Replication and the genetic code

• When a cell divides, an exact copy of the DNA must be

produced so that each of the daughter cells receives a
copy. This process of copying the DNA is called replication.
DNA replication
• 1- The DNA double helix unwinds from one end and the two strands separate as
the hydrogen bonds between the bases break catalyzed by the action of DNA
helicase.
• 2- Free DNA mononucleotides line up alongside both DNA strands and hydrogen
bonds form between the complementary base pairs catalyzed by DNA
polymerase( A with T and C with G).
• 3- The enzyme DNA ligase catalyses the linking of the adjacent nucleotides by
condensation reaction (phosphodiester bond) to form a complementary strand.

• In this way each strand of DNA acts as a template on which a new

strand is built and, overall, two complete DNA molecules are
formed.
• These are identical to each other and to the original DNA molecule.
• Each of the two DNA molecules now contains one ‘old/original’
strand and one ‘new’ strand. This process is therefore known as
semi-conservative replication.
 Process of DNA replication

Parent molecule New strand

ssDNA ssDNA

Addition of
New strand Parent molecule
Denaturation New nucleotides ssDNA ssDNA

Parent molecule
Parent molecule Parent molecule Parent molecule Parent molecule
dsDNA ssDNA ssDNA ssDNA ssDNA

DNA
DNA helicase DNA ligase
polymerase
 Process of DNA replication

1- The DNA double helix unwinds from one end and the two strands
separate as the hydrogen bonds between the bases break catalyzed
by the action of DNA helicase.
 Process of DNA replication

Template strand

New strand
Already added ACG
 DNA Polymerase Parental strand
Adds nucleotides New strand = “Template”
5‘ end 3‘
One by one end

Nitrogenous base

 The parental strand is Sugar

used as a template Phosphate

 To know which nucleotide

is to be added next
By complementary base pairing
They join the parent strand by
3‘ end
Hydrogen bonds

A T
T A
C G dTTP)
Will be added 5‘ end
G C
 Process of DNA replication

Template strand

New strand
Already added ACG
Parental strand
New strand = “Template”
5‘ end 3‘
end

Nitrogenous base

DNA ligase NEEDS the 3’OH Sugar

New strand and
from the last added nucleotide
Phosphate
template strand
to add another nucleotide are antiparallel
Via making a phosphodiester
bond

3‘ end

A T
T A
C G
5‘ end
G C
 Process of DNA replication

Template strand

New strand
Already added
Parental strand ....ACGT
New strand = “Template” Parental strand
New strand = “Template”

DNA
Polymerase

A T
T A
C G
G C
 Process of DNA replication Template strand

Replication of two DNA strands “manually” New strand

5' 3'
aatgtagtagtaaactggtgaaatgcaaggtatttattgctaaggaagatgctggaga
ttacatcatcatttgaccactttacgttccataaataacgattcgttctacgacctct
3’ 5’
1. Separation of the double strands
5' 3'
aatgtagtagtaaactggtgaaatgcaaggtatttattgctaaggaagatgctggaga DNA Helicase
breaks hydrogen
bonds between
paired bases
ttacatcatcatttgaccactttacgttccataaataacgattcgttctacgacctct
3’ 5’
 Process of DNA replication Template strand

Replication of two DNA strands “manually” New strand

5' 3'
aatgtagtagtaaactggtgaaatgcaaggtatttattgctaaggaagatgctggaga
ttacatcatcatttgaccactttacgttccataaataacgattcgttctacgacctct
3’ 5’
1. Separation of the two strands
5' 3'
aatgtagtagtaaactggtgaaatgcaaggtatttattgctaaggaagatgctggaga DNA Helicase
breaks hydrogen
bonds between
paired bases
ttacatcatcatttgaccactttacgttccataaataacgattcgttctacgacctct
3’ 5’

