DNA

Nabeel
Salah
replication
Basheer
Banat35 6
10/10/20

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Genetics Lecture 6
10/10/2010
Done by : Salah Banat

Before I start I would like to give you small announcement

concerning the first exam . The first exam will be on 26/10 instead
of 18 .
As you see , I will talk in this lecture and the coming lecture and may
be the third lecture on DNA replication but I hope to finish DNA
replication in 2 lectures.
These are specific objectives that you have to ask yourself by the
end of these 2-3 lectures .
The outlines of the lectures of DNA replication :
1-I will talk about the mechanism .
2-The general characteristics of DNA replication.
3-General concepts , then I will talk about the tools of DNA
replication , the process , the mechanism of DNA replication , and
finally I will talk about mutation and repair of those mutations.
So these are the outlines of what I’m going to talk .
1st I’m going to talk about general concepts of DNA replication.
DNA replication is very important because it helps for the transfer of
genetic information from parents to children . SO it must be accurate
without any mistake , If there is any mistake , mutation will take

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place and then will change the whole transfer of genetic
informations .
So from parental DNA to exactly daughter cells , the genetic
information must be transferred with high accuracy rate .
So what’s going on in transferring genetic information DNA from
Here is the major reaction that will take place , we will have the :
- DNA strand which is composed of number of deoxynucleoside
mono phosphate units as the template . ( dNMP )n ….. n = The
number of (deoxynucleoside mono phosphate)’s .
- and also you need substrate in order to built the new synthesized
strand according to the template , the substrate that I want are the
dNTP’s , they aren’t mono phosphate nucleotides , and they aren’t
ribo nucleotide mono phosphate , they are deoxynucleoside
triphosphates .
Could you give me an example of dNTP’s ?
dATP , dGTP , dTTP , but dUTP is not correct ! why ?
because UTP is not founded in DNA , okay ?!
So when the template and the substrate interact, the result will be :
elongated DNA strand . ( dNMP )n +1 + PPi(pyrophosphate)
* The PPi here is from the dNTP .
And what happened as you will see in a moment , that there will be
formation of 3’-5’ phosphodiester bond , and that’s catalyzed by a
group of enzymes .

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DNA replication is not a simple process , it’s multi process and
require alot of tools and enzymes .
See slide ‘’7’’ >> This exactly what happened , suppose you have
this template ( on the left ) , and this is the newly elongated DNA or
the newly synthesized DNA ( on the right ) .
The phosphodiester bond that will be formed between the free OH
group at the 3’ end of the template and alpha phosphate ( p #1 ) of
the substrate , and a PPi will be generated ( go out from the
substrate ) , and the enzyme that will catalyze this reactant is called
DNA polymerase ,, ending up with the addition of 1 deoxynucleoside
monophosphate to the strand ( template ) that you started with .
Now remember that the process takes place in the 5’-3’ direction of
the new DNA strand although we add the deoxynucleoside
monophosphate to the 3’ free OH group of the new synthesized DNA
, so it is in the 5’ – 3’ in the new strand .
What we need for DNA replication :
1. Template , and the template here is the DNA that is going to be
replicated , and it must be processed , because we cannot use a
double stranded DNA ,, it must unwinded , and we have to use the
template as a single stranded DNA that already comes from double
stranded DNA .
2. We need substrate ,, deoxynucleoside triphosphate .
3. primer .
4. DNA-dependent DNA polymerase (DDDP) .

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DNA polymerase cannot initiate DNA replication ( de novo)by it self
it requires as I mentioned earlier free 3’ OH group and that is
provided by what is called primer .
Primer is stretched oligo-nucleotide or oligo-deoxynucleotide that
provides the free 3’ OH groups . In case of DNA replication in most
of organisms , it is an RNA oligo-ribonucleotide , and DNA
polymerase will use that olige-ribonucleotide RNA primer to start
the DNA replication. In some other organisms the primer could be
protein .

Could a protein provides free 3’ OH group or free riboxyl group ??

If that protein has serine , tyrosine or thrioneen , and the OH group
is free >> it could act as primer .
We need enzymes , and the most important enzyme is DNA
polymerase as you will see in a moment , and it’s called DNA
dependant , because it uses DNA as a template , also you are going
to see RNA dependant DNA polymerase .
Not only this enzyme which is DNA polymerase ; but also DNA
replication requires addition of proteins as you will see . ( battery of
proteins ) .
These are general properties of DNA replication :
- it’s semi conservative replication , and that is highly significant ,
because of the transferring of genetic information accurately as you
will see .

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- DNA replication is bi directional , it goes in two directions ;; in
opposite directions in order to finish tha DNA replication In a short
time .
- Also as you will see , it is semi-continuous ; because one DNA
strand will be synthesized in a continuous form , and the other
strand will be synthesized is discontinuous form , and because of the
continuity of synthesizing of one strand and the discontinuity of
synthesizing in the other strand, it’s called semi continuous . So it’s
not continuous , it’s not discontinuous ,, it’s semi continuous .
And about the semi conservation ,, it’s not conservative , it’s not
distributive >> It’s semi conservative , which means that in the
first generation , the parental DNA will be separated and in the
daughter DNA always you will have one of the parental DNA’s , and
in the 2nd generation you will see some of the daughter DNA and the
parental DNA ,,, but if it’s conservative always the 2 parental DNA
will not be separated from each other .

