Topic 4a: Protein synthesis

FSC BIO 134

Biology 2
Foundation in Science
Dr Chew

Chew Weiyun
Learning outcomes
• Explain the semi-conservative replication of DNA
• interpret experimental evidence for this process.
• Explain the mechanism of DNA replication.

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DNA is semi-conservative?
In the 1950s, no-one knew exactly how DNA replicated. Three
possibilities were suggested:
- conservative replication, in which one completely new double helix
would be made from the old one
- semi-conservative replication, in which each new molecule would
contain one old strand and one new one
- dispersive replication, in which each new molecule would be made of
old bits and new bits scattered randomly through the molecules.

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Chew Weiyun
Experiments by Meselson & Stahl
• There were three basic models for DNA
replication that had been proposed by
the scientific community after the
discovery of DNA's structure

• Most biologist believed that semi-

conservative model is the answer but
they need proofs.

• That’s where Meselson & Stahl came in.

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Experiments by Meselson & Stahl
• They use E. coli bacteria as a model system.

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Experiments by Meselson & Stahl
1. When grown on medium containing heavy 15N, the bacteria took up the
nitrogen and used it to synthesize new biological molecules, including
DNA.
2. After many generation, nitrogenous bases of the bacteria's DNA were all
labeled with heavy 15N.
3. Then, the bacteria were switched to medium containing a “light” 14N
isotope, and allows it to grow for several generations.
1. They know how often the bacteria cells divide
4. Extract and purify the DNA.
5. Measured the density of the DNA by using density gradient
centrifugation.

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Experiments by Meselson & Stahl
6. Molecule are separated based on the
density, forming a density gradient.
(Heavy at the bottom, light at the top)

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Predictions

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Results?

Try to draw out the possible DNA

structure of each generation with
the label of DNA strand of N15 and
N14.
Note: Gen 0 is given above as
example.

Please write down the conclusion of

the experiment.

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Conclusion?

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Conclusion

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 DNA Replication is semi-conservative.
DNA Replication
Simplified mechanism of DNA replication.

Chew Weiyun
 DNA Replication is semi-conservative.
DNA Replication
Simplified mechanism of DNA replication.

Chew Weiyun
 DNA Replication is semi-conservative.
DNA Replication
Simplified mechanism of DNA replication.

Chew Weiyun
DNA Replication
Starting DNA replication
How do DNA polymerases and other replication factors know where to
begin?

• origins of replication – short stretches of DNA that have a specific sequence

of nucleotides.
• Origin is about 245 base pairs long
• Mostly A/T base pairs (easier to separate, fewer hydrogen bonds)

• E. coli has single origin of replication

• Human can have 100,000 origin of replication.

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DNA Replication
Starting DNA replication

• How does the replication bubble starts?

• Does it starts on both direction at the

same time?

• Did you see the semi-conservative of

DNA replication?

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Chew Weiyun
DNA Replication
Imagine DNA replication as a GAME.

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DNA Replication
DNA replication - THE GAME
rules of play
• DNA can only be made in 5’ to 3’ direction.
• DNA cannot initiate the synthesis of a polynucleotide by itself
• Can only add DNA nucleotides to the end of an already existing chain of
nucleotides (DNA / RNA).
• Newly synthesis DNA strand will be complementary to the existing DNA
strand (A-T, C-G)
• The end of the DNA have to be “capped”, if not DNA will unwind.
• Each DNA nucleotide have to be activated before they can be joined to
form DNA strand. (two extra phosphate group)
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DNA Replication
Enzyme involved in DNA replication
Introducing the game characters.
Protein Function
Helicase Unwinds parental double helix at replication forks

Single-strand binding Binds to and stabilizes single-stranded DNA until it is

protein used as a template

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DNA Replication
Enzyme involved in DNA replication
Introducing the game characters.
Protein Function
Topoisomerase Relieves over winding strain ahead of replication forks
by breaking, swiveling, and rejoining DNA strands

Primase Synthesizes an RNA primer at 5′ end of leading strand

and at 5′ end of each Okazaki fragment of lagging
strand

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DNA Replication
Enzyme involved in DNA replication
Introducing the game characters.
Protein Function
DNA polymerase III Using parental DNA as a template, synthesizes new
DNA strand by adding nucleotides to an RNA primer or
a pre-existing DNA strand

DNA polymerase I Removes RNA nucleotides of primer from 5′ end and

replaces them with DNA nucleotides added to 3′ end of
adjacent fragment

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DNA Replication
Enzyme involved in DNA replication
Introducing the game characters.
Protein Function
DNA ligase Joins Okazaki fragments of lagging strand; on leading
strand, joins 3′ end of DNA that replaces primer to rest
of leading strand DNA

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 5’ 3’

DNA Replication
DNA
The game board.