2. Complement both strands from 5’ to 3’

5' 3' A T
aatgtagtagtaaactggtgaaatgcaaggtatttattgctaaggaagatgctggaga T A
ttacatcatcatttgaccactttacgttccataaataacgattcgttctacgacctct
3’ 5’ C G
G C
5' 3'
aatgtagtagtaaactggtgaaatgcaaggtatttattgctaaggaagatgctggaga
ttacatcatcatttgaccactttacgttccataaataacgattcgttctacgacctct
3’ 5’
 Role of intracellular enzymes
• DNA polymerase:
Adds new nucleotides to new strand semi-
conservative replication pairing complementary
bases;

• DNA helicase:
Unwinds the two DNA strands; breaks hydrogen
bonds between bases.
• DNA ligase:
Joins phosphate group of new nucleotide to
(deoxyribose) sugar of growing strand forms
phosphodiester bond.
DNA replication = making 2 new DNA
molecules from one
 Multiple sites

Two replication forks make a

replication eye/bubble
 Multiple sites

Many sites for DNA replication (replication forks)

in Eukaryotes
DNA replication only happens during the S phase of the cell cycle
Eukaryotes have LONG chromosomes
Replication speed: 50 nucleotides per second
3000 nucleotides per minute
Longest chromosome of Drosophila (fly)
6.5 x 107 (650 000 000) nucleotides.
If only one replication fork, time needed is 150 DAYS
This chromosome is replicated in 4 minutes !!
The replication of this chromosome uses 54 000 replication forks
 *(b) Describe the process of DNA replication(5)

• 1. reference to semi-conservative replication ;

• 2. DNA strands {unwinds/ separate } ; by DNA helicase
• 3. (mono)nucleotides line up along (both) strands ; NOT RNA OR
one strand only
IGNORE bases line up
• 4. reference to complementary pairing between bases ;by DNA
polymerase.
• 5. reference to hydrogen bonds formed (between bases) ; NOT
between nucleotides in the same strand
• 6. reference to formation of phosphodiester bonds between
adjacent mononucleotides ;by DNA ligase
• 7. through a condensation reaction;
• 8. name of an enzyme involved in DNA replication ;
 Explain the meaning of the term
semiconservative replication.(2)
• 1. (new) DNA is synthesised and both original
strands are copied\act as templates, which
increases the number of (DNA) {molecules /
double helices},
IGNORE DNA is replicated

• 2. {new} DNA molecules each contain one

{parent old/original } strand and one new
strand ;
 Explain the importance of semi-conservative replication in the
production of new cells.(2)

• because it results in genetically identical

(daughter) cells (1)
• that will have the same {structure / function}
(as the parent cell)(1)

Video of semiconservative replication

Mechanisms of replication
Three models of DNA Replication??
• semiconservative replication would produce two copies/
molecules that each contained one of the original strands
and one new strand.
• Conservative replication would leave the two original
template DNA strands together in a double helix and
would produce a copy/molecules composed of two new
strands containing all of the new DNA base pairs.
• Dispersive replication would produce two
copies/molecules of the DNA, both containing distinct
regions of DNA composed of either both original strands
or both new strands.
Three models for DNA replication?

Old DNA

NewDNA

Conservative ?
Dispersive ?
Semi-conservative ?
Three models for DNA replication?

Old DNA

NewDNA
Three models for DNA replication?

Old DNA

NewDNA

Conservative ?
Dispersive ?
Semi-conservative ?
Three models for DNA replication?

Old DNA

NewDNA
Three models for DNA replication?

Old DNA

NewDNA

Conservative ?
Dispersive ?
Semi-conservative ?
Three models for DNA replication?

Old DNA

NewDNA
 Three models for DNA replication?

Which of the three models is right ?

Experiments in the late 1950s

Matthew Meselson and Franklin Stahl

It has been called "the most beautiful

experiment in biology." Meselson and Stahl
decided the best way to tag the parent DNA
would be to change one of the atoms in the
parent DNA molecule. Since nitrogen is found
in the nitrogenous bases of each nucleotide,
they decided to use an isotope of nitrogen
(heavy) to distinguish between parent and
newly copied DNA.
 Three models for DNA replication?

