The flow of Genetic information

DNA Replication is to keep the genes from a generation to a generation

Replication in DNA is semi-conservative which means :

We have 2 strands, When they separate we’ll have each strand connect to a new strand.

So we need to “revert it” back to tertiary then secondary then primary

 DNA Replication
 DNA is a double-helical molecule
Watson and Crick Predicted Semi-conservative Replication of
DNA
 The mechanism: Strand separation, followed by copying of each
strand.
Each separated strand acts as a template for the synthesis of a
new complementary strand.
 Each strand of the helix must be copied in complementary
fashion by DNA polymerase
DNA replication takes place in a semiconservative manner. That
is, a parent double helix forms two daughter double helices, each
composed of one parent DNA stand and on newly synthesized
stand.

First we separate them then each strand acts as a template

Each DNA (double strand) gives 2 copies.

The replication is induced by DNA polymerase.

 Semiconservative DNA
Replication
Two identical new copies of
the DNA double helix are
produced during replication
Each new strand is
complementary to its old
template strand
In each new helix, one strand
is the old template and the
other is newly synthesized.

Original DNA was labeled by a radioactive substance called N15 & The daughter was labeled using N14
Semiconservative
DNA Replication
 • E. coli genome size = 4.6 X 106 bp
• Bacteria have circular chromosome with single origin of replication.
• Replication rate is ~1000 base pairs per second.
• Duplicate chromosome in 38 minutes.
• Eukaryotes have larger genomes 3 X 109 bps
• Rate of Eukaryote chromosome replication is slower
• But because eukaryote chromosomes have multiple origins of
replication, it takes about the same amount of time to replicate
complete genome.

Eukaryote

prokaryote

Difference of DNA replication in eukaryotes and prokaryotes :

6
In prokaryotes it’s simple, its’ DNA is circular and it’s usually 4.6x10 base pair

Even though eukaryotes are a lot larger, the time of replication for eukaryotes and prokaryotes is the
same, how?

Its because in prokaryotes replication happens on 1 region “Single original point” and it replicates around
1000 base pair per second (Fast rate)

While eukaryotes have multiple origin of replication. So the replication is completed around the same
time. (Slow rate)
DNA Replication
 Stages of DNA Replication
 Initiation: perpriming complex
 Elongation: RNA primer and DNA polymerase
 Termination
Initiation
DNA should unfold before the beginning of replication. This process
is performed by a group of proteins called the prepriming complex:

• The first important protein is a small enzyme called DNAa protein.

•20-50 monomers of this enzyme will recognize and bind to a
consensus sequence called the origin of replication (a region on
DNA rich in the nucleotides Adenosine ad Thymidine).
• DNAa then causes a local opening or local melting to appear.
•This process is energy consuming; the energy is obtained by DNAa
form of ATP.

What do we expect seeing in the initiation? “What is the core of the initiation”

Unfolding

DNAa Protein binds to the consensus sequence that is recognized by DNAa proteins

Consensus sequence are fixed and are usually adenine and thymine rich

When DNAa are bound, they form the prepriming complex. Which make the DNA loose which results in
an opening of some sort “Local opening”
 Initiation
•Once the strands are separated, a
protein called single strand binding
protein (SSB protein) will bind to each
strand preventing their refolding into a
double helical form.
• The binding of SSB proteins does not
require energy.
•Another function of SSB proteins is to
protect the single DNA strands from the
action of endonucleases. Since the single
DNA strand is a substrate for these
enzymes.

Helicase
Is an enzyme that uses energy (ATP) to separate the two DNA strands
apart beginning at the local opening formed by DNAa protein. Its action is
focused at the replication fork.

SSB bind to the single strands that are created from the opening of DNA , they prevent the refolding of
the DNA “Keep 2 strands separation” as well as protecting the single strand from enzymes called
endonucleases which cut the single strand.

Another Enzyme is Helicase which separates the 2 DNA strands. It requires energy.

Helicase function “completes” the function of DNAa protein.

The shape above is called : Replication fork.

 Endonucleases and Exonucleases

We have endonucleases and exonucleases.

Exonucleases cleave from the end.

Endonucleases : Cleave within. Ex: To form nicks.

 Priming and Replication
DNA polymerase III is unable to form a nucleotides sequences from
scratch it requires a primer or an already existing nucleotide
which is RNA primer
•The starting point for DNA polymerase is a short segment of RNA
known as an RNA primer.
•The primer is RNA strand complementary to the DNA template
synthesized by an enzyme known as RNA polymerase or Primase.
•The RNA primer consists of about 10 ribonucleotides
complementary to the sequence on the DNA strand.

•The DNA polymerase (once it has reached its starting point as

indicated by the primer) then adds nucleotides one by one in an
exactly complementary manner, A to T and G to C.

Once we have the opening, we start priming & replication

We have an enzyme called DNA Polymerase 3, it’s the enzyme that forms the new strand.

