Lecture 2 - Mechanism of DNA replication | Molecular Biology (DNA) by ILLL, DU - Biotechnology Engineering (BT) PDF Download

 Page 1


           Mechanism of DNA replication 
 
1 
Institute of Lifelong Learning, University of Delhi 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Subject : Molecular Biology 
Lesson : Mechanism of DNA replication  
Lesson Developer : Dr. Devi Lal  
College / Department : Department of Zoology , Ramjas 
College 
 
 
 
  
 
 
 
 
 
 
 
Page 2


           Mechanism of DNA replication 
 
1 
Institute of Lifelong Learning, University of Delhi 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Subject : Molecular Biology 
Lesson : Mechanism of DNA replication  
Lesson Developer : Dr. Devi Lal  
College / Department : Department of Zoology , Ramjas 
College 
 
 
 
  
 
 
 
 
 
 
 
           Mechanism of DNA replication 
 
2 
Institute of Lifelong Learning, University of Delhi 
 
Table of Contents   
 
Chapter: Mechanism of DNA replication 
 Topic         Page No.                                                       
Introduction…………………………………………………………………………………………………………..1 
Raw materials for DNA synthesis………………………………………………………….  
Mechanism/model for DNA replication…………………………………………………………. 
DNA replication follows semi-conservative model……………………………… 
Enzymes involved in DNA replication…………………………………………………………. 
DNA polymerases………………………………………………………….………………………………………… 
DNA polymerases in eukaryotes………………………………………………………….……………………… 
DNA polymerases in prokaryotes………………………………………………………….…………………….. 
Family of DNA polymerases………………………………………………………….…………………………….. 
Sliding clamps and clamp loader………………………………………………………….……… 
DNA ligase………………………………………………………….………………………………………………… 
Primase………………………………………………………….…………………………………………………………. 
DNA helicases………………………………………………………….…………………………………………… 
Single stranded DNA binding protein (SSBs) ………………………………………… 
Topoisomerases………………………………………………………….………………………………………. 
General principles of DNA replication…………………………………………………..  
Summary………………………………………………………….………………………………………………… 
Exercise/ Practice………………………………………………………….…………………………………. 
Glossary………………………………………………………….……………………………………………………… 
References/ Bibliography/ Further Reading……………………………………… 
Web links………………………………………………………….………………………………………………… 
Answers………………………………………………………….…………………………………………………… 
 
Page 3


           Mechanism of DNA replication 
 
1 
Institute of Lifelong Learning, University of Delhi 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Subject : Molecular Biology 
Lesson : Mechanism of DNA replication  
Lesson Developer : Dr. Devi Lal  
College / Department : Department of Zoology , Ramjas 
College 
 
 
 
  
 
 
 
 
 
 
 
           Mechanism of DNA replication 
 
2 
Institute of Lifelong Learning, University of Delhi 
 
Table of Contents   
 
Chapter: Mechanism of DNA replication 
 Topic         Page No.                                                       
Introduction…………………………………………………………………………………………………………..1 
Raw materials for DNA synthesis………………………………………………………….  
Mechanism/model for DNA replication…………………………………………………………. 
DNA replication follows semi-conservative model……………………………… 
Enzymes involved in DNA replication…………………………………………………………. 
DNA polymerases………………………………………………………….………………………………………… 
DNA polymerases in eukaryotes………………………………………………………….……………………… 
DNA polymerases in prokaryotes………………………………………………………….…………………….. 
Family of DNA polymerases………………………………………………………….…………………………….. 
Sliding clamps and clamp loader………………………………………………………….……… 
DNA ligase………………………………………………………….………………………………………………… 
Primase………………………………………………………….…………………………………………………………. 
DNA helicases………………………………………………………….…………………………………………… 
Single stranded DNA binding protein (SSBs) ………………………………………… 
Topoisomerases………………………………………………………….………………………………………. 
General principles of DNA replication…………………………………………………..  
Summary………………………………………………………….………………………………………………… 
Exercise/ Practice………………………………………………………….…………………………………. 
Glossary………………………………………………………….……………………………………………………… 
References/ Bibliography/ Further Reading……………………………………… 
Web links………………………………………………………….………………………………………………… 
Answers………………………………………………………….…………………………………………………… 
 