Meselson and Stahl’s experiment

• The scientists cultured the bacterium, Escherichia coli, for several generations in a
culture medium containing nutrients made with the heavy isotope of nitrogen 15N.
• The bacteria incorporated the 15N into their nucleotides and then into their DNA so
that all the DNA contained 15N. They extracted the bacterial DNA and centrifuged
it. The DNA settled at a low point in the tube (because all DNA molecules contained
two strands both made from 15N)
• The 15N bacteria were washed, then transferred to a medium containing the
normal, lighter form of nitrogen, 14N, and were allowed to divide once more
• When extracts of DNA from this first generation culture were centrifuged, the DNA
was shown to have a mid-point density since each new DNA molecule contained
one newly synthesized DNA strand containing 14N (the light isotope) and an original
DNA strand containing N15 (the heavy isotope)
• When DNA extracts were taken from the second generation grown in 14N, the DNA
settled at mid points and high points in the centrifuge tube. This was conclusive
evidence for the semi-conservative hypothesis
 Three models for DNA replication?

Bacteria:
One generation = 20 minutes
Fast DNA replication
No ethical issues
Small in size

All DNA contains…

15
N ONLY
centrifugation

The nitrogen atoms in the food source (ammonium

chloride) that Meselson and Stahl supplied to the bacteria
were the heavy isotope, nitrogen-15 (15N). The bacteria
used the 15N to make their DNA. They were left in it long
enough for them to divide many times, so that nearly all
of their DNA contained only 15N atoms, not nitrogen-14
(14N). This DNA would be heavier than DNA containing
14N.
 Three models for DNA replication?
Some of these bacteria were then transferred
to a food source in which the nitrogen atoms
were all 14N.
Some were left there just long enough for their
DNA to replicate once – about 20 minutes.
Others were left long enough for their DNA to
replicate two times.
DNA was then extracted from each group of
bacteria.
The samples were placed into a solution of
caesium chloride and spun in a centrifuge. The
heavier the DNA was, the closer to
the bottom of the tube it came to rest.

Centrifugation
The actual results

Can you
predict the
third
generation?
?????
 Three models for DNA replication?

Old DNA 15N

NewDNA 14N New DNA is lighter than old DNA

 Three models for DNA replication?

Old DNA 15N

NewDNA 14N New DNA is lighter than old DNA

 Three models for DNA replication?

Old DNA 15N

NewDNA 14N New DNA is lighter than old DNA

 Three models for DNA replication?

Old DNA 15N

NewDNA 14N New DNA is lighter than old DNA

 Three models for DNA replication?

Old DNA 15N

NewDNA 14N New DNA is lighter than old DNA

 Three models for DNA replication?

Old DNA 15N

NewDNA 14N New DNA is lighter than old DNA

 Three models for DNA replication?

Old DNA 15N

NewDNA 14N New DNA is lighter than old DNA

 Three models for DNA replication?

Old DNA 15N

NewDNA 14N New DNA is lighter than old DNA

 Three models for DNA replication?

Old DNA 15N

NewDNA 14N New DNA is lighter than old DNA

 Three models for DNA replication?

Old DNA 15N

NewDNA 14N New DNA is lighter than old DNA

• C;
• D;
• nucleus ;
Stage 1
• 1. only one bond drawn in lower half of tube;
Stage 2
• 2. one only bond drawn (higher than the one drawn in stage 1) ;
Stage 3
Diagram
• 3. {1 / 2} molecules shown with one light and one heavy strand ;
• 4. {1 / 2} molecules shown with two light strands;
Test tube
• 5. 2 bands shown in roughly correct position (middle to upper half of test tube) ;
• 6. bands should be of (roughly) equal width ;
[consequential error from stage 2 should apply for both marking points 5 and 6]
Explain how Meselson and Stahl’s experiment provides
evidence for the accepted theory for the replication of
DNA.
• 1. (Meselson and Stahl’s experiment was designed) to distinguish
between different types of replication;
• 2. at the start of the experiment cells were grown in (medium with)
{ heavy nitrogen / 15N } ;
• 3. only a heavy DNA band was observed ;
• 4. the cells were then transferred to (medium with) { light nitrogen / 14N
};
• 5. after one round of replication an intermediate DNA band was
observed ;
• 6. after two rounds of replication intermediate and light bands were
observed ;
• 7. showing that replication was { semiconservative / one strand of
original DNA and one strand of newlyjsynthesised DNA } ;
 Review of DNA Replication

