6 - DNA Replication

Introduction to Biology
Emine Kandemiş
Bahcesehir University
Besiktas, Istanbul
1
DNA Replication
2
The accurate replication of genomic DNA is essential to the lives
of all cells and organisms.
Each time a cell divides

• its entire genome must be duplicated,
• complex enzymatic machinery is required to copy the large
DNA molecules.
3
DNA Replication
DNA replication is a semiconservative process.
DNA polymerase  the central enzyme
4
Semiconservative DNA Replication
• The DNA replication model is known as the

semiconservative model, because each
progeny molecule retains (“conserves”) one of
the parental strands.
5
Parental strands are shown in red, and the newly synthesized strands are shown in blue.
6
DNA Polymerases
Polymerase I is principally involved in repair of damaged DNA.
Both prokaryotic and eukaryotic cells contain multiple different

DNA polymerases.
DNA polymerases play distinct roles in DNA replication and

repair.
7
DNA synthesis catalyzed by DNA polymerase
8
The Replication Fork
Replication of E. Coli DNA
The structure of a DNA replication fork.
9
Since the two strands of double-helical DNA run in opposite (antiparallel)

directions, continuous synthesis of two new strands at the replication fork
would require that one strand be synthesized in the 5' to 3' direction while
the other is synthesized in the opposite (3' to 5') direction.
DNA polymerase catalyzes the polymerization of dNTPs only in the 5' to 3'
direction.
10
Synthesis of leading and lagging strands of DNA
One strand of DNA is synthesized in a continuous manner.
The other is formed from short (1-3 kb), discontinuous pieces of DNA
11
small pieces of newly synthesized DNA
Okazaki fragments
Okazaki fragments are joined by the action of DNA ligase,

forming an intact new DNA strand.
The continuously synthesized strand
the leading strand
12
Since DNA polymerase requires a primer and cannot initiate
synthesis de novo, how is the synthesis of Okazaki fragments
initiated?
Short fragments of RNA serve as primers for DNA replication
Initiation of Okazaki fragments with RNA primers 13

In contrast to DNA synthesis, the synthesis of RNA can initiate de novo,

and an enzyme called primase synthesizes short fragments of RNA.
Okazaki fragments are then synthesized via extension of these RNA

primers by DNA polymerase.
To form a continuous lagging strand of DNA, the RNA primers must

eventually be removed from the Okazaki fragments and replaced
with DNA.
14
Removal of RNA primers and joining of Okazaki fragments

15
In prokaryotes, RNA primers are removed by the action of
polymerase I.
In addition to its DNA polymerase activity, polymerase I acts as an

exonuclease that can hydrolyze DNA (or RNA) in either the 3' to 5' or 5'
to 3' direction.
The action of polymerase I as a 5' to 3' exonuclease removes

ribonucleotides from the 5' ends of Okazaki fragments, allowing them
to be replaced with deoxyribonucleotides to yield fragments consisting
entirely of DNA.
16
In eukaryotic cells, RNA primers are removed by the combined action

of RNase H.
RNase H degrades the RNA strand of RNA-DNA hybrids, and 5' to 3'
exonucleases.
The resulting gaps are then filled by polymerase δ and the DNA
fragments are joined by DNA ligase, yielding an intact lagging strand.
17
Roles of DNA polymerases in E. coli and mammalian cells
18
Eukaryotic cells contain 3 DNA polymerases:
1. α
2. β replication of nuclear DNA
3. ε
DNA polymerase γ is localized to mitochondria.
19
Polymerases + primase + proteins
at the replication fork
20
In eukaryotes;
• clamp-loading proteins replication factor C [RFC]
• sliding-clamp proteins  proliferating cell nuclear antigen [PCNA]
RFC and PCNA form a complex and this complex specifically recognizes
and binds DNA at the junction between the primer and template.
21
Helicases catalyze the unwinding of

parental DNA, coupled to the hydrolysis
of ATP, ahead of the replication fork.
Single-stranded DNA-binding proteins

stabilize the unwound template DNA,
keeping it in an extended single-stranded
state.
Action of helicases and singlestranded DNA-
binding proteins
22
As the strands of parental DNA unwind, the DNA ahead of the replication fork is
forced to rotate.
Unchecked, this rotation would cause circular DNA molecules to become

twisted around themselves, eventually blocking replication.
Action of topoisomerases
during DNA replication
23
Topoisomerases
Topoisomerases, enzymes that catalyze the reversible breakage and rejoining of

DNA strands.
There are two types of these enzymes:
• Type I topoisomerases break just one strand of DNA
• Type II topoisomerases introduce simultaneous breaks in both strands.
24
Although eukaryotic chromosomes are composed of

linear rather than circular DNA molecules, their
replication also requires topoisomerases; otherwise,
the complete chromosomes would have to rotate
continually during DNA synthesis.
25
Type II topoisomerase is needed to
• unwind DNA
• unravel newly replicated circular DNA molecules that become
intertwined with each other.
Topoisomerase II
• appears to be involved in mitotic chromosome condensation (in
eukaryotic cells).
• is required for the separation of daughter chromatids at mitosis.
• functions to untangle newly replicated loops of DNA in the
chromosomes of eukaryotes.
26
Model of the E. coli replication fork
27
In eukaryotic cells,
• Nucleosomes are also disrupted during DNA replication.
• The histones bound to the parental chromatin are divided between

the two daughter strands of DNA,
• New histones are then added to reassemble nucleosomes by

additional proteins (chromatin assembly factors) that travel along
with the replication fork
28
The Fidelity of Replication
• The accuracy of DNA replication is critical to cell reproduction.
• DNA polymerase increases the fidelity of replication.
• DNA polymerase
1. helps to select the correct base for insertion into newly synthesized
DNA.
2. has proofreading activity.
29
In eukaryotic cells
1- polymerase δ
2- polymerase ε replicative DNA polymerases
• have an exonuclease activity that can

hydrolyze DNA in the 3' to 5'
E.Coli direction.
1- polymerase III This 3' to 5' exonuclease operates in the

reverse direction of DNA synthesis, and
participates in proofreading newly
synthesized DNA
30
Proofreading by DNA polymerase
31
Origins and the Initiation of Replication
The replication of both prokaryotic and eukaryotic DNAs starts at sites 
origins of replication
Origin replication serves as binding sites for proteins .
Replication always begins at a unique site on the bacterial chromosome.
Origin of replication in E. coli
32
Single origins are sufficient to direct the replication of bacterial.
Viral genomes and multiple origins are needed to replicate the

much larger genomes of eukaryotic cells.
– E. coli (4 x 106 base pairs)  from a single origin in approximately 30
minutes
– If mammalian genomes (3