Dna Replication
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- Pages: 62
Dr. Maha Ismail 10/09/09
1
Nucleic acids Nucleic Acids are macromolecules which store and
express all the information necessary for building and maintaining life.
The main function of DNA (Deoxyribonucleic Acid) is to
keep the genetic information which reside in the sequence of nucleotides.
Any change from the original sequence of nucleotides is
mutation
10/09/09
2
DNA Structure The DNA Molecule consist of two polynucleotide
chains (strands) held together in an antiparallel manner by hydrogen bonds formed between specific pairs of bases [Adenine-Thymine] [Guanosine-Cytosine]. Thus the bases sequence (code) in one strands
determines the code (complementarity). 10/09/09
of
the
other
strands
3
10/09/09
4
DNA Structure The joined anti-parallel strands are twisted
about each other in the shape of a righthanded double helix. Indeed DNA is often depicted as a twisted
ladder in which rungs are bases pairing and sides are deoxyribose-phosphate chains.
10/09/09
5
DNA Structure The double helix structure is mainly stabilized
by hydrogen bonds between bases pairs. Since the hydrophobic bases are stacked inside and the hydrophilic ribose-phosphate chains are on the outside. In eukaryotic the DNA is found in the nucleus, , not free. It is mixed with proteins, while in prokaryotes the DNA is found free in the cytoplasm. 10/09/09
6
There is normal turnover of the cells (cell
division), and the zygote must also divide & differentiate to develop into a human. In all these conditions the genetic informations must be passed to the daughter cell, & to the offspring
10/09/09
7
So, all the cells of the body contain the
same genetic makeup, but certain cells express different genes. Not all the of the genome is expressed in the cell & different cells express different genes. B- cell in express the gene responsible for the synthesis of insulin 10/09/09
8
DNA Replication Each time a cell divides into two daughter cells,
all the DNA molecule must be duplicated. Duplication of an old DNA molecule into two new DNA molecules is called Replication.
DNA replication is the synthesis of DNA from
DNA – the reproduction of DNA.
The objective of DNA Replication is to conserve
the genetic information of the same organism. 10/09/09
9
DNA Replication The DNA replication is carried out with high accuracy
and fidelity.
It is a semi conservative process (Each daughter DNA
molecule is an exact copy of its parent molecule, consisting of one old and one new DNA strand).
The whole process takes about 9 hours in mammals.
10/09/09
10
10/09/09
11
10/09/09
12
DNA Replication Requirements 1. Template ( mother DNA molecule).
RNA –Primer. 3. The 4 Nucleotides (A,G,T,C) ( Building blocks), which are used in the form of triphosphates, dNTP. 4. Proteins, including enzymes & the other accessory proteins. The key enzyme in replication is DNA Polymerase (Pol) 2.
10/09/09
13
Steps of DNA Replication There at least five steps in DNA replication: 1. Identification of the origin of replication . 2. Unwinding of DNA. 3. Formation of Replication fork. 4. Formation of Replication bubbles. 5. Reformation of chromatin.
10/09/09
14
Identification of the origin of replication. 1 The starting point / origin of replication should be defined. The origin of replication ( Ori) is usually near an area rich in
A – T bases. This (Ori) has a specific sequence & is recognized by a specific protein called O –protein . This stage is only recognized in Prokaryotes, as in higher
animals is less well understood. 10/09/09
15
.Unwinding of DNA. 2 This stage is necessary in order for the DNA to
be accessible for replication. The unwinding process is a co-operative work
i.e. meaning different mechanisms work cooperatively to unwind, separate the double strands & to keep them separated:
10/09/09
16
.Unwinding of DNA. 2 1. The binding of the O- protein to the origin result in
local unwinding of the adjacent A & T rich area. 2. The enzyme HELICASE is responsible for breaking the helical structure, to provide a single stranded template.
10/09/09
17
10/09/09
18
Unwinding of DNA. 2 3. The Topoisomerase prevents supercoiling of the strands.
10/09/09
19
4. Single stranded binding proteins (SSBP), they bind each single strand separately. They enhance the activity of helicase and to bind to the single stranded template until replication is completed.
10/09/09
20
Formation of Replication. 3 fork One of the new strand is formed from
5 to 3 & the other is from 3 to 5. The main enzyme needed in
polymerization is DNA polymerase, which need RNA primer to start polymerization.