It binds to the single strand but it needs a starting step before binding, this step is called “Primer”

Primer : Short segment that is made from RNA (Contains ribonucleotide and Uracil)

The primer is really important because the DNA polymerase needs to identify it first then it’ll start
synthesis.

Q:The starting point of the DNA polymerase to function is?

A: RNA Primer.

Primer is synthesized by RNA polymerase or Primase

Q: When DNA synthesis is complete, will the RNA primer still be there?

A: It’s removed by Polymerase I .

Replication fork movement is to the inside

The synthesis is always 5’  3’

5’ – 3’  Leading strand (It’s synthesis is continuous, I need RNA primer once)

3’ – 5’  Lagging strand (Needs more than one RNA Primer and it’s synthesis is fragmented “ Okazaki
fragment “ )

‫ لكن دائما تذكر الجملة‬.. 5 ‫ ل‬3 ‫’ لكن باالتجاه المعاكس عشان هيك اعتبرت انه‬3 – ’5 ‫احنا بتبعت اوكازاكي اللي عملناه انه صنعنا‬
‫اللي محطوط تحتها خطين‬

At some point RNA primer is removed causing gaps so the cells rebuild them

Elongation and replication

• Primase Complex Synthesizes short RNA primers.
•The class of enzymes which perform DNA synthesis are called the polymerase
enzymes. The polymerases can only read DNA in a 3` →5` direction  they
synthesize DNA only in 5` to 3` direction.
• The DNA double helix has the following polarity:

Leading strand 3` → 5`
lagging strand is 5` → 3`
DNA polymerase uses
the parent strands as
template and synthesizes
a new 5` → 3` strand

 Leading strand

Original : 3’  5’

Replica : 5’  3’
  Lagging Strand (Okazaki)

Original 5’  3’

Replica : 3’  5’
 DNA Replication is Semidiscontinuous
Synthesis along the leading strand is continuous and is carried out by
polymerase III. On the other hand, synthesis along the lagging strand
is discontinuous and is produced in fragments called Okazaki
fragments. and takes place via two enzymes: polymerase III and
polymerase I.
DNA Replication
 Replication of lagging strand by Okazaki fragments.
-The enzyme responsible for synthesis of these fragments is polymerase II
- It requires an RNA primer
-the synthesis takes place from 5` → 3`, in an opposite direction to that in
which the replication fork progresses.
-As the replication fork spreads and more of the double helix is unfolded, an
RNA primer is added to the lagging strand at the replication fork, the
polymerase III then proceeds in adding deoxynudeotides to the OH end of
the RNA primer, until it reaches another primer.
- A gap or a nick between the Okazaki fragments and the RNA primer.
The nick is recognized by polymerase I, that removes the ribonucleotides of
the primer and replace them by deoxynucleotides complementary to the
parent DNA strand
-This forms a discontinuous strand with stretches of DNA separated by
gaps or nicks.
-Another enzyme, ligase, seals these nicks by forming phosphodiester
bonds. The polymerase I also replaces the RNA primer on the leading strand
with deoxynucleotides.

Deoxyribonucleotide or deoxynucleotide
Replication of lagging strand by Okazaki fragments.
 Enzyme When does it work? Function Need Energy?
Topoisomerase, gyrase Initiation Remove the supercoiling
to make the DNA straight
DNAa protein Prepriming Initiation protein opening Yes
complex
SSB protein single strand Initiation Binds to the single No
binding protein strand to :
1- Prevent refolding
2- Protect it from
endonucleases
Helicase (DNAb) Initiation separates the 2 DNA Yes
strands “Replication
fork”
DNA Polymerase III Priming (Replication) Forms the new strand.
RNA Primer Priming (Replication) starting “building”
point of the DNA
polymerase III
DNA polymerase I Priming (Replication) Removes RNA primer
and replaces it by
deoxynucleotides “fills
the gaps”
Ligase Priming (Replication) Seals the nicks (gaps)
forms phosphodiester
bonds)
DNA Polymerase II Priming (Replication) Proof reading
 The Enzymology of DNA Replication
• If Watson and Crick were right, then there should be an
enzyme that makes DNA copies from a DNA template
• In 1957, Arthur Kornberg and colleagues demonstrated the
existence of a DNA polymerase -
• Three DNA polymerases in E. coli
- DNA polymerase I – DNA repair and participates in synthesis
of lagging strand
- DNA polymerase II – DNA repair
- DNA polymerase III – major polymerase involved in DNA
replication.
- Elongation involves DnaB helicase unwinding, SSB binding to
keep strands separated.
- Primase Complex synthesizes short RNA primers.
- DNA polymerase grinding away on both strands
- Topoisomerase II (DNA gyrase) relieves supercoiling that
remains

What does DNA repair mean?

