MOLECULAR GENETICS

INTRODUCTION
• Cells
• Fundamental working units of every living system.
• Every organism is composed of one of two radically
different types of cells:
• prokaryotic cells
• eukaryotic cells which have DNA inside a nucleus.
• Prokaryotes and Eukaryotes are descended from
primitive cells and the results of
3.5 billion years of evolution.
 Cont-------
• All Cells have common Cycles
• Born, eat, replicate, and die
 Common features of organisms

• Chemical energy is stored in ATP

• Genetic information is encoded by DNA
• Information is transcribed into RNA
• There is a common triplet genetic code
• some variations are known, however
• Translation into proteins involves ribosomes
• Shared metabolic pathways
• Similar proteins among diverse groups of organisms
 All Life depends on 3 critical molecules

1.DNAs (Deoxyribonucleic acid)

• Hold information on how cell works
2.RNAs (Ribonucleic acid)
• Act to transfer short pieces of information to different parts of
cell
• Provide templates to synthesize into protein
3.Proteins
• Form enzymes that send signals to other cells and regulate gene
activity
• Form body’s major components
 DNA, RNA, and the Flow of Information

The central dogma

• Formulated by Francis Crick
• Defines three major steps in the processing of
genetic information
• 1. replication
• 2. Transcription
• 3. Translation
The central dogma
 DNA Replication

• Replication is the duplication of DNA content

before mitosis, i.e., the two DNA copies are
divided equally between the new daughter
cells. Each old strand will be copied into a
complementary new strand.
• DNA replication is the process by which the
genetic material is copied prior to distribution
into daughter cells
 Cont----
• The original DNA strands are used as templates
for the synthesis of new strands
• It occurs very quickly, very accurately and at the
appropriate time in the life cycle of the cell

• DNA replication relies on the complementarities

of DNA strands
• The AT/GC rule or Chargaff’s rule
 Cont-------
• This process is called semi-conservative
because it conserves only half of the original
(parent) DNA molecule in the two daughter
DNA molecules. One strand in each daughter
molecule is completely new.
• which means that each daughter cell will
receive a DNA strand from the mother cell and
a complementary newly synthesized strand.
 Cont----
Basic requirements for replication
A. Substrates
• The four deoxyribonucleosides triphosphate
• dATP
• dGTP
• dCTP
• dTTP
 Cont----
• B. Template
• For DNA replication to occur the two chains
have to unwind and separate.
• The separated strands serve as template for
the synthesis of the new daughter strands.
• A template is required to direct the addition
of the appropriate complimentary nucleotide
to the newly synthesised DNA strand.
 Cont----
• C. Enzymes
• DNA synthesis is catalyzed by enzymes called DNA
dependent DNA polymerase
• DNA polymerase I, is responsible for gap filling by nick
translation of the RNA primers used during DNA
replication into DNA sequence.
• DNA polymerase II, has proofreading and DNA repair
function.
• DNA polymerase III, is the major enzyme responsible
for DNA replication.
 Cont----
DNA helicase
• Unwinds the double helix into two single-
stranded DNA.
• It breaks down hydrogen bonds between the
bases at expense of ATP. Two ATP molecules
are consumed for each base pair broken. Once
separated, the single stranded DNA formed is
stabilized by proteins called DNA-binding
proteins.
 Cont----
The single-stranded DNA binding proteins
(SSB)
• that prevent reannealing of DNA helix by
binding the single stranded DNA created by
helicase without interfering with DNA
polymerase function.
 Cont----
DNA gyrase
• Removes positive supercoils and introduces
negative supercoils.
Topoisomerases
• Catalyzes the relaxation of supercoiled DNA by
breaking just one strand of DNA.
 Primase
• DNA-dependent RNA polymerase that
synthesizes short RNA primers (5-200 bases) with
the help of DNA binding protein (primosome).
• RNA primer complementary and anti-parallel to
the DNA is synthesized by primase.
• The DNA polymerase III requires a primer to
elongate at the beginning of replication. The
primer has a free 3'-OH to accept deoxy-
ribonucleotides polymerized by DNA polymerase
III.
 Cont----
DNA ligase
• joins the free 3'-OH on C3 of one end to 5'-OH
of phosphate on C5 of the other end of DNA.
 STAGES OF REPLICATION