10/09/09
21
.Formation of Replication fork. 3 DNA polymerase requires: 1. Energy. 2. RNA primer, with a free 3’OH group for
attachment of the new nucleotides. 3. DNA as a template.
10/09/09
22
Formation of Replication. 3 fork Primer formation need PRIMASE enzyme. The Primase enzyme initiate the synthesis of an
RNA molecule. This RNA primer length varies between 20 -200 nucleotides. The Primase + Helicase form a complex called PRIMOSOME 10/09/09
23
10/09/09
24
.Formation of Replication fork. 3 The new nucleotide is added to the 3’ end of a primer & new
synthesis is said to occur in a 5’ to 3’ direction.
The polymerization process involve the nucleophilic attack
by the 3’ hydroxyl group of the existing nucleotide, This hydroxyl group serves as the acceptor of the α – phosphate group of the first entering dNTP with the release of pyrophosphate and forming phosphodiester bond.
10/09/09
25
10/09/09
26
10/09/09
27
Replication fork Replication of the double – stranded
DNA is bidirectional, from the replication origin
10/09/09
28
10/09/09
29
DNA Replication Since the DNA strands are antiparallel, and
replication proceed only in the 5' to 3' direction, one strand named the leading strand forms a continuous copy, while the other named lagging strand forms discontinuous short DNA strings called Okazaki fragments.
10/09/09
30
. Okazaki fragments This lagging strands is synthesized in segments
and each segment is preceded by an RNA primer. Each segment is called Okazaki fragment, its
length varies from 150-250 nucleotide and in each replication fork there is up to 250 okazaki fragments.
10/09/09
31
10/09/09
32
10/09/09
33
10/09/09
34
.Formation of Replication bubbles. 4 A number of replication forks form
replication bubbles.
10/09/09
35
10/09/09
36
Action of DNA polymerase 1. Addition of nucleotides. DNA polymerase III initiate and elongate the chain by adding nucleotides complementary to that in the single stranded template, in 5 – 3 direction. All 4 deoxynucleotides should be present, if one is absent synthesis will stop if the nucleotide is depleted.
10/09/09
37
10/09/09
38
Action of DNA polymerase 2. Proofreading of newly synthesized DNA: It edit its mistakes. Misreading can cause mutations.
10/09/09
39
Action of DNA polymerase 3. The RNA primer sequence is not part of the DNA structure so it must be removed. DNA polymerase I will remove the RNA primer and fill the gap.
10/09/09
40
Action of DNA Polymerase The DNA polymerase works on both strands
simultaneously 1. Addition or polymerization of nucleotides. 2. Proofreading. 3. Excision and repair activity.
10/09/09
41
10/09/09
42
:In Summary The replication process is: Semi-discontinuous. Semi- conservative. Asymmetrical, two directions.
10/09/09
43
10/09/09
44
Packaging of DNA, Reformation of. 5 .chromatin In Eukaryotic cells, the DNA is packaged in
Chromatin within the nucleus. DNA is surrounded by histones forming the basic structural units of DNA [ Nucleosomes} Nucleosomes forms of chromatin (less densely
packaged) and then to chromosomes.
10/09/09
45
10/09/09
46
10/09/09
47
Question 1 What is the action of the following 1. 2. 3. 4.
10/09/09
drugs: Adenine Arabinoside. Azidothymidine (AZT). Actinomycin D. Nalidixic acid
48
Question 2 Compare and contrast between
Eukaryotic and prokaryotic replication.
10/09/09
49
10/09/09
50
Substrate analogues: Adenine Arabinoside is an antiviral, Cystine Arabinoside antileukemic, they alter the DNA structure & it become more prone to breakage
2. Azidothymidine (AZT), antiviral, used for treatment of HIV, the azido group in the 3 position will unacceptable primer
10/09/09
51
3. Inhibitors that act directly with DNA Actinomycin D, used for cancer, its structural ring intercalate between the bases of DNA. 4. Inhibitors of replicative enzymes. Nalidixic acid, inhibit topoisomerase, used in UTI.
10/09/09
52
10/09/09
53
10/09/09
54
10/09/09
55
10/09/09
56
10/09/09
57
10/09/09
58
10/09/09
59
DeoxyriboNucleic Acid
The Genome is the complete set of
instructions to create and maintain an organism alive. DNA molecules are support for the Genome of all living organisms.