           Mechanism of DNA replication 
 
3 
Institute of Lifelong Learning, University of Delhi 
 
Introduction 
The mid of nineteenth century was dominated by the work of Charles Darwin. Much 
attention was focused on his work “On the Origin of Species”. The beginning of twentieth 
century saw the rediscovery of Mendel’s law of heredity.  Mid twentieth century was marked 
by important discoveries that changed the perspective of molecular biologists. These 
discoveries, that led to the origin of modern era in molecular biology were the landmark 
works of Erwin Chargaff; Rosalind Franklin and Maurice Wilkins; James Watson and Francis 
Crick. This work established the structure of DNA, the key macromolecule that directs life. 
Once the structure of DNA was known, the scientists started looking for the mechanisms 
that are responsible for duplication of this important molecule. The accurate replication of 
DNA is important to pass correct genetic information from one generation to next. The 
discovery that DNA replication is semi-conservative helped in understanding how DNA 
replicates. Following this various enzymes were isolated that were known to play key role in 
this process. Subsequently it was found that the process of DNA replication is same in 
prokaryotes and eukaryotes employing same set of enzymes with some variation. In this 
unit we will describe the functions of enzymes involved in DNA replication and basic 
mechanism of this process.  
Raw material for DNA synthesis 
The synthesis of DNA requires certain raw materials. These are deoxyribonucleotides (dATP, 
dCTP, dGTP and dTTP), primer, and the enzyme (DNA polymerase) that can faithfully copy 
the template DNA. The primer is a short stretch of RNA that has the complementarity with 
the template. A primer has a free 3’-OH that is extended by the DNA polymerase by 
addition of the nucleotides. The addition of nucleotides takes place by formation of 
phosphodiester bond. This reaction follows S
N
2 mechanism in which the exposed 3’-OH of 
the primer attacks the a-phosphate group of the nucleotide, leaving pyrophosphate which is 
subsequently hydrolyzed by pyrophosphatase. The hydrolysis of pyrophosphate provides the 
energy for the reaction to proceed. The overall reaction is: 
(NMP)
n
+ NTP (NMP)
n+1
+ P—P 2Pi
Mononucleotide chain Incoming nucleotide 
triphosphate
pyrophosphate Growing nucleotide 
chain
 
Page 4


           Mechanism of DNA replication 
 
1 
Institute of Lifelong Learning, University of Delhi 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Subject : Molecular Biology 
Lesson : Mechanism of DNA replication  
Lesson Developer : Dr. Devi Lal  
College / Department : Department of Zoology , Ramjas 
College 
 
 
 
  
 
 
 
 
 
 
 
           Mechanism of DNA replication 
 
2 
Institute of Lifelong Learning, University of Delhi 
 
Table of Contents   
 
Chapter: Mechanism of DNA replication 
 Topic         Page No.                                                       
Introduction…………………………………………………………………………………………………………..1 
Raw materials for DNA synthesis………………………………………………………….  
Mechanism/model for DNA replication…………………………………………………………. 
DNA replication follows semi-conservative model……………………………… 
Enzymes involved in DNA replication…………………………………………………………. 
DNA polymerases………………………………………………………….………………………………………… 
DNA polymerases in eukaryotes………………………………………………………….……………………… 
DNA polymerases in prokaryotes………………………………………………………….…………………….. 
Family of DNA polymerases………………………………………………………….…………………………….. 
Sliding clamps and clamp loader………………………………………………………….……… 
DNA ligase………………………………………………………….………………………………………………… 
Primase………………………………………………………….…………………………………………………………. 
DNA helicases………………………………………………………….…………………………………………… 
Single stranded DNA binding protein (SSBs) ………………………………………… 
Topoisomerases………………………………………………………….………………………………………. 
General principles of DNA replication…………………………………………………..  
Summary………………………………………………………….………………………………………………… 
Exercise/ Practice………………………………………………………….…………………………………. 
Glossary………………………………………………………….……………………………………………………… 
References/ Bibliography/ Further Reading……………………………………… 
Web links………………………………………………………….………………………………………………… 
Answers………………………………………………………….…………………………………………………… 
 