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 - It’s high accurate = no mistakes  probability of a mistake will be
(1/10^9) which is very low , although of that , it happens , because
during replications we have proteins and enzymes to repair these
mistakes and correct them .
So these are very important concepts and general characteristics
concerning DNA replication - semi conservative - bi directional
- semi continuous - high fidelity and highly accurate.
Slide ( 10 )
This is the semi conservative theory ( Below ) . These are the
parental DNA molecules in the semi conservative model , in the 1st
generation the 2 parentally strands will be separated and will be
included in the daughter DNA strands .

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In a conservatives model of DNA replication , the parental DNA will
stay as it is , they will not be separated and newly 2 DNA strands
will be synthesized .
In the case of distributive , parts of the parental DNA will be
included in the newly synthesized daughters DNA , it was found by
evidence and experiments that the model of DNA replication for
human being’s genetic transferring from one generation to another ,
, the DNA replication is semi conservative .
Who could you think about how the scientists reached to the
conclusion that DNA replication is semi conservative ?
A student answers: by using radioactive phosphate to mark the new
strands .
DR : the concept is correct , yes they use radioactive indeed.
The radioactive nitrogen ( the heavy nitrogen) (N15) , versus (N14)
( normal ) , and then they took a liquid after they but the (N15) all

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over the media for the bacteria to grow , then they stop using N15,
and put the bacteria in N14( normal ) , and they started taking
liquids in the 1st generation and the 2nd generation , and they
separated the DNA and with time by using ultra centrifugation and
chloride and separating the DNA and looking the constituents of the
N15 versus N14 they found it is a semi conservative model .

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But why did they use nitrogen ? another student answers , but I
couldn’t hear what she said !
DR : Yes because the nitrogen will be distributed all over the DNA in
the nitrogen bases in the pyrimidin’s and purine’s , they will use
N15 to synthesize new deoxynucleotides , so they could separate the
heavy nitrogen from the normal nitrogen .
Now concerning the bi directional replication : the replication will
go in opposite directions in both prokaryotes and eukaryotes , and it
was found that DNA replication will not start at any place in the DNA
or in the chromosomes , but it will initiate or start at regions called
“origin of replication” in prokaryotes or ‘’autonomous replication
sequence’’ in eukaryotes , and those origins or sites have a
consensus sequences in which the DNA replication will start from .

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And once it starts a replication fork will be formed in both directions
and the DNA replication will extend in opposite directions .
Now , there is 1 portion of replication in prokaryotes and there are
multi origin of replication in eukaryotes . Why we have multiple
replication sequences in eukaryotes comparable to prokaryotes ??
Student : The size of the DNA .
DR : The size , because the eukaryotic genome is very huge
compared to the prokaryotes .
( The figure below )
This is the replication fork , that I’m talking about this is the double
stranded DNA , and in order for a replication fork to form ; the 2
strands must be unwind , and these are the newly synthesized DNA
( referring to them ) , and this is the direction of replication ( also ) ,
now I want you to remember that the DNA replication is from the
5’3’ direction . The synthesize of the newly synthesized DNA goes
from 5’3’ direction because DNA polymerase only could do
polymerization in that direction and we don’t have any DNA
polymerase to do in the 3’5’ direction .
And these direction i’m talking about are related to the newly
synthesized DNA , so I’m talking about the direction of synthesizing
the new DNA strand in the 5’3’ direction .

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And this is the replication in prokaryotes , The replication process
starts from the origin, and proceeds in two opposite directions. It is
named q replication. ( It seems like q ) .

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Concerning the replication in eukaryotics , I mentioned it has multi
sequences to start replication . It is not at 1 site , and it has specific
consensus sequence . The space between two adjacent origins is
called the replicon ,( a functional unit of replication ) .
About the origin of starting replication , In eukaryotic it is called
autonomous replication sequences, while in prokaryotic it is called
origin of replication , it’s 1 origin of replication in prokaryotics and
it is multi in eukaryotics .

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The replication forks will not form simultaneously , they will be
formed in sequential mode . So suppose that this (referring to one of
the forks above ) starts and goes in this direction( one of the tow ) ,
the other goes in the opposite direction. Once it reachs the ARS , the
2nd replication fork will start doing the replication .
While we are talking about DNA replication , we will see that one
strand will be synthesized in a continuous fashion and it’s called the
leading strand and it’s synthesized in the 5’3’ direction , and
another strand will be synthesized in a discontinuous small
fragments, each small fragment is called “okazaki fragment” , and
it’s called lagging strand ( The sum of the okazaki’s ) .
Why it is in discontinuous ?
Because if they are not synthesized in a discontinuous they will be
unable to synthesize in the 3’5 direction , so the DNA strand must
take some formation in order for the lagging strand to be
synthesized into small fragments and then those small fragments
will be connected with each other ,, and remember>> each
fragment must be synthesized in the 5’3’ direction because we
don’t have enzymes that will synthesize in 3’5’ direction .
( There will be a lot of 5’ to 3’ small directions of replication , in the
hollow 3’ to 5’ direction of replication ) .
( Slide 23 )