How to win?
To create perfect copy of DNA.

Now let’s play!

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DNA Replication
7 6
8 5

3
4
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DNA Replication
1) Topoisomerase Relieves over winding strain of DNA ahead.
2) Helicase unwinds the parental double helix.
3) Molecules of single-strand binding protein stabilize the unwound
template strands.
4) The leading strand is synthesized continuously in the 5′ to 3′
direction by DNA polymerase III.
5) Primase begins synthesis of the RNA primer for the Okazaki
fragment.

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DNA Replication
6) DNA polymerase III is completing synthesis of Okazaki fragment.
When it reaches the RNA primer on earlier Okazaki fragment, it will
detach and begin adding DNA nucleotides to the 3′ end of the
fragment primer in the replication fork.
7) DNA polymerase I removes the primer from the 5′ end of Okazaki
fragment, replacing it with DNA nucleotides added one by one from
3′. After the last addition, the backbone is left with a free 3’ end.
8) DNA ligase joins the 3’ end of fragment to the 5’ end of Okazaki
fragment.

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DNA Replication
Addition of a nucleotide to a
DNA strand
• DNA polymerase catalyzes
the addition of a nucleotide
to the 3′ end of a growing
DNA strand, with the
release of two phosphates.

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DNA Replication
Leading strand
1. After RNA primer is made,
DNA polymerase III starts to 1
synthesise the leading strand.

2. The leading strand is elongated

continuously in the 5’ → 3’
direction as the fork
progresses.
2

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DNA Replication
Lagging strand
1. Primase joins RNA
nucleotides into a
primer. 1
2. DNA polymerase III adds
DNA nucleotides to the
primer, forming Okazaki
fragment 1.

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DNA Replication
Lagging strand 3
3. After reaching the next
RNA primer to the right,
DNA polymerase III
detaches.
4. Fragment 2 is primed.
Then DNA polymerase III
adds DNA nucleotides, 4
detaching when it
reaches the fragment 1
primer.
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DNA Replication
Lagging strand
5. DNA polymerase I replaces
the RNA with DNA, adding
nucleotides to the 3′ end 5
of fragment 1 (and, later,
of fragment 2).
6. DNA ligase forms a bond
between the newest DNA
and the DNA of fragment
1. 6
7
7. The lagging strand in this
region is now complete.

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 4
3

Chew Weiyun 6 7 8
DNA Replication
Proofreading of DNA
The mistakes in the game >> defeats
• When DNA polymerases copying the results, errors can occur.
• Resulted mutation.

• At the same time, it is the job of the DNA polymerases to proofread

each nucleotide against its template while growing/elongating strand.

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DNA Replication
Proofreading of DNA
➢Polymerase adds an incorrect
nucleotide to the new strand of DNA

➢Polymerase detects that bases are

mispaired.

➢Polymerase uses 3’ → 5’ exonuclease

activity to remove incorrect
nucleotide.
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DNA replication
Mismatch repair
The bad guys (/girls) in the game >> defeats
• The mutagens / carcinogens
• Radiation (e.g. UV radiation, X-Rays)
• harmful chemicals (e.g. Benzoyl Peroxide)
• Infectious agents (e.g. Human
Papillomavirus, HPV, H. pylori)

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DNA replication
Mismatch repair
With bad guys (/girls), you will have the unsung heroes/heroines
• At certain degrees, DNA can fix the damages.
• There is four ways to do it, depending on the degree of damages.
• Direct reversal
• Excision repairs
• Base excision repairs
• Nucleotide excision repairs
• Double-stranded break repairs.

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DNA replication
Mismatch repair
• Direct reversal
• Some DNA-damaging chemical reactions can be directly "undone" by
enzymes in the cell.

