THE 3 COMPONENTS OF A NUCLEOTIDE: 1. THE SUGAR

RNA DNA

Ribose, found in RNA, is a "normal" sugar, with one oxygen atom attached to each carbon
atom. Deoxyribose, found in DNA, is a modified sugar, lacking one oxygen atom (hence
the name "deoxy").
THE 3 COMPONENTS OF A NUCLEOTIDE: 2. THE BASE

There are two structural groups of

nitrogen-containing bases in nucleotides.

PYRIMIDINES (smaller – 6 atoms) –

Cytosine, Thymine, Uracil

PURINES (larger – 9 atoms) – Adenine and

Guanine

A, G, C and T are found in DNA

A, G, C and U are found in RNA
 THE 3 COMPONENTS OF A NUCLEOTIDE: 3. THE PHOSPHATE
The potential energy stored in these
(d)NTPs drives the polymerisation reaction
in a growing strand of nucleic acid.
The phosphate group of a nucleotide comes from nucleoside
triphosphates, or “activated” nucleotides.
• ATP is an example of an activated nucleotide.
The N-containing base in this case is adenine (A), but it can
also be C, G or U, as in CTP, GTP etc. or collectively, NTPs.
Note that the nucleoside on the left has a ribose sugar, and
NTP is correct for this, but if referring to a nucleoside/nucleotide
of DNA, you use the abbreviation dNTPs –
deoxyribonucleoside triphosphates. (Also nucleosides contain
only a sugar and a nitrogen base, whereas nucleotides
contain a sugar, a nitrogen base and a phosphate group)
dNTP’s - Deoxyribonucleoside
triphosphates

Deoxyribose
 THE SYNTHESIS OF DNA
From nucleotide monomers to nucleic acid polymers

Nucleic acids (DNA/RNA) form when nucleotides

polymerize through phosphodiester linkages which is the
result of a condensation reaction!
 How do nucleic acid polymers form?

Activated nucleotide• The potential energy stored in activated nucleotides (see

top left slide!) is released when inorganic pyrophosphate
(PPi) is removed by hydrolysis before the polymerising
condensation reaction occurs.

• The condensation reaction causes nucleotide

polymerization through the formation of phosphodiester
linkages between the hydroxyl group (OH) of the 3’
carbon of one nucleotide, and the phosphate on the
5’carbon of the next nucleotide.

• A single water molecule (H2O) and inorganic

pyrophosphate are consequently released.

• This gives rise to DNA or RNA.

 Pyrophosphate and water molecule
released

Activated nucleotide
Polymerisation forms single strands of DNA and RNA – (Nucleic acid)

• Nucleic acids are polar (or directional) molecules. Why?

They have an unlinked phosphate group on the 5’ carbon,

and an unlinked hydroxyl group on the 3’ carbon,

• Polymerisation occurs in the 5’  3’ direction.

 THE PRIMARY STRUCTURE OF DNA & RNA IS THE SAME
Note that at this level of organisation they only differ in one of their pyrimidines
(thymine in DNA, Uracil in RNA& sugars (ribose and deoxyribose).

Summary of PRIMARY STRUCTURE

In both, nucleotide monomers join to form nucleic acid

polymers.
• Each monomer is made up of a 5-carbon sugar, a phosphate group and
a nitrogenous base.

• Nucleotide monomers are joined together by phosphodiester bonds. This

links the 3’carbon of one sugar to 5’ carbon of the next sugar.

• This condensation reaction uses energy from nucleoside triphosphates.

• Polymerisation always happens in the 5’ to 3’ direction.

 How do nucleic acid polymers form?

Activated nucleotide• The potential energy stored in activated nucleotides (see

top left slide!) is released when inorganic pyrophosphate
(PPi) is removed by hydrolysis before the polymerising
condensation reaction occurs.

• The condensation reaction causes nucleotide

polymerization through the formation of phosphodiester
linkages between the hydroxyl group (OH) of the 3’
carbon of one nucleotide, and the phosphate on the
5’carbon of the next nucleotide.

• A single water molecule (H2O) and inorganic

pyrophosphate are consequently released.