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Concerning about okazaki fragments , in eukaryotics they are
smaller than that in prokaryotic , averaging from 100-150 , but in
the prokaryotics , it is from 1000-2000 , that’s why they are called
semi continuous .
A Student asks an excellent question : the primer is RNA but it
synthesize DNA ? Is it logical to have RNA while we are doing DNA
replication ?
during this process >> yes .. it’s logical . While the end result ,
those RNA primers , there will be retinoid and the gaps instead of
them will be built by DNA , according to the template , and as a
result , the lagging strand will be pure of DNA and nothing RNA is
found in the leading nor lagging .
Now , I will talk about the enzyme machinery that is required for the
process of DNA replication , this is a sample , this is of what is known
so far about the proteins that are required for DNA replication , and
many other proteins will be discovered in few chapters . when I
studied this topic before 30 years only 1 or 2 proteins were known
but now , dozens of proteins are known and after 20-30 years many
dozens of proteins will be known and remember that we or the
scientists only know 50% of the function of the genes that are
already known .

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Now this table as I mentioned:

This is a sample and each one of these proteins is coming from a

gene and each has a function , for example the DNA A protein , it
will recognize origin of the replication .
but what is origin of replication ?
it’s a specific DNA sequence .
DNA B protein : the function of this protein is to unwind the double
stranded DNA , once the DNA A recognize the origin of replication ,
DNA B comes to open the double stranded DNA .
Why do we want to unwind ?
Because each single strand will be used as a template to exactly
transferring the genetic information with a fidelity and high

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accuracy from the parent to daughter . so unwinding is very
important .
DNA polymerase (DNA POL) that means it’s going to polymerize
deoxynucleotide into poly nucleotide or into DNA , RNA polymerase
synthesize RNA . DNA G protein sometime it’s called primase that
means it synthesize primer , as the cell cannot start synthesizing
DNA from nothing , but the cell could synthesize RNA from
nucleotides . it doesn’t require free 3’ OH group like DNA . usually
the primer is an oligo-ribonucleotide , it margins from 1-20 ribo
nucleotides , and as I mentioned earlier for the leading strand we
need only 1 primer but for the lagging strand we need multi RNA’s
primers .

SSB stand for single stranded binding protein , what do you think
the function of this SSB ?
DR : to stabilize the single stranded DNA and prevent it from
winding , and to protect it from nucleases because some nucleases
will come and attack the single stranded DNA , so presence of those
SSB on the single stranded DNA will protect them from digestion ,
So it has 2 important things : protection and stabilization .
DNA topoisomerase : there are many types of DNA topoisomerase
which means enzymes that will unchain the conformation of the
DNA . The DNA is double stranded right ?
It is stabilized from one side and the other one is open , if I have a
rope I would show it to you !

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Now , DNA will unwind by DNA G [ make sure about this point bcs
DNA G is to synthesis the primer , maybe he meant DNA B ] , I cannot
reopen it again , and this is called the positive super coil , which
means that the number of turns is more than 10 , and when this
happens DNA replication stops ! so  topoisomerase will come and
bind the positive super coil and it will break the the 2 strands of
DNA and relief the positive super coil and change the positive super
coil to negative super coil . this is one type of topoisomerase , and
there are 2 types of topoisomerase :
Type 1 : break in one strand and changes the negative super coil into
relaxed DNA .
Type 2 : makes 2 breaks and changes positive super coil into
negative super coil .

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 Type I topoisomerases cut one strand of double-stranded DNA, relax the strand, and
re-anneal the strands.

Type II topoisomerases cut both strands of the DNA helix simultaneously in order to
unwind it.
Wikipedia.com

So the topoisomerase relief super coil constrain .

Now . I will talk in more details about DNA polymerase , indeed we
have 5 types of DNA polymerase :
1- DNA-pol I
2- DNA-pol II
3- DNA-pol III

And as you know DNA-pol is very important .

After Arthur Kornberg discovered DNA-pol I , how did they discover
the DNA-pol II & DNA-pol III ???? who have an idea ?
DR : what happened is that after they discovered DNA-pol I they
made a mutant of DNA-pol I ( micro organism that doesn’t have
DNA-pol I ) and they found that this mutant was able to live and
grow even DNA-pol I is not there , after that they found another
DNA-pol and named it DNA-pol II , after that they did another
mutation for DNA-pol I & DNA-pol II and try to grow the culture
that have double mutants (DNA-pol I & DNA-pol II ) , and it was
able to grow also . so they found another DNA-pol and named it
DNA-pol III .

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o a5eeran 5ala9na el mo7a9’ara  thnx to 7amzeh el rashdan ll
mosa3adeh  .
o forgive me 4 any mistake ……. Wella 27keelko don’t forgive
me l2no el 7ag mesh 3aleako el 7ag 3ale fara3’ elko :P
7amloone jmeeleh Kaman shwai :P

Done by :

Sama group _Correction team .

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