DNA organization, synthesis,

mutation & repair

Biomedical Sciences for Optometry I

(OPT 2034)

Dr. Christinal Teh

christinaltehpw@segi.edu.my
 Learning Objectives & Outcomes

• At the end of this lecture, all students should be able

to explain
1. DNA organization & synthesis
2. DNA mutation & repair
 Introduction: Genetic material

• The genetic information of an organism is kept in the

form of DNA
• In some lower life-forms, such as prokaryotic cells : as
plasmids (circular)
• DNA is “copied” during cell replication (mitosis) to its
daughter cells via “DNA replication”
• It is very important that these genetic information is
copied precisely (high fidelity); there are mechanism
(DNA repair) if mistakes were to occur - mutation
• Two distinct type of nucleic acids:
a) deoxy-ribo-nucleic acid (DNA)
b) ribo-nucleic acid (RNA)
 Nucleotide structure
Nucleic acid are polymers (polynucleotides)
- Made of nucleotides

Purine

Pyrimidine

Nucleotide

Nucleoside
 Nitrogenous Bases

Purine

Pyrimidine

DNA RNA
 RNA and DNA
 Different pentose sugar in RNA & DNA
Adenosine
 Nucleotide
 Phosphate esters of nucleosides
 Base + pentose sugar + phosphoric acid
 Esterification at 5th or 3rd hydroxyl group of the pentose sugar.
 Structure of DNA

Sequence is
deoxyribose- important
phosphate
backbone

4
 phosphodiester bond

• connects between two deoxy-ribose : 3’-hydroxyl

group of one nucleotide to the 5’-hydroxyl group of
adjacent nucleotide with 1 phosphate group

• By convention: read from 5’ to 3’

• These phosphodiester bonds can be cleaved:
In DNA: deoxyribo-nucleases
In RNA: ribo-nucleases
 Watson & Crick Model
 Features:
1. Right handed double helix
2. Anti-parallel
3. Coiled around the same axis
4. Base-pairing (perpendicular
to axis of symmetry)
5. Hydrogen bonds between the bases
6. Hydrophobic interactions
(deoxyribose-phosphate backbone is
hydrophilic – facing outside; the
bases are hydrophobic – stacked
inside)
 Base pairing
Purines Pyrimidines

Adenine Thymine
(Urasil)

Cytosine Guanine

1. Anti-parallel
2. No. of H-bonds
Adenine Uracil
 DNA Melting temperature (Tm)
• Hydrogen bonds between the bases
can be disrupted; by modifying the
pH of the solution or applying heat
(temperature).
• Melting temp (Tm) = Temp that ss
denature half of the DNA 2

• Tm can be monitored at 260 nm

 ssDNA has higher relative abs than
dsDNA
• It is because DNA can denature,
ds 1
genetic manipulation can be
performed (eg. PCR)
• GC bonds are stronger than AT bonds
 The law of semiconservative replication
• The replication (synthesis) of DNA
follows the law of semi
conservative replication
• Two strands of DNA helix separates
to form as a template to produce
two daughter copies
• The original strand is “conserved”
in the respective copied strands
• Examples discussed here refers to
prokaryotic system (eukaryotic
system almost the same or more
complex)
 The beginning of replication
• DNA replication begins at a unique nucleotide sequence
(“origin of replication”); AT-rich regions
1

[A] small prokaryotic circular DNA [B] Long eukaryotic DNA

 No spoons, just forks

• The replication fork is the region where

the synthesis of DNA begins

• The opening of the fork is facilitated by:

i) DnaA
Which recognize the AT rich region,
melting (opening) the region – ATP-
dependent
ii) Single-stranded DNA binding (SSB)
proteins
Bind to ssDNA - To keep the ssDNA
open for polymerase to attach.
iii) E: Helicase
Acts like a wedge, forcing the strands 1. To keep the ssDNA open for
polymerase to attach
apart – ATP-dependent 2. To prevent nucleases
 Direction of replication
• NEW strand is synthesized : 5’  3’
• Two templates, opposing direction
1: Advancing the fork (leading)
2: Away from the fork (lagging)
• 1: Synthesized continuously
2: Synthesized discontinuously
(Okazaki fragments)
• RNA Primer
replication cannot start without this. They are short ss template.
With free 3’ end for additional nucleotide.
E: Primase (syn. the primers) 10 bases (T is replaced by U)
• E: DNA polymerase 3 (DNA Pol3)
Fx: copy the DNA template, addition of new dNTPs (substrate)
1
2

4
 DNA Polymerase III Function (adding)
• DNA Pol III “hooks” onto the
template, and start reading
and synthesizing

5’3’ polymerase activity

 DNA Polymerase III Function (proof reading)
• Mistakes in synthesis is
corrected with

3’5’ exonuclease activity

• By hydrolysis (phosphodiester
bond breaks)
• Then replace with the correct
nucleotide
Cant leave it dangling
 The DNA Polymerase I
• The problem with Lagging strand is that, at one point,
DNA Pol III will meet the head of the primer, synthesis
stops
• These are then mended by DNA Pol I
• Similar to DNA Pol III; Pol I has:-
5’3’ polymerase activity (syn.)
3’  5’ exonuclease activity (proofreading)
+ 5’3’ exonuclease activity (removing primer)
Thus removing the (Us) in the primer, replace with (A)
• Remove one nucleotide at a time; until complete
• Finale; E: Ligase
Joining the 5’3’ phosphodiester bond
 DNA Repair
• Mistakes in the DNA, caused by:-
a) incorrect base pairing (even though, proofreading)
b) insertion
c) radiation (UV light)
i. Base dimers
ii. Double strand breaks
• If not repaired  damage, mutation, cell death, cancer
• Repair involve:
(i) recognition
(ii) excision
(iii) replacement
(iv) ligation
 DNA damage & repair (examples)
• Here, we discuss two examples of DNA damage and
the mechanism involved in its repair:-
• A) Damage caused by UV light
• B) Damage caused by base alteration
 UV induced DNA Damage

• Exposure to UV light can fuse (covalent

joining) of neighboring thymine bases
(pyrimidine); forming a thymine dimer
• Result in: unable to replicate
• Recognition & Excision:
E: UV-specific endonuclease
Releasing an oligonucleotide
• Replacement & Ligation:
sister strand as template
DNA polymerase – syn and fill the gap
DNA ligase – to fuse the remaining nick
 Base alteration induced DNA
damage
• The cytosine base in a DNA can deaminate (-NH2)
to form uracil spontaneously
• Deaminating/alkylating agents: Adenine 
hypoxanthine; Guanine  xanthine
• The recognition & excision:-
i) Uracil-N-glycosylase – uracil is excised
ii) Apyrmidinic endonuclease – Cut the 5’ end
iii) Deoxyribose phosphate lyase – Removes
the ribose-P
• Repair & Ligation:
i) DNA Polymerase - replace the missing nucleotide
ii) DNA Ligase – ligate the end
 Assignment
1. With the aid of diagram, briefly describe the competitive and
non-competitive inhibition. (10 marks)

2. Briefly explain how does galactose gain entry into the

glycolytic pathway. (5 marks)

3. Briefly explain the DNA damage by base alteration and its

repair mechanisms. (5 marks)