The methyl-bearing guanine, if not fixed, will pair with thymine

Chew Weiyun (T) rather than cytosine (C) during DNA replication.
DNA replication
Mismatch repair
• Excision repairs
• Damage to one or a few bases of DNA is often fixed by removal (excision) and
replacement of the damaged region.
• Base excision repair
• just the damaged base is removed.
• Glycosylases → enzyme for base excision repair.

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DNA replication
Mismatch repair | Base excision repair

glycosylase detects and

Chew Weiyun removes deaminated cytosines.
DNA replication
Mismatch repair | Base excision repair

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DNA replication
Mismatch repair
• Excision repair
• Nucleotide excision repair, as in the mismatch repair we saw above, a patch
of nucleotides is removed.
• This pathway detects bases that have been modified by bulky chemical
groups, UV radiation, and etc.

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DNA replication
Mismatch repair | Nucleotide excision repair

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DNA replication
Mismatch repair | Nucleotide excision repair

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DNA replication
Mismatch repair
• Double-stranded breaks
• Can happen during environmental factors such as high-energy radiation,
cause the double-stranded DNA breaks into two.
• Dangerous, massive loss of gene if not repaired.
• Non-homologous end joining, the two broken ends of the chromosome are
simply glued back together.
• “messy” and typically loss of few nucleotides.
• Sometime, produce mutation.
• Homologous recombination, information from the homologous chromosome
that matches the damaged one is used to repair the break.
• Usually from a sister chromatid
• “cleaner”, usually will not cause mutations.
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DNA replication
Mismatch repair | Double-stranded break repair

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DNA replication
Mismatch repair | Double-stranded break repair

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DNA replication
Game ended?

Not yet!!

How to stop an DNA replication?

If the DNA strand is not “capped” it will unwind itself like a shoe lace!!

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DNA replication
Telomeres
DNA: End game
• Unlike bacterial chromosomes, the chromosomes of eukaryotes are
linear (rod-shaped), meaning that they have ends.
• These ends pose a problem for DNA replication.

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DNA replication
Telomeres
DNA: End game

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DNA replication

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 Results in the lost of information.
Chew Weiyun Solution?
DNA replication
Telomeres
DNA: End game
• To prevent the loss of genes as chromosome ends wear down, the
tips of eukaryotic chromosomes have specialized DNA “caps”
called telomeres.
• Telomeres consist of hundreds or thousands of repeats of the same
short DNA sequence
• Varies between organisms
• 5'-TTAGGG-3' in humans and other mammals.

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DNA replication
Telomeres
DNA: End game
• telomeres need to be protected from a cell's
DNA repair systems / triggering cell death
• They have single-stranded overhangs, which “look
like” damaged DNA.

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DNA replication
Telomeres
DNA: End game
• For human, the single-stranded overhangs
bind to complementary repeats in the nearby
double-stranded DNA
• Forming protective loops
• Protein associated with the telomere ends also
help protect them
• Prevent from activating cell death / cell arrest.

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DNA replication
Telomeres
DNA: End game
Protective functions of Telomeres
• Prevent daughter molecule
from activating DNA repair
system / triggering Cell death.
• Acts as a buffer zone that
provides some protection
against the organism’s genes
shortening.

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DNA replication
Telomerase
• an enzyme that extends the telomeres of chromosomes.
• Some cells have the ability to reverse telomere shortening
• RNA-dependent DNA polymerase
• Can make DNA using RNA as template.
• Usually active in germ cells (make sperms and eggs), not somatic
cells.

How does telomerase work?

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DNA replication
Telomerase
1. The enzyme binds to a special RNA molecule that contains a
sequence complementary to the telomeric repeat.
2. It extends (adds nucleotides to) the overhanging strand of the
telomere DNA using this complementary RNA as a template.

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DNA replication
Telomerase

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DNA replication
Telomerase

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DNA replication
Telomerase

many cancer cells have shortened telomeres, and telomerase

Chew Weiyun is active in these cells
DNA replication | Case study
Xeroderma pigmentosum
• Caused by mutations affecting the nucleotide
excision repair pathway.
• thymine dimers and other forms of UV damage
can't be repaired.
• Extremely sensitive to UV light

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DNA replication | Case study
Xeroderma pigmentosum
• They can develop severe sunburns from just a
few minutes in the sun.
• Can get skin cancer by the age of 10 unless
they avoid the sun.

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Thank you and all the best!!

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