• This gives rise to DNA or RNA.

Khanacademy.com
What are genes made of?

DNA or PROTEIN?
 A BIT OF HISTORY:
DNA, the information carrying molecule

• Since the 1800s it was known that chromosomes are a complex of DNA and proteins, but which
carries the hereditary information was unknown i.e. GENES?

• The most popular hypothesis was that protein carried genes. Why?

Because there is almost limitless variation in the structure and function of proteins, compared to DNA.
DNA is a macromolecule based on a repetitive four nucleotide code: A, G, C, T.
DNA in the 1950s – Answering long-standing questions

The Hershey-Chase Experiment (1952)

Their aim:
To determine whether hereditary, genetic
information is carried on DNA or
proteins?
 Hershey-Chase Experiment (1952)
(refer to pages of textbook: 5th ed. 375-376; 6th ed. 361-362)

DNA contains
phosphorous but not
sulphur Proteins contains
sulphur, but not
phosphorous and
 THE SECONDARY STRUCTURE OF DNA & RNA IS DIFFERENT

Secondary structure
Single-stranded molecules of RNA and DNA each form
secondary structures, but they do it differently.

• Complementary base pairing occurs in both.

• RNA remains single stranded and folds into a stem-and-loop structure.

• In DNA two single strands “bond” to form a double helix.

 THE SECONDARY STRUCTURE OF DNA & RNA IS DIFFERENT

DNA forms a DOUBLE HELIX RNA forms a HAIRPIN that has

STEM & LOOP regions

Note: Complementary base pairing occurs in both nucleic acids, so there are
double-stranded regions in RNA but this is formed from bonding between short
sections of the single-stranded molecule.
 COMPLEMENTARY BASE PAIRING IN DNA

• A DNA molecule is made up of two linear polymers that run in opposite

directions, only certain bases pair up and are held together inside the helix.

• Antiparallel - the polarities of the two

nucleic acid strands run in opposite
directions.

• Hydrogen bonds between bases hold the

strands together.

• Complementary base pairing occurs:

G always pairs with C; A always pairs with T.

 DNA double helix

• Hydrophobic bases are tightly

packed inside the helix.

• Phosphates and sugars form the

backbone of the helix where they
interact with polar solvents e.g.
water, that surround the molecule.
 Complementary base pairing via hydrogen bonds

• G-C pairing is stronger as there

are three hydrogen bonds

• A-T pairing has only two H-bonds.

• Why only purine-pyrimidine pairs?

Why purine-pyrimidine pairs only?
DNA in the 1950s – Answering longstanding questions

Discovery of the DNA double helix (1953)

 Photo 51

The Nobel Prize in Physiology or Medicine 1962

Awarded jointly to Francis Crick, James Watson and Maurice Wilkins

"for their discoveries concerning the molecular structure of nucleic acids and its
significance for information transfer in living material."
 Photo 51

In January 1953, Wilkins changed the course of DNA history by disclosing, without
Franklin's permission or knowledge, her Photo 51 to competing scientist James Watson,
who was working on his own DNA model with Francis Crick at Cambridge.

Upon seeing the photograph, Watson said, "My jaw fell open and my pulse began to race,"
according to author Brenda Maddox, who in 2002 wrote a book about Franklin
titled Rosalind Franklin: The Dark Lady of DNA.
Replicating and Reproducing cells
By the end of this section you should understand and be able to explain the following:

• The components of nucleotides.

• How nucleic acids are formed

• The primary and secondary structure of DNA and RNA

• Differences between DNA and RNA (with regards to primary and

secondary structure)
DNA REPLICATION IN THE CELL:

Getting the process started. DNA Replication

L1. Origin of replication and the opening and

unwinding of DNA
L1. Synthesising the leading strand

L2.Synthesising the lagging strand

L3. Replicating the end of the chromosome

(Telomere)
DNA REPLICATION IN THE CELL:

Getting the process started. DNA Replication

L1. Origin of replication and the opening and

unwinding of DNA

Origin of replication
 Replication bubble

DNA REPLICATION IN THE CELL:

Getting the process started.