           Mechanism of DNA replication 
 
3 
Institute of Lifelong Learning, University of Delhi 
 
Introduction 
The mid of nineteenth century was dominated by the work of Charles Darwin. Much 
attention was focused on his work “On the Origin of Species”. The beginning of twentieth 
century saw the rediscovery of Mendel’s law of heredity.  Mid twentieth century was marked 
by important discoveries that changed the perspective of molecular biologists. These 
discoveries, that led to the origin of modern era in molecular biology were the landmark 
works of Erwin Chargaff; Rosalind Franklin and Maurice Wilkins; James Watson and Francis 
Crick. This work established the structure of DNA, the key macromolecule that directs life. 
Once the structure of DNA was known, the scientists started looking for the mechanisms 
that are responsible for duplication of this important molecule. The accurate replication of 
DNA is important to pass correct genetic information from one generation to next. The 
discovery that DNA replication is semi-conservative helped in understanding how DNA 
replicates. Following this various enzymes were isolated that were known to play key role in 
this process. Subsequently it was found that the process of DNA replication is same in 
prokaryotes and eukaryotes employing same set of enzymes with some variation. In this 
unit we will describe the functions of enzymes involved in DNA replication and basic 
mechanism of this process.  
Raw material for DNA synthesis 
The synthesis of DNA requires certain raw materials. These are deoxyribonucleotides (dATP, 
dCTP, dGTP and dTTP), primer, and the enzyme (DNA polymerase) that can faithfully copy 
the template DNA. The primer is a short stretch of RNA that has the complementarity with 
the template. A primer has a free 3’-OH that is extended by the DNA polymerase by 
addition of the nucleotides. The addition of nucleotides takes place by formation of 
phosphodiester bond. This reaction follows S
N
2 mechanism in which the exposed 3’-OH of 
the primer attacks the a-phosphate group of the nucleotide, leaving pyrophosphate which is 
subsequently hydrolyzed by pyrophosphatase. The hydrolysis of pyrophosphate provides the 
energy for the reaction to proceed. The overall reaction is: 
(NMP)
n
+ NTP (NMP)
n+1
+ P—P 2Pi
Mononucleotide chain Incoming nucleotide 
triphosphate
pyrophosphate Growing nucleotide 
chain
 
           Mechanism of DNA replication 
 
4 
Institute of Lifelong Learning, University of Delhi 
 
Mechanism/model for DNA replication 
Three basic models have been proposed for DNA replication (Fig. 1). These are:  
1) Conservative Model: This model assumes that the parent DNA strands serve as the 
template for synthesis of complementary strand. After replication is finished, both 
the parent strands are retained.  
2) Semi-conservative Model: In this type of replication model, the parent DNA 
strands separate and serve as a template for synthesis of new complementary 
strand. Thus the daughter DNA produced after replication has one parental strand 
and one newly formed strand. This is so far the correct model for DNA replication 
which has been proved in the experiments of Meselson and Stahl.  
3) Dispersive Model: In this model, the parent DNA strands are broken and serve as 
template for the production of new DNA double helix.  
 
Conservative 
replication
Semi-conservative 
replication
Dispersive 
replication
 
Fig 1. Three models that were proposed to explain the possible ways in which the DNA 
replicates. Out of these models semi-conservative replication model was supported from 
the experimental evidences. (Source: Author) 
Page 5


           Mechanism of DNA replication 
 
1 
Institute of Lifelong Learning, University of Delhi 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Subject : Molecular Biology 
Lesson : Mechanism of DNA replication  
Lesson Developer : Dr. Devi Lal  
College / Department : Department of Zoology , Ramjas 
College 
 
 
 
  
 
 
 
 
 
 
 
           Mechanism of DNA replication 
 
2 
Institute of Lifelong Learning, University of Delhi 
 
Table of Contents   
 
Chapter: Mechanism of DNA replication 
 Topic         Page No.                                                       
Introduction…………………………………………………………………………………………………………..1 
Raw materials for DNA synthesis………………………………………………………….  
Mechanism/model for DNA replication…………………………………………………………. 
DNA replication follows semi-conservative model……………………………… 
Enzymes involved in DNA replication…………………………………………………………. 
DNA polymerases………………………………………………………….………………………………………… 
DNA polymerases in eukaryotes………………………………………………………….……………………… 
DNA polymerases in prokaryotes………………………………………………………….…………………….. 
Family of DNA polymerases………………………………………………………….…………………………….. 
Sliding clamps and clamp loader………………………………………………………….……… 
DNA ligase………………………………………………………….………………………………………………… 
Primase………………………………………………………….…………………………………………………………. 
DNA helicases………………………………………………………….…………………………………………… 
Single stranded DNA binding protein (SSBs) ………………………………………… 
Topoisomerases………………………………………………………….………………………………………. 
General principles of DNA replication…………………………………………………..  
Summary………………………………………………………….………………………………………………… 
Exercise/ Practice………………………………………………………….…………………………………. 
Glossary………………………………………………………….……………………………………………………… 
References/ Bibliography/ Further Reading……………………………………… 
Web links………………………………………………………….………………………………………………… 
Answers………………………………………………………….…………………………………………………… 
 