10/09/09
60
10/09/09
61
10/09/09
62
1
Nucleic acids Nucleic Acids are macromolecules which store and
express all the information necessary for building and maintaining life.
The main function of DNA (Deoxyribonucleic Acid) is to
keep the genetic information which reside in the sequence of nucleotides.
Any change from the original sequence of nucleotides is
mutation
10/09/09
2
DNA Structure The DNA Molecule consist of two polynucleotide
chains (strands) held together in an antiparallel manner by hydrogen bonds formed between specific pairs of bases [Adenine-Thymine] [Guanosine-Cytosine]. Thus the bases sequence (code) in one strands
determines the code (complementarity). 10/09/09
of
the
other
strands
3
10/09/09
4
DNA Structure The joined anti-parallel strands are twisted
about each other in the shape of a righthanded double helix. Indeed DNA is often depicted as a twisted
ladder in which rungs are bases pairing and sides are deoxyribose-phosphate chains.
10/09/09
5
DNA Structure The double helix structure is mainly stabilized
by hydrogen bonds between bases pairs. Since the hydrophobic bases are stacked inside and the hydrophilic ribose-phosphate chains are on the outside. In eukaryotic the DNA is found in the nucleus, , not free. It is mixed with proteins, while in prokaryotes the DNA is found free in the cytoplasm. 10/09/09
6
There is normal turnover of the cells (cell
division), and the zygote must also divide & differentiate to develop into a human. In all these conditions the genetic informations must be passed to the daughter cell, & to the offspring
10/09/09
7
So, all the cells of the body contain the
same genetic makeup, but certain cells express different genes. Not all the of the genome is expressed in the cell & different cells express different genes. B- cell in express the gene responsible for the synthesis of insulin 10/09/09
8
DNA Replication Each time a cell divides into two daughter cells,
all the DNA molecule must be duplicated. Duplication of an old DNA molecule into two new DNA molecules is called Replication.
DNA replication is the synthesis of DNA from
DNA – the reproduction of DNA.
The objective of DNA Replication is to conserve
the genetic information of the same organism. 10/09/09
9
DNA Replication The DNA replication is carried out with high accuracy
and fidelity.
It is a semi conservative process (Each daughter DNA
molecule is an exact copy of its parent molecule, consisting of one old and one new DNA strand).
The whole process takes about 9 hours in mammals.
10/09/09
10
10/09/09
11
10/09/09
12
DNA Replication Requirements 1. Template ( mother DNA molecule).
RNA –Primer. 3. The 4 Nucleotides (A,G,T,C) ( Building blocks), which are used in the form of triphosphates, dNTP. 4. Proteins, including enzymes & the other accessory proteins. The key enzyme in replication is DNA Polymerase (Pol) 2.
10/09/09
13
Steps of DNA Replication There at least five steps in DNA replication: 1. Identification of the origin of replication . 2. Unwinding of DNA. 3. Formation of Replication fork. 4. Formation of Replication bubbles. 5. Reformation of chromatin.
10/09/09
14
Identification of the origin of replication. 1 The starting point / origin of replication should be defined. The origin of replication ( Ori) is usually near an area rich in
A – T bases. This (Ori) has a specific sequence & is recognized by a specific protein called O –protein . This stage is only recognized in Prokaryotes, as in higher
animals is less well understood. 10/09/09
15
.Unwinding of DNA. 2 This stage is necessary in order for the DNA to
be accessible for replication. The unwinding process is a co-operative work
i.e. meaning different mechanisms work cooperatively to unwind, separate the double strands & to keep them separated:
10/09/09
16
.Unwinding of DNA. 2 1. The binding of the O- protein to the origin result in
local unwinding of the adjacent A & T rich area. 2. The enzyme HELICASE is responsible for breaking the helical structure, to provide a single stranded template.
10/09/09
17
10/09/09
18
Unwinding of DNA. 2 3. The Topoisomerase prevents supercoiling of the strands.
10/09/09
19
4. Single stranded binding proteins (SSBP), they bind each single strand separately. They enhance the activity of helicase and to bind to the single stranded template until replication is completed.
10/09/09
20
Formation of Replication. 3 fork One of the new strand is formed from
5 to 3 & the other is from 3 to 5. The main enzyme needed in
polymerization is DNA polymerase, which need RNA primer to start polymerization.