When DNA polymerase 3 is doing the replication process, sometimes there will be mistakes and we need
proof reading

So if a strand is A T G and it’s complementary is T A C “Mutation” this means we need DNA Repair

Point mutation : mutation on a single nucleotide

Deletion : Nucleotide isn’t added

 DNA Polymerase also has proof reading function
• The polymerization reactions have an error rate of 1 mistake for every
100,000 base pairs incorporated (1 X 10-5 errors per base)
• Polymerases have proof reading functions. As they synthesize DNA,
they double check for any incorrectly paired bases.
• If any mismatches are found, they are removed and replaced by the
correct base This means that polymerase III has 3` → 5`
exonuclease activity or it proofreads DNA from 3` → 5`.
• Therefore proof reading function helps eliminate errors which could
lead to detrimental mutations.
• However proof reading exonuclease has error rate of 1 mistake for
every 100 base pairs (1 X 10-2 errors per base)
• Overall error rate is 1 X 10-7 errors per base.
• Polymerase I has a 5` → 3` exonuclease activity in order to remove
the RNA primer. In addition to that, it also possesses 3` → 5`
exonuclease activity which aims at proofreading the DNA sequence that
has replaced the RNA primer.

Proof reading “exonuclease activity” of polymerase III  3’ – 5’

Proof reading “exonuclease activity” of polymerase I :-

5’ – 3’ : RNA Primer removal (Synthesis activity)

3’ – 5’ : Proof reading activity (Exonuclease activity)

Polymerase action
 DNA polymerase I has 5’ to 3’ exonuclease activity that removes RNA
primer. Also has 5’ to 3’ DNA polymerase activity to fill in the gap.
(proofreading 3’-5’ exonuclease activity)
Ligase connects loose ends. Used NAD+ in phosphoryltransfer reaction,

DNA Polymerase I/ Ligase Required to Join

Okazaki Fragments

Q1 If the strand is 5’ AAG 3’

How will the proof reading be?

GAA 

AAG 

Q2 : The primary enzymes that are used in the joining for the okazaki fragment?

A: DNA Polymerase I & Ligase

•Polymerase I has a 5`→
3` exonuclease activity in
order to remove the RNA
primer. In addition to that,
it also possesses 3`→ 5`
exonuclease activity which
aims at proofreading the
DNA sequence that has
replaced the RNA primer.
DNA Replication
 Enzymes that relief supercoiling (Super-twisting )
During the replication the two strand of the DNA should be away
from each other
Supercoiling should be removed  so the DNA have to rotate
opposite of the direction of the coiling but this method costs
energy and lead to another supercoiling
 Topoisomerase enzymes solve this problem. There are two types:
 Topoisomerase I works only on one strand of the DNA and cut that
strand, then allows the other strand to pass through to solve the
supercoil, then it connects the strand again [nuclease activity and
ligase activity]
Topoisomerase II works on the two strands of the DNA at the
same time, and it is more efficient than topoisomerase I.
Topoisomerase II cuts both strands of the DNA, allows the other
one to move through it, and basically it will relieve supercoil.

topoisomerase remove the tertiary structure “supercoiling”

topoisomerase I  works one strand only by cutting it and then connecting it

so it has cutting activity and ligase activity

topoisomerase 2  2 strands at the same time and is more efficient and complex, removes the
supercoiling in a better way.
Supercoiling During DNA strand separation
Enzymes that Relief Supercoiling
 DNA Replication in Eukaryotes
• Occurs similarly to what occurs in prokaryotes.
• Multiple origins of replication
• Replication is slower than in prokaryotes.
• 5 different DNA polymerases in Eukaryotes.

Eukaryotic DNA Polymerases

 Alpha (Pol  )– Primer synthesis and DNA repair
 Beta – DNA repair
 Gamma – Mitochondrial DNA replication
 Delta (Pol  )– Leading and lagging strand synthesis, and DNA
repair
 Epsilon (Pol  ) – Repair and gap filling on lagging strand.

Generally speaking we must have the DNA polymerase I & III

 Termination of Replication
• Termination occurs at ter (terminus )region of
E. coli chromosome.
• ter region rich in Gs and Ts, signals the end of
replication.
• Terminator utilization substance (Tus) is a
protein which binds to ter region.
• Tus prevents replication fork from passing by
inhibiting helicase activity.

Termination happens when there is a specific sequence of the deoxyribonucleotides on the DNA

Usually full of guanine and thymine.

The End
 DNA Repair
• A fundamental difference from RNA,
protein, lipid, etc.
• All these others can be replaced, but DNA
must be preserved
• Cells require a means for repair of missing,
altered or incorrect bases, bulges due to
insertion or deletion, UV-induced pyrimidine
dimers, strand breaks or cross-links
• Two principal mechanisms: methods for
reversing chemical damage and excision
repair.
 Repair of UV
Induced Thymine
Dimers
 General excision-
repair pathway

•Excision-repair systems
scan DNA duplexes for
mismatched bases, excise
the mispaired region and
replace it
Slide 34

Repair of damage resulting

from the deamination of
cytosine
• Deamination of cytosine to uracil
is one of most common forms of
DNA damage
• DNA glycosylases cleave bases at
N-glycosidic linkages. Leaving
sugar-phosphate backbone.
• Endonuclease identifies abscent
base and sugar phosphate.
• Gap then filled in by DNA
polymerase and ligase.