Page 1
Models of DNA synthesis
Institute of Lifelong Learning, University of Delhi
Lesson: Models of DNA synthesis
Lesson Developer: Dr. Devi Lal
College/ Department: Department of Zoology, Ramjas College,
University of Dekhi
Reviewer: Dr. Neeraj Sood
college/ Department : Department of Zoology, Dyal Singh College,
University of Delhi
Page 2
Models of DNA synthesis
Institute of Lifelong Learning, University of Delhi
Lesson: Models of DNA synthesis
Lesson Developer: Dr. Devi Lal
College/ Department: Department of Zoology, Ramjas College,
University of Dekhi
Reviewer: Dr. Neeraj Sood
college/ Department : Department of Zoology, Dyal Singh College,
University of Delhi
Models of DNA synthesis
2
Table of Contents
Chapter: Models of DNA synthesis
Topic Page No.
Introduction……………………………………………………………………………………………………..3
Initiation of DNA replication ………………………………………………………………………………………………………….3.
The Replicon Model………….………………………………………………………………………………………………………………………….3
Regulation of DNA replication
Regulation of DNA replication in E. coli……………………………………………………..4
Regulation of DNA replication in eukaryotes……………………………………………4
Overview of DNA replication
Overview of DNA replication in prokaryotes…………………………………………….5
Overview of DNA replication in eukaryotes……………………………………………..8
End replication problem
Telomeres and Telomerases…………………………………………………………………….9
Role of Telomere binding proteins………………………………………………………….11
Priming protein……………………………………………………………………………………….12
Replication model for other circular DNAs
Rolling circle replication………………………………………………………………………….12
D-loops and mitochondrial DNA replication…………………………………………..13
Summary………………………………………………………….…………………………………………………….…14
Exercise/ Practice………………………………………………………….………………………………………….15
Glossary………………………………………………………….………………………………………………………….19
References/ Bibliography/ Further Reading…………………………………………….19
Web links………………………………………………………….……………………………………………………….20
Answers………………………………………………………….………………………………………………………….20
Page 3
Models of DNA synthesis
Institute of Lifelong Learning, University of Delhi
Lesson: Models of DNA synthesis
Lesson Developer: Dr. Devi Lal
College/ Department: Department of Zoology, Ramjas College,
University of Dekhi
Reviewer: Dr. Neeraj Sood
college/ Department : Department of Zoology, Dyal Singh College,
University of Delhi
Models of DNA synthesis
2
Table of Contents
Chapter: Models of DNA synthesis
Topic Page No.
Introduction……………………………………………………………………………………………………..3
Initiation of DNA replication ………………………………………………………………………………………………………….3.
The Replicon Model………….………………………………………………………………………………………………………………………….3
Regulation of DNA replication
Regulation of DNA replication in E. coli……………………………………………………..4
Regulation of DNA replication in eukaryotes……………………………………………4
Overview of DNA replication
Overview of DNA replication in prokaryotes…………………………………………….5
Overview of DNA replication in eukaryotes……………………………………………..8
End replication problem
Telomeres and Telomerases…………………………………………………………………….9
Role of Telomere binding proteins………………………………………………………….11
Priming protein……………………………………………………………………………………….12
Replication model for other circular DNAs
Rolling circle replication………………………………………………………………………….12
D-loops and mitochondrial DNA replication…………………………………………..13
Summary………………………………………………………….…………………………………………………….…14
Exercise/ Practice………………………………………………………….………………………………………….15
Glossary………………………………………………………….………………………………………………………….19
References/ Bibliography/ Further Reading…………………………………………….19
Web links………………………………………………………….……………………………………………………….20
Answers………………………………………………………….………………………………………………………….20
Models of DNA synthesis
3
Introduction
The previous unit describes the enzymes and the general principle of DNA replication. The
enzymes and general machinery for replication is more or less same in both prokaryotes
and eukaryotes with slight differences. In this unit we will learn about the modes of DNA
replication in both prokaryotes and eukaryotes. Apart from this we also describe the role of
telomerase in maintaining the length of linear chromosomes of eukaryotes.
Initiation of DNA replication
As already described, for replication to take place both the strands of a double helix unwind
by the action of helicases and each strand serves as the template for the synthesis of
complementary strand. These sites from where the unwinding of DNA starts are known as
origin of replication. In case of prokaryotes, there is generally a single origin of replication
while eukaryotes have multiple origins of replication. These origins are generally at internal
regions of the chromosomes. These internal regions bind a number of protein factors that
direct the initation of DNA replication.