The process of replication can be divided in to

three stages.
• Initiation
• Elongation
• Termination
 Cont----
• Initiation
• DNA replication is initiated by the binding of
DnaA proteins to the DnaA box sequences
• This binding stimulates the cooperative binding
of an additional 20 to 40 DnaA proteins to form a
large complex.
• This causes the DNA to twist and the puts torque
on the nearby AT-rich region to denature and
form a replication bubble
 Cont----
– AT base pairs are held together by only
2 H bonds
– CG base pairs are held together by 3 H
bonds
– Therefore, AT-rich regions of DNA
denature more easily than CG-rich
regions of DNA
 Cont----
• In the next step, DnaB (also called helicase)
binds to each strand of the separated double
helix. It’s job is to move along the DNA,
progressively expand the replication fork in
both directions.
•  DNA helicase separates the two DNA strands
by breaking the hydrogen bonds between
them
 Cont----
• This generates positive supercoiling ahead
of each replication fork so another enzyme,
topoisomerase, travels ahead of the
helicase and alleviates these supercoils
• Single-strand binding proteins (SSBPs) are
also needed to bind to the separated DNA
strands and keep them apart
– Otherwise, the strands would simply reanneal
 Cont----
• After the helicase, gyrase, and SSBPs are in
place, short (10 to 12 nucleotides) RNA primers
are synthesized by DNA primase
• These short RNA strands start, or prime, DNA
synthesis because DNA polymerase, the
enzyme that copies DNA, cannot start a new
strand on its own
• The RNA primers are later removed and
replaced with DNA
 Cont----
• Elongation
• DNA polymerase III begins the process of elongation
by adding free deoxy-ribonucleotides to the free 3'-
OH end of the RNA primer in the 5'3' direction in
a continuous manner (the leading strand) to the
end of the DNA molecule or till reaching the
flanking replication fork.
• This is done according to the base pairing rule: A-T
and G-C using the 3'5' DNA strand as a template.
 Cont----
• The other 5'3' the lagging strand of DNA, lags
behind for a while until at least 1000 to 5000
nucleotides in prokaryotes (but only 150 - 250
nucleotides in eukaryotes) are exposed, where
primase put a primer at the upper end of it at
the replication fork, i.e., in 5'3' direction.
• DNA polymerase III covers the downstream(away
from the replication fork) sequence by
synthesizing at least 1000 DNA nucleotides.
 Cont----
• This process is repeated when a new enough
stretch of DNA is again exposed due to the
continuous progress of the leading strand,
where, primase puts a new primer that is
elongated by DNA polymerase III until it
reaches the previous RNA primer, and so on.
Each area enough for the DNA polymerase III
to work on the lagging strand is called Okazaki
fragment.
 Cont----
• Thus, one strand is built towards the
replication fork 5’3’, i.e., the Leading strand,
while the other strand is built in the opposite
direction but also in 5'3', i.e., Lagging
strands.
• Leading strand
• One RNA primer is made at the origin
• DNA pol III attaches nucleotides in a 5’ to 3’ direction as
it slides toward the replication fork
 Cont----
Lagging strand
• Synthesis is also in the 5’ to 3’ direction
–However it occurs away from the replication fork
• Many RNA primers are required
• DNA pol III uses the RNA primers to synthesize
small DNA fragments (1000 to 2000 nucleotides
each)
–These are termed Okazaki fragments after their
discoverers
 Cont----
• Finally, the RNA primers are removed by the
5'3' nick translation by DNA polymerase I
activity with a replacement into DNA
nucleotides.
• The 5'-end of DNA fragments built by DNA
polymerase III is linked to the 3'-end of the
DNA fragment built by the DNA polymerase I
by ATP-dependent ligase
 Cont----
• All DNA polymerases, whether bacterial or
eukaryotic, share 2 very important limitations:
• 1. They cannot initiate DNA synthesis on their
own. They require that an RNA primer be laid
down on the DNA first by DNA primase.
• 2. They can only “grow” a new DNA chain in
the 5’ to 3’ direction.
 Cont----
• Proofreading
• DNA replication exhibits a high degree of fidelity.
• Mistakes during the process are extremely rare
• There are several reasons why fidelity is high:
• 1. Instability of mismatched pairs
– Complementary base pairs have much higher stability
than mismatched pairs
– This feature only accounts for part of the fidelity
– It has an error rate of 1 per 1,000 nucleotides
 Cont----
• 2. Configuration of the DNA polymerase active
site
– DNA polymerase is unlikely to catalyze bond
formation between mismatched pairs
• 3. Proofreading function of DNA polymerase
– DNA polymerases can identify a mismatched
nucleotide and remove it from the daughter strand
– The enzyme uses its 3’ to 5’ exonuclease activity to
remove the incorrect nucleotide
 Cont----
• C. Termination
• A specific protein, ter binding protein, binds
termination sequences and prevents helicase
from further unwinding of DNA and facilitates
the termination of replication.
 5' 3'