Replication bubble

Replication bubble

Replication bubble
DNA REPLICATION IN THE CELL:

Getting the process started.

Origin of replication
Replication proceeds in the 5 prime to
3 prime direction from each starting
point
DNA Replication Fork
 DNA REPLICATION:

Opening and unwinding the DNA

1
 DNA REPLICATION:

Opening and unwinding the DNA

2
 DNA REPLICATION:

Opening and unwinding the DNA

2
 DNA REPLICATION:

Priming the DNA: First step in DNA synthesis

4 3

2
Why does DNA replication require an RNA primer?
Why does DNA replication require an RNA primer?

• Complementary base pairing

occurs:

G always pairs with C; A always pairs

with T.
Why does DNA replication require an RNA primer?

This primer even provides DNA polymerase

with a hydroxyl group that can be linked with
a deoxyribonucleotide to form a
phosphodiester bond
 DNA REPLICATION:

Synthesising the leading strand

 DNA REPLICATION:

Synthesising the leading strand

 DNA REPLICATION:

Synthesising the leading strand

 CHROMOSOMES UNRAVELLED

Histone Octamer
(H2A, H2B, H3, and H4)

• Chromatin is the most basic level of structural organisation of a chromosome.

• Chromatin is made of double-stranded DNA (dsDNA) and histone proteins

that form nucleosomes linked by short stretches of DNA.

• Nucleosomes: 8 globular histone proteins with 146 base pairs of dsDNA

wrapped around them. Between each nucleosome there is linker DNA which
is usually 20 – 80 bps long.

• Another histone, H1 holds the linker DNA and nucleosome together.

What happens to the nucleosomes
during DNA replication?

Chromatin disassembly on old DNA:

• During DNA replication, histone
proteins are removed from the
parental chromatin strand ahead of
the DNA replication machinery

• 225–285 bp of naked DNA remains

behind the replication fork.

Chromatin re-assembly on new DNA:

• Histones H3/H4 are first deposited
to form the H3/H4 heterotetramer
on DNA, followed by addition of two
heterodimers of histones H2A/H2B
by histone binding proteins called
“histone chaperones” on the naked DNA
Why does DNA replication require an RNA primer?

This primer even provides DNA polymerase

with a hydroxyl group that can be linked with
a deoxyribonucleotide to form a
phosphodiester bond
 DNA REPLICATION:

Synthesising the leading strand

 DNA REPLICATION:

Synthesising the leading strand

 DNA REPLICATION:

Synthesising the lagging strand

 DNA REPLICATION:

Synthesising the lagging strand

 DNA REPLICATION:

Synthesising the lagging strand

 DNA REPLICATION:

Synthesising the lagging strand

 DNA REPLICATION:

Synthesising the lagging strand

 DNA REPLICATION:

Synthesising the lagging strand

 DNA REPLICATION:

Synthesising the lagging strand

Page 383 of
the textbook

https://www.yourgenome.org/video/dna-replication
 DNA REPLICATION:

Synthesising the lagging strand

3D animation of DNA replication

https://www.yourgenome.org/video/dna-replication
DNA REPLICATION:

Replisome
 THE DYNAMIC CELL

An amazing 3D animation of the cell showing DNA replication and separation of

chromatids, with details of what is going on at the kinetochore.
9 mins - DNA starts at 3min; single chromosome at 6.10 mins; kinetochore at 7 mins.
Well worth watching!
https://www.ted.com/talks/drew_berry_animations_of_unseeable_biology
 Origin of Replication
Prokaryotes vs. Eukaryotes

Single origin of replication multiple origins of replication

Origin of Replication: PROKARYOTES
 DNA REPLICATION

How do you make new strands of DNA from existing ones?

Three hypotheses have been put forward:

• conservative,
• semiconservative,
• dispersive.

Refer to pages 363-373 of Freeman, 6th edition.

 Three models of DNA replication.