           Mechanism of DNA replication 
 
3 
Institute of Lifelong Learning, University of Delhi 
 
Introduction 
The mid of nineteenth century was dominated by the work of Charles Darwin. Much 
attention was focused on his work “On the Origin of Species”. The beginning of twentieth 
century saw the rediscovery of Mendel’s law of heredity.  Mid twentieth century was marked 
by important discoveries that changed the perspective of molecular biologists. These 
discoveries, that led to the origin of modern era in molecular biology were the landmark 
works of Erwin Chargaff; Rosalind Franklin and Maurice Wilkins; James Watson and Francis 
Crick. This work established the structure of DNA, the key macromolecule that directs life. 
Once the structure of DNA was known, the scientists started looking for the mechanisms 
that are responsible for duplication of this important molecule. The accurate replication of 
DNA is important to pass correct genetic information from one generation to next. The 
discovery that DNA replication is semi-conservative helped in understanding how DNA 
replicates. Following this various enzymes were isolated that were known to play key role in 
this process. Subsequently it was found that the process of DNA replication is same in 
prokaryotes and eukaryotes employing same set of enzymes with some variation. In this 
unit we will describe the functions of enzymes involved in DNA replication and basic 
mechanism of this process.  
Raw material for DNA synthesis 
The synthesis of DNA requires certain raw materials. These are deoxyribonucleotides (dATP, 
dCTP, dGTP and dTTP), primer, and the enzyme (DNA polymerase) that can faithfully copy 
the template DNA. The primer is a short stretch of RNA that has the complementarity with 
the template. A primer has a free 3’-OH that is extended by the DNA polymerase by 
addition of the nucleotides. The addition of nucleotides takes place by formation of 
phosphodiester bond. This reaction follows S
N
2 mechanism in which the exposed 3’-OH of 
the primer attacks the a-phosphate group of the nucleotide, leaving pyrophosphate which is 
subsequently hydrolyzed by pyrophosphatase. The hydrolysis of pyrophosphate provides the 
energy for the reaction to proceed. The overall reaction is: 
(NMP)
n
+ NTP (NMP)
n+1
+ P—P 2Pi
Mononucleotide chain Incoming nucleotide 
triphosphate
pyrophosphate Growing nucleotide 
chain
 
           Mechanism of DNA replication 
 
4 
Institute of Lifelong Learning, University of Delhi 
 
Mechanism/model for DNA replication 
Three basic models have been proposed for DNA replication (Fig. 1). These are:  
1) Conservative Model: This model assumes that the parent DNA strands serve as the 
template for synthesis of complementary strand. After replication is finished, both 
the parent strands are retained.  
2) Semi-conservative Model: In this type of replication model, the parent DNA 
strands separate and serve as a template for synthesis of new complementary 
strand. Thus the daughter DNA produced after replication has one parental strand 
and one newly formed strand. This is so far the correct model for DNA replication 
which has been proved in the experiments of Meselson and Stahl.  
3) Dispersive Model: In this model, the parent DNA strands are broken and serve as 
template for the production of new DNA double helix.  
 
Conservative 
replication
Semi-conservative 
replication
Dispersive 
replication
 
Fig 1. Three models that were proposed to explain the possible ways in which the DNA 
replicates. Out of these models semi-conservative replication model was supported from 
the experimental evidences. (Source: Author) 
           Mechanism of DNA replication 
 
5 
Institute of Lifelong Learning, University of Delhi 
 
DNA replication follows semi-conservative model 
Meselson and Stahl (1958) in a landmark discovery proved that DNA replication is semi-
conservative i.e. the two strands of duplex DNA separate and each strand serves as a 
template for the production of complementary strand. The success of their experiment was 
in part due to the use of density gradient centrifugation a technique in which molecules are 
separated according to their densities. These workers allowed culture of E. coli to grow on a 
medium with heavy isotope of nitrogen 
15
N. Thus, E. coli grown in the presence of 
15
N would 
incorporate this heavy nitrogen in their DNA making it denser (
15
N-
15
N). After growing in 
15
N 
medium the bacteria were transferred to the medium with light isotope 
14
N and allowed to 
grow for two more generations. DNA was isolated from the culture grown in 
15
N medium 
and also from first and second generation grown on 
14
N medium. They subjected the 
isolated DNA to cesium chloride density dependent centrifugation. The DNA isolated from 
the culture grown in 
15
N consisted of dense DNA which settled to the bottom of the 
centrifuge tube (having salt solution of higher density) giving a single band. The DNA 
isolated from first generation culture also gave a single band higher than the dense DNA 
(because the density of this DNA was intermediate as it consisted of one light and one 
heavy strand, 
15
N-
14
N). The DNA from second generation yielded two bands of different 
densities. One band was of the same density as that of first generation while the second 
band was of low density (Fig. 2). This would have been possible only if DNA replication is 
semi-conservative. When the E. coli culture was transferred to 
14
N medium, after first round 
of cell division the bacteria has incorporated light isotope of nitrogen in their DNA. So after 
first generation, the bacteria has one DNA strand made of heavy isotope and other of light 
isotope yielding a DNA of intermediate density (
15
N-
14
N). When this bacterial culture was 
allowed to grow for second generation in the same medium having 
14
N, the daughter DNA 
would again incorporate the light isotope of nitrogen yielding two bands one with 
intermediate density (
15
N-
14
N) and other of lighter density (
14
N-
14
N).