10/09/09
21
.Formation of Replication fork. 3 DNA polymerase requires: 1. Energy. 2. RNA primer, with a free 3’OH group for
attachment of the new nucleotides. 3. DNA as a template.
10/09/09
22
Formation of Replication. 3 fork Primer formation need PRIMASE enzyme. The Primase enzyme initiate the synthesis of an
RNA molecule. This RNA primer length varies between 20 -200 nucleotides. The Primase + Helicase form a complex called PRIMOSOME 10/09/09
23
10/09/09
24
.Formation of Replication fork. 3 The new nucleotide is added to the 3’ end of a primer & new
synthesis is said to occur in a 5’ to 3’ direction.
The polymerization process involve the nucleophilic attack
by the 3’ hydroxyl group of the existing nucleotide, This hydroxyl group serves as the acceptor of the α – phosphate group of the first entering dNTP with the release of pyrophosphate and forming phosphodiester bond.
10/09/09
25
10/09/09
26
10/09/09
27
Replication fork Replication of the double – stranded
DNA is bidirectional, from the replication origin
10/09/09
28
10/09/09
29
DNA Replication Since the DNA strands are antiparallel, and
replication proceed only in the 5' to 3' direction, one strand named the leading strand forms a continuous copy, while the other named lagging strand forms discontinuous short DNA strings called Okazaki fragments.
10/09/09
30
. Okazaki fragments This lagging strands is synthesized in segments
and each segment is preceded by an RNA primer. Each segment is called Okazaki fragment, its
length varies from 150-250 nucleotide and in each replication fork there is up to 250 okazaki fragments.
10/09/09
31
10/09/09
32
10/09/09
33
10/09/09
34
.Formation of Replication bubbles. 4 A number of replication forks form
replication bubbles.
10/09/09
35
10/09/09
36
Action of DNA polymerase 1. Addition of nucleotides. DNA polymerase III initiate and elongate the chain by adding nucleotides complementary to that in the single stranded template, in 5 – 3 direction. All 4 deoxynucleotides should be present, if one is absent synthesis will stop if the nucleotide is depleted.
10/09/09
37
10/09/09
38
Action of DNA polymerase 2. Proofreading of newly synthesized DNA: It edit its mistakes. Misreading can cause mutations.
10/09/09
39
Action of DNA polymerase 3. The RNA primer sequence is not part of the DNA structure so it must be removed. DNA polymerase I will remove the RNA primer and fill the gap.
10/09/09
40
Action of DNA Polymerase The DNA polymerase works on both strands
simultaneously 1. Addition or polymerization of nucleotides. 2. Proofreading. 3. Excision and repair activity.
10/09/09
41
10/09/09
42
:In Summary The replication process is: Semi-discontinuous. Semi- conservative. Asymmetrical, two directions.
10/09/09
43
10/09/09
44
Packaging of DNA, Reformation of. 5 .chromatin In Eukaryotic cells, the DNA is packaged in
Chromatin within the nucleus. DNA is surrounded by histones forming the basic structural units of DNA [ Nucleosomes} Nucleosomes forms of chromatin (less densely
packaged) and then to chromosomes.
10/09/09
45
10/09/09
46
10/09/09
47
Question 1 What is the action of the following 1. 2. 3. 4.
10/09/09
drugs: Adenine Arabinoside. Azidothymidine (AZT). Actinomycin D. Nalidixic acid
48
Question 2 Compare and contrast between
Eukaryotic and prokaryotic replication.
10/09/09
49
10/09/09
50
Substrate analogues: Adenine Arabinoside is an antiviral, Cystine Arabinoside antileukemic, they alter the DNA structure & it become more prone to breakage
2. Azidothymidine (AZT), antiviral, used for treatment of HIV, the azido group in the 3 position will unacceptable primer
10/09/09
51
3. Inhibitors that act directly with DNA Actinomycin D, used for cancer, its structural ring intercalate between the bases of DNA. 4. Inhibitors of replicative enzymes. Nalidixic acid, inhibit topoisomerase, used in UTI.
10/09/09
52
10/09/09
53
10/09/09
54
10/09/09
55
10/09/09
56
10/09/09
57
10/09/09
58
10/09/09
59
DeoxyriboNucleic Acid
The Genome is the complete set of
instructions to create and maintain an organism alive. DNA molecules are support for the Genome of all living organisms.
10/09/09
60
10/09/09
61
10/09/09
62
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