The Replicon Model
In order to explain the regulation of DNA replication in bacteria, François Jacob, Sydney
Brenner and François Cuzin in 1963 proposed the replicon model. A replicon was defined
as the DNA that is replicated from a particular origin of replication. According to this model,
two basic elements are required to initiate the DNA replication: replicator and initiator. A
replicator is defined as a cis-acting genetic element that is required to direct the initiation
of DNA replication. All known replicators consist of a binding site for the initiator protein and
AT-rich stretch of DNA. The origin of replication generally is a part of replicator. E. coli has a
single replicator (OriC) that consisted of two repeated motifs, 9-mer motif that is repeated
five times and 13-mer motif that is repeated three times (Fig. 1 (A)). The 9-mer motif
serves as the binding site for the initiator protein and 13-mer motif is the site of unwinding
DNA. In case of yeast, three sequence motifs are present: A, B1 and B2 (Fig. 1 (B)). While
A and B1 serve as the binding site for initiator, B2 represents the site from where the
unwinding of DNA starts. The overall structure of a replicator from different organism is
found to be same despite sequence diversity.
Page 4
Models of DNA synthesis
Institute of Lifelong Learning, University of Delhi
Lesson: Models of DNA synthesis
Lesson Developer: Dr. Devi Lal
College/ Department: Department of Zoology, Ramjas College,
University of Dekhi
Reviewer: Dr. Neeraj Sood
college/ Department : Department of Zoology, Dyal Singh College,
University of Delhi
Models of DNA synthesis
2
Table of Contents
Chapter: Models of DNA synthesis
Topic Page No.
Introduction……………………………………………………………………………………………………..3
Initiation of DNA replication ………………………………………………………………………………………………………….3.
The Replicon Model………….………………………………………………………………………………………………………………………….3
Regulation of DNA replication
Regulation of DNA replication in E. coli……………………………………………………..4
Regulation of DNA replication in eukaryotes……………………………………………4
Overview of DNA replication
Overview of DNA replication in prokaryotes…………………………………………….5
Overview of DNA replication in eukaryotes……………………………………………..8
End replication problem
Telomeres and Telomerases…………………………………………………………………….9
Role of Telomere binding proteins………………………………………………………….11
Priming protein……………………………………………………………………………………….12
Replication model for other circular DNAs
Rolling circle replication………………………………………………………………………….12
D-loops and mitochondrial DNA replication…………………………………………..13
Summary………………………………………………………….…………………………………………………….…14
Exercise/ Practice………………………………………………………….………………………………………….15
Glossary………………………………………………………….………………………………………………………….19
References/ Bibliography/ Further Reading…………………………………………….19
Web links………………………………………………………….……………………………………………………….20
Answers………………………………………………………….………………………………………………………….20
Models of DNA synthesis
3
Introduction
The previous unit describes the enzymes and the general principle of DNA replication. The
enzymes and general machinery for replication is more or less same in both prokaryotes
and eukaryotes with slight differences. In this unit we will learn about the modes of DNA
replication in both prokaryotes and eukaryotes. Apart from this we also describe the role of
telomerase in maintaining the length of linear chromosomes of eukaryotes.
Initiation of DNA replication
As already described, for replication to take place both the strands of a double helix unwind
by the action of helicases and each strand serves as the template for the synthesis of
complementary strand. These sites from where the unwinding of DNA starts are known as
origin of replication. In case of prokaryotes, there is generally a single origin of replication
while eukaryotes have multiple origins of replication. These origins are generally at internal
regions of the chromosomes. These internal regions bind a number of protein factors that
direct the initation of DNA replication.
The Replicon Model
In order to explain the regulation of DNA replication in bacteria, François Jacob, Sydney
Brenner and François Cuzin in 1963 proposed the replicon model. A replicon was defined
as the DNA that is replicated from a particular origin of replication. According to this model,
two basic elements are required to initiate the DNA replication: replicator and initiator. A
replicator is defined as a cis-acting genetic element that is required to direct the initiation
of DNA replication. All known replicators consist of a binding site for the initiator protein and
AT-rich stretch of DNA. The origin of replication generally is a part of replicator. E. coli has a
single replicator (OriC) that consisted of two repeated motifs, 9-mer motif that is repeated
five times and 13-mer motif that is repeated three times (Fig. 1 (A)). The 9-mer motif
serves as the binding site for the initiator protein and 13-mer motif is the site of unwinding
DNA. In case of yeast, three sequence motifs are present: A, B1 and B2 (Fig. 1 (B)). While
A and B1 serve as the binding site for initiator, B2 represents the site from where the
unwinding of DNA starts. The overall structure of a replicator from different organism is
found to be same despite sequence diversity.
