Genetics, Lecture 6 (Lecture Notes)

8/8/2019 Genetics, Lecture 6 (Lecture Notes)

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DNA replication

Nabeel Basheer

Salah Banat

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Genetics Lecture 6

10/10/2010

Done by : Salah Banat

Before I startI 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 Im going to talk .

1stIm going to talk aboutgeneral 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 whats 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 synthesizedstrand according to the template , the substrate that I want are the

dNTPs , they arent mono phosphate nucleotides , and they arent

ribo nucleotide mono phosphate , they are deoxynucleoside

triphosphates .

Could you give me an example of dNTPs ?

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 thats catalyzed by a

group of enzymes .


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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 its called DNA

dependant , because it uses DNA as a template , also you are goingto 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 :

- its 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, its called semi continuous . So its

not continuous , its not discontinuous ,, its semi continuous .

And about the semi conservation ,, its not conservative , its not

distributive >> Its 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 DNAs , and

in the 2nd generation you will see some of the daughter DNA and the

parental DNA ,,, but if its conservative always the 2 parental DNA

will not be separated from each other .


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- Its 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 characteristicsconcerning 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 beincluded 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 beings 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 1stgeneration and the 2nd generation , and they

separated the DNA and with time by using ultra centrifugation andchloride 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

couldnt hear what she said !

DR : Yes because the nitrogen will be distributed all over the DNA in

the nitrogen bases in the pyrimidins and purines , 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 .

And once it starts a replication fork will be formed in both directions

and the DNA replication will extend in opposite directions .


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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 Im 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

53 direction . The synthesize of the newly synthesized DNA goes

from 53 direction because DNA polymerase only could do

polymerization in that direction and we dont have any DNA

polymerase to do in the 35 direction .

And these direction im talking about are related to the newly

synthesized DNA , so Im talking about the direction of synthesizing

the new DNA strand in the 53 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 U replication. ( It seems like U ) .


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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 , its 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 theARS , 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 its called the

leading strand and its synthesized in the 53 direction , and

another strand will be synthesized in a discontinuous small

fragments, each small fragment is called okazaki fragment , and

its called lagging strand ( The sum of the okazakis ) .

Why it is in discontinuous ?

Because if they are not synthesized in a discontinuous they will be

unable to synthesize in the 35 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 53 direction because we

dont have enzymes that will synthesize in 35 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 )

Concerning about okazaki fragments , in eukaryotics they are

smaller than that in prokaryotic , averaging from 100-150 , but in


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the prokaryotics , it is from 1000-2000 , thats 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 .. its 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 thescientists 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 ?

its 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 its going to polymerize

deoxynucleotide into poly nucleotide or into DNA , RNA polymerase

synthesize RNA . DNA G protein sometime its 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 doesnt 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 RNAsprimers .

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 intorelaxed DNA .

Type 2 : makes 2 breaks and changes positive super coil into

negative super coil .


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So the topoisomerase relief super coil constrain .

Now . I will talk in more details aboutDNA polymerase , indeed we

have 5 types of DNA polymerase :

1- DNA-pol I2- DNA-pol II3- 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 ofDNA-pol I ( micro organism that doesnt 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 itDNA-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 wasable to grow also . so they found another DNA-pol and named it

DNA-pol III .

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


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o a5eeran 5ala9na el mo7a9ara thnx to 7amzeh el rashdan ll

mosa3adeh .

o forgive me 4 any mistake . Wella 27keelko dont 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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Genetics, Lecture 6 (Lecture Notes)