Topoisomerases (Gyrase),
Remove supercoiling

Helicase
PPI

ATP, GTP, CTP, TTP

Primase

Single-strand
binding protein 5'

5'
DNA polymerase III

PPi

dATP
dGTP
dCTP
dTTP DNA polymerase I

Leading strand
3' DNA ligase 5'
5' 3'
RNA Primer Lagging strand
 Mechanisms of repair of DNA Damage

• Introduction
• Despite proof reading and mismatch repair
during replication, some mismatch bases do
persist
• In addition DNA can be damaged by mutagens
produced in cells or from the environment.
• If the damage is not repaired mutation can
happen leading to cancer.
 Repair mechanisms
• 1.Recognition of the distorted part of DNA
• 2. Removal or excision of the damaged region
of the DNA strand
• 3. Filling the gap left by the excision of the
damaged DNA by DNA polymerase
• 4. Sealing the nick in the strand that has
undergone repair by Ligase
 Systems of repair of DNA Damage

• 1. Excision repair:
• Used to correct many types of DNA damage that
affects only one strand. It has three mechanisms
as follows,
• A. Nucleotide excision repair of thymine-thymine
(or pyrimidine) dimer
• B. Mismatch Repair:
• C. Base excision repair or correction of
deamination of cytosine to uracil:
A. Nucleotide excision repair of thymine-thymine (or pyrimidine)
dimer:

• Thymine-thymine dimer is covalent linkage of

two adjacent thymine bases in a single strand
and occurs spontaneously, or due to chemical,
radiation, or ultraviolet (UV) light damage to
DNA segment.
• These dimers prevent DNA polymerase from
replicating DNA strand beyond the site of the
dimer.
• There are two ways of correcting such damage:
 Cont----
• 1. DNA photolyase enzyme is photo-
reactivated by photons of UV or blue spectral
region of light to cleave the dimer into its
original bases.
• 2. UV-specific endonuclease:
• This enzyme recognizes the dimer and makes
a nick in the affected DNA strand 8 nucleotides
away from the dimer site to the 5'-side.
 Cont----
• DNA polymerase I fills the gap with new
nucleotides using the healthy strand as a template
in the 5'3' direction by nick translation.
• Then, UV-specific endonuclease cut the freed
damaged sequence4 nucleotides from the dimer
site away to the 3'-side. Then, the damaged piece
of DNA diffuses away.
• Then, DNA ligase joins the 3' end of the new DNA
and the 5' end of the original DNA.
5' 3'
3' 5'
UV-specific
endonuclease
5' 3'
3' 5'

DNA polymerase I

5' 3'
3' 5'

DNA polymerase I
5' 3'
3' 5'

DNA polymerase I

5' 3'
3' 5'

DNA ligase
5' 3'
3' 5'
 B. Mismatch Repair:
• Mismatch, e.g., G-T, is due to copying errors
during replication that may also lead to 1 - 5
bases unpair loops.
• The mismatch is recognized by a group of
proteins called MutS, MutL and MutH that
identify the parent DNA strand by its
methylation at GATC sequences.
 Cont----
• Once identified the mismatch, they cut the
defective DNA strand at the 3’ side of the
mismatch dimer.
• A special exonuclease (exonuclease 1)
hydrolyzes DNA in 3'5' direction to a few
nucleotides 5' the mismatch and releases free
DNA nucleotides.
 Cont----
• A new DNA is synthesized to fill the gap by
DNA polymerase III
• DNA ligase joins the 3'-end of the new DNA
and the 5'-end of the DNA ahead of it.
• Defects in these Mut proteins lead to specific
types of hereditary types of cancer
3' 5'
G
5' T 3'

MutL
MutH
MutS
3' 5'
G
5' T 3'

Exonuclease 1
3' 5'
G
5' 3'

DNA polymerase III

3' 5'
G
5'
C
3'

DNA polymerase III

5' 5'
G
C
3' 3'
DNA ligase

3' 5'
G
C
5' 3'
C. Base excision repair or correction of deamination of cytosine to
uracil:

• Oxidative deamination of cytosine into uracil,

adenine into hypoxanthine and guanine into
xanthine occurs spontaneously, or due to
chemical or radiation damage to a single base.
• Since uracil, hypoxanthine and xanthine are
foreign to DNA; they are recognized and
removed from the DNA strand by specific-
DNA glycosylase
 Cont----
• AP endonuclease cuts the deoxyribose-
phosphate backbone of the affected strand at 5'
end of the site and removes a few nucleotides.
• Then, DNA polymerase I excise the residual
deoxyribose phosphate unit and inserts
cytosine, as dictated by the presence of guanine
on the undamaged complementary strand.
• Then, the deoxyribose-phosphate backbone is
rejoined by a Ligase.
2. Recombinational double strand breaks repair:

• The mechanism of repair of double DNA

strand breaks that are due to ionizing
radiation, chemotherapy and oxidative free
radicals.
• Two proteins are involved; Ku and DNA-
dependent protein kinase. Both of them
attach to each end of the double strand break,
activate one another other, unwind the duplex
 Cont----
• (Ku has helicase activity) and search for the
closest DNA sequence of minimum
complementarity in the opposite strands.
• The extranucleotide tails are degraded into
free nucleotides by exonuclease.
• The gaps are filled by DNA polymerase III and
DNA ligase seals the free ends.
 Ku

DNA-dependent
protein kinase

Exonuclease

DNA ligase DNA polymerase III

 DNA alteration (Mutations)

• Definition:
• Mutation is the change of base sequence of
nucleotides in the genetic code due to
replacement, deletion (removal) or insertion
(addition) of one or more bases resulting in
altered gene product and/or regulation, or a
change in gene copy number, or a structural or
numerical abnormality in chromosomes.
 Cont----
• Causes:
• Physical (most common) such as: UV, X and 
radiations.
• Chemical carcinogens such as anticancer base
analogs and alkylating agents.
• Environmental pollutants-derived oxidative free
radical such as nitrous acid.
• Genomic instability, errors of DNA replication and
defective repair.
 Types of DNA mutation or alteration:

• a) Single base alteration (point mutation):

• Deamination, e.g., adenine into hypoxanthine and
cytosine into uracil.
• Depurination or depyrimidination, i.e., removal of
a purine or a pyrimidine base.
• Alkylation of a base (mostly guanine) by covalent
addition of an alkyl radical.
• Insertion or deletion of a nucleotide.
• Base-analog incorporation.
 Cont----
B) Two bases alteration:
• UV-induced thymine–thymine dimer.
• Cross linkage caused by bifunctional alkylating
agent.
c) Strand breakage: Single-strand breaks are
less harmful than double- strand breaks in the
phosphodiester backbone. It is caused by:
 Cont----
• Ionizing radiation.
• Radioactive disintegration of incorporated
element.
• Oxidative free radical formation.
d) Cross-linkage:
• Two bases in same strand or opposite strands.
• DNA cross-linkage to histones or other
proteins.
 A. Point mutations:
Definition and types:
• It is a single base change that can be
Transition mutation: a purine base is changed
to another purine base,e.g., adenine into
guanine or a pyrimidine base to another
pyrimidine base, e.g., thymine into cytosine.
Or, transversion mutation: a purine base is
changed into a pyrimidine base and vice versa.
Fate (or effect) of point mutation:
Silent mutation
• the changed base leads to a codon, which
produces the same amino acid.
• In this case, the change lies in the third base of
the codon, which has several alternative
names for same amino acid.
 Missense mutation
• The change occurred either in first or second
base of the codon producing a different amino
acid.
• The effect of missense mutation on the protein
produced is dependent on the position and
nature of the replacement amino acid. This
protein could be normally functioning, partially
functioning, non-functioning or not produced
at all because of defective RNA processing.
 Cont----
• Example is Sickle cell anemia or hemoglobin S
with normal two  chains and abnormal two 
chains leading to rapid hemolysis due to
mutant glutamate at position 6 into valine.
• Non-sense mutation, i.e., the altered base
results in a non-sense termination codon. This
leads to pre-mature stopping of protein
synthesis
 Frame shift mutation
• Shift in the trinucleotide reading frame e.g., in the BAX gene
in ovarian cancer resulting in:
• Truncated protein if a non-sense codon is developed due to
the shift.
• Garbled translation into totally different protein after the
shift point.
• A protein may not be produced at all because mRNA is
degraded.
• Insertion or deletion of three bases produces a protein with
an extra or lacking an amino acid. It has a moderate effect
on the produced protein.