Models of DNA synthesis
4
13-mer repeated motifs 9-mer repeated motifs
245 bp
100 bp
A element B1 element B2 element
(A)
(B)
Fig. 1. The structure of replicator from (A) E. coli showing 13-mer motif (repeated three
times) and 9-mer motif (repeated five times) and (B) yeast showing three sequence motifs:
A, B1 and B2 (Source: Author).
Initiator is defined as a trans-acting protein that recognizes and binds with the initiator
and recruits other proteins required for replication. Some initiators also function to distort
DNA that results in initial unwinding of DNA duplex. Initiator proteins are the only sequence-
specific DNA binding proteins involved in replication. All these proteins are ATP binding
proteins that use energy from ATP hydrolysis. The initiator protein of E. coli is DnaA while
in case of eukaryotes it is a six-protein complex known as origin recognition complex
(ORC).
Regulation of DNA replication
DNA replication should be tightly regulated to ensure that each chromosome is replicated
only once per cycle. Both prokaryotes and eukaryotes have evolved certain mechanisms
that keep a check on DNA replication. While prokaryotes like E. coli depend on methylated
state of their DNA, eukaryotes have tightly regulated cell cycle that ensures that DNA
replication takes place only in S-phase of cell cycle.
Regulation of DNA replication in E. coli
Page 5
Models of DNA synthesis
Institute of Lifelong Learning, University of Delhi
Lesson: Models of DNA synthesis
Lesson Developer: Dr. Devi Lal
College/ Department: Department of Zoology, Ramjas College,
University of Dekhi
Reviewer: Dr. Neeraj Sood
college/ Department : Department of Zoology, Dyal Singh College,
University of Delhi
Models of DNA synthesis
2
Table of Contents
Chapter: Models of DNA synthesis
Topic Page No.
Introduction……………………………………………………………………………………………………..3
Initiation of DNA replication ………………………………………………………………………………………………………….3.
The Replicon Model………….………………………………………………………………………………………………………………………….3
Regulation of DNA replication
Regulation of DNA replication in E. coli……………………………………………………..4
Regulation of DNA replication in eukaryotes……………………………………………4
Overview of DNA replication
Overview of DNA replication in prokaryotes…………………………………………….5
Overview of DNA replication in eukaryotes……………………………………………..8
End replication problem
Telomeres and Telomerases…………………………………………………………………….9
Role of Telomere binding proteins………………………………………………………….11
Priming protein……………………………………………………………………………………….12
Replication model for other circular DNAs
Rolling circle replication………………………………………………………………………….12
D-loops and mitochondrial DNA replication…………………………………………..13
Summary………………………………………………………….…………………………………………………….…14
Exercise/ Practice………………………………………………………….………………………………………….15
Glossary………………………………………………………….………………………………………………………….19
References/ Bibliography/ Further Reading…………………………………………….19
Web links………………………………………………………….……………………………………………………….20
Answers………………………………………………………….………………………………………………………….20
Models of DNA synthesis
3
Introduction
The previous unit describes the enzymes and the general principle of DNA replication. The
enzymes and general machinery for replication is more or less same in both prokaryotes
and eukaryotes with slight differences. In this unit we will learn about the modes of DNA
replication in both prokaryotes and eukaryotes. Apart from this we also describe the role of
telomerase in maintaining the length of linear chromosomes of eukaryotes.
Initiation of DNA replication
As already described, for replication to take place both the strands of a double helix unwind
by the action of helicases and each strand serves as the template for the synthesis of
complementary strand. These sites from where the unwinding of DNA starts are known as
origin of replication. In case of prokaryotes, there is generally a single origin of replication
while eukaryotes have multiple origins of replication. These origins are generally at internal
regions of the chromosomes. These internal regions bind a number of protein factors that
direct the initation of DNA replication.
The Replicon Model
In order to explain the regulation of DNA replication in bacteria, François Jacob, Sydney
Brenner and François Cuzin in 1963 proposed the replicon model. A replicon was defined
as the DNA that is replicated from a particular origin of replication. According to this model,
two basic elements are required to initiate the DNA replication: replicator and initiator. A
replicator is defined as a cis-acting genetic element that is required to direct the initiation
of DNA replication. All known replicators consist of a binding site for the initiator protein and
AT-rich stretch of DNA. The origin of replication generally is a part of replicator. E. coli has a
single replicator (OriC) that consisted of two repeated motifs, 9-mer motif that is repeated
five times and 13-mer motif that is repeated three times (Fig. 1 (A)). The 9-mer motif
serves as the binding site for the initiator protein and 13-mer motif is the site of unwinding
DNA. In case of yeast, three sequence motifs are present: A, B1 and B2 (Fig. 1 (B)). While
A and B1 serve as the binding site for initiator, B2 represents the site from where the
unwinding of DNA starts. The overall structure of a replicator from different organism is
found to be same despite sequence diversity.
Models of DNA synthesis
4
13-mer repeated motifs 9-mer repeated motifs
245 bp
100 bp
A element B1 element B2 element
(A)
(B)
Fig. 1. The structure of replicator from (A) E. coli showing 13-mer motif (repeated three
times) and 9-mer motif (repeated five times) and (B) yeast showing three sequence motifs:
A, B1 and B2 (Source: Author).
Initiator is defined as a trans-acting protein that recognizes and binds with the initiator
and recruits other proteins required for replication. Some initiators also function to distort
DNA that results in initial unwinding of DNA duplex. Initiator proteins are the only sequence-
specific DNA binding proteins involved in replication. All these proteins are ATP binding
proteins that use energy from ATP hydrolysis. The initiator protein of E. coli is DnaA while
in case of eukaryotes it is a six-protein complex known as origin recognition complex
(ORC).
Regulation of DNA replication
DNA replication should be tightly regulated to ensure that each chromosome is replicated
only once per cycle. Both prokaryotes and eukaryotes have evolved certain mechanisms
that keep a check on DNA replication. While prokaryotes like E. coli depend on methylated
state of their DNA, eukaryotes have tightly regulated cell cycle that ensures that DNA
replication takes place only in S-phase of cell cycle.
Regulation of DNA replication in E. coli
Models of DNA synthesis
5
In E. coli DNA replication is tightly controlled by two proteins: the initiator DnaA; and
SeqA. Just before DNA replication, the A within every GATC sequence throughout the E. coli
genome are methylated by Dam methytransferase. The initiator, DnaA with ATP bound to
it, binds with these methylated sites and recruits other proteins required for replication
initiation. The bound ATP is hydrolyzed to ADP after the initiation process, inactivating
DnaA, so that it cannot initiate fresh round of DNA replication. Moreover, the rate of
exchange of ATP with ADP is slow, further delaying the activation of DnaA.
Once DNA replication takes place, the methylated sites are converted to hemi-methylated
state (methyl group on only one DNA strand). This state of hemi-methylation is detected by
a protein SeqA which binds to GATC sequences present near the OriC. This binding prevents
the methylation by Dam methytransferase and also prevents the binding of the initiator
DnaA, thus inhibiting the initiation of DNA replication. So, the two mechanisms exists in E.
coli that prevent the unnecessary DNA replication making it a controlled phenomenon.
Regulation of DNA replication in eukaryotes
Like E. coli, mechanism also exists in eukaryotes that make sure that DNA is replicated once
per cell cycle. These mechanisms prevent the entry into mitosis with incompletely replicated
chromosomes or with extra chromosomes. However, the situation in eukaryotes is more
complex as compared to prokaryotes because of the presence of many origin of replication.
So tightly regulating the replication initiation from these origins is a challenge. In case of
eukaryotes the process of replication initiation takes place in two phases: replicator
selection and origin activation. The replication selection involves the identification of DNA
sequences that will direct initiation. Replication selection occurs in G1 phase and involves
the formation of pre-replicative complexes (pre-RCs). The assembly of pre-RCs involves
four different proteins. The first protein is the initiator, ORC (origin recognition complex)
which recognizes and binds with the replicator and recruits Cdc6 and Cdt1, helicase
loading proteins. These three proteins then recruit the helicase Mcm2-7 complex. Pre-
RCs assemble in inactivated form and therefore no unwinding of DNA takes place until the
cell reaches the S-phase of cell cycle.
The activation of pre-RCs is mediated by two protein kinases: cyclin dependent kinase
(Cdk) and Dbf4-dependent kinase (Ddk). These kinases are inactivated in G1 phase
and therefore no activation of pre-RCs takes place during this phase. The kinases get
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