Important things to remember

To ‘copy’ each strand of the double-stranded DNA
To “copy” = to complement

DNA Helicase breaks DNA Polymerase DNA Ligase joins

hydrogen bonds adds DNA adjacent nucleotides
between paired bases mononucleotides in the new strand
Separating the One by one along both together by
strands strands (by hydrogen phosphodiester bonds
bonds) ( through a
condensation
reaction)

Both strands act as TEMPLATE

DNA replication is semi-conservative

Role of DNA
How to know which amino acid is needed?
Out of 20 possibilities

When we can only use nucleotides

Out of only 4 nucleotides ???

We need a CODE

The Genetic code

Cracking the code
 The nature of the genetic code
• 1-Triplet code
Each adjacent group of three bases codes for an amino
acid.
In the genetic code, one base does not simply code for one
amino acid. There are only four bases, so if this were the
case proteins could contain only four different amino acids,
instead of the 20 amino acids found commonly in proteins.
If double 4x4 = 16 possible combinations…still not enough
If triplet 4x4x4=64 possible combination
 The GENETIC CODE

Four different bases are used to be translated into a combination of 20 different amino acids

A C G U
One base Not enough
41 = Four combinations

AA AC AG AU
Combinations CA CC CG CU
of two bases GA GC GG GU Not enough
UA UC UG UU
42 = 16 combinations

AAA AAC AAG AAU

Combinations ACA ACC ACG ACU Enough
of three bases .......
UUA UUC UUG UUU + EXTRA
43 = 64 combinations
 The GENETIC CODE

Second base in the codon  The mRNA is read “3 bases at the time”
Three mRNA bases = one CODON

 One codon always codes for

First base in the codon (5’ end)

Third base in the codon (3’end)

the same amino acid

 One amino acid can be encoded by

one codon (Tryptophane)
or more than one codons (the 19 others)
The genetic code is degenerate

AUG = Start Codon

UAA – UAG – UGA = Stop Codons

 The GENETIC CODE

2-The genetic code is non-overlapping

 2- Non overlapping:

Each triplet is discrete, each base is only used

once in a triplet.

 The genetic code is non-overlapping

Read a codon, do not use any
of the 3 bases again
• to
minimise
the effect
of
mutations
3-Degenerate code:
Several triplets code for the same/single amino acids
which means that there are more codes than {needed / number of amino acids}
arranging the four bases in triplets gives 64 possible combinations and there are 20 amino acids .
Which reduces/ minimises the effect of point mutations.

Also punctuation : 3 stop codons (meaning no more amino acids can be added to the
{polypeptide chain / protein} - signal or end of translation)
DEGENARATE CODE
The GENETIC CODE
 The GENETIC CODE
4-Universal code:
The same codons specify the same amino acid in all genes and in all organisms.

The genetic code is (almost)* (almost)* all organisms use the same
universal genetic code

GFP Gene from jellyfish

expressed in plant root

Jellyfish is green fluorescent Can produce human insulin in bacteria

Due to GFP protein Human gene cloned into bacteria
Green Fluorescent Protein Transgenic organisms are possible

GFP Gene from jellyfish

expressed in mice
* Mitochondria in humans
use a slightly different
genetic code
Explain the nature of the genetic code.

• 1. triplet code / 3 bases to each code ;

• 2. reference to adenine, thymine, guanine and
cytosine ;
• 3.each triplet of bases codes for one amino
acid ;
• 4. the code is not overlapping ;
• 5. the code is universal ;
• 6. the code is degenerate ;