Keep in mind that all bases (A, G, C, T) have to be accessible in order to be copied.
 DNA REPLICATION IS SEMICONSERVATIVE

We now know that DNA replication is semiconservative i.e. all double-

stranded DNA comprises one template strand (old strand) bound to a
newly synthesized strand.
 DNA REPLICATION:

Replicating the telomere and the associated problem

• See pages 385 – 387 of

course text (Freeman et al.,
5th edition),

and/or

• The Khan Academy article

on RUConnected:
Telomeres and Telomerase
 DNA REPLICATION: Telomere: -
literally the end
End replication problem part of the
chromosome.
 DNA REPLICATION:

End replication problem

 DNA REPLICATION:

Telomere replication
 DNA REPLICATION:

Telomere replication
 DNA REPLICATION:

Telomere replication
 Some things to know
and think about.

Do not treat this as a complete list of what you must know

– it is a guide ONLY!

• Do you know the names and functions of the enzymes involved in DNA
synthesis (replication)?
• What is the function of an RNA primer in DNA synthesis, and why is it
necessary?
• Presented with a diagram that shows an ‘origin of replication’ and a
replication fork in dsDNA, how would you distinguish between the leading
and lagging strands?
• Which strand do you think would have more work for DNA ligase to do?
Explain why.
 Useful video resources from Khan Academy

Introduction to nucleic acids and nucleotides (6 mins)

https://www.khanacademy.org/science/ap-biology/gene-expression-and-regulation/dna-and-rna-structure/v/introduction-to-nucleic-acids-and-nucleotides?modal=1

DNA (13 mins)

Overview of DNA bases, complementary base pairing, and the structure of the double helix.
https://www.khanacademy.org/science/ap-biology/gene-expression-and-regulation/dna-and-rna-structure/v/dna-deoxyribonucleic-acid?modal=1

Antiparallel structure of DNA strands (10 mins)

DNA is composed of two strands of nucleotides held together by hydrogen bonding. The strands each run from 5' to 3' and run in antiparallel,
or opposite, directions from one another.
https://www.khanacademy.org/science/ap-biology/gene-expression-and-regulation/replication/v/antiparallel-structure-of-dna-strands

Leading and lagging strands in DNA replication (10 mins)

Roles of DNA polymerase, primase, ligase, helicase and topoisomerase in DNA replication. An explanation of leading and lagging strands.

https://www.khanacademy.org/science/ap-biology/gene-expression-and-regulation/replication/v/leading-and-lagging-strands-in-dna-replication
Other useful resources

DNA Structure (6 mins)

https://www.youtube.com/watch?v=o_-6JXLYS-k

DNA Replication animation (3 mins)

https://www.youtube.com/watch?v=TNKWgcFPHqw
 Some things to know
and think about.

Do not treat this as a complete list of what you must know

– it is a guide ONLY!

• Historically it was thought that proteins carried hereditary information. Why

was this the case?
• What questions did Franklin’s Photo 51 contribute to answering?
• DNA replication is described as semiconservative. Explain what this means
and be able to draw a simple diagram to support your explanation.
• What are the other two models of DNA replication, and what was the
experiment that demonstrated that they were not correct?
How is DNA extracted?
How is DNA extracted?

Charles Darwin, tree-of-Life sketch from notebook B, 1837. Reproduced by kind permission of the syndics of
Cambridge university Library. ms.DaR.121:p36.
E.g. Chloroplast genome
with different genes

Charles Darwin, tree-of-Life sketch from notebook B, 1837. Reproduced by kind permission of the syndics of
Cambridge university Library. ms.DaR.121:p36.
Gladiolus carneus
Chloroplast DNA
• MatK

• psbA-trnH

• trnS-trnG

• rpl32-trnL

• trnQ-rps16

Chloroplast genome
Chloroplast genome
 A BIT OF HISTORY:
DNA, the information carrying molecule

• Knowing the base pairing rules led Watson & Crick to realise that DNA acts
as a template for its own replication e.g. if A is present at position 6 of the
template strand, then T must be added at that position in the new strand etc.

Significance?

• It maintains the integrity of genetic information across many, many

replication events,

• from cell to cell during cell division (growth),

• and down through generations of organisms via the formation and fusion
of gametes (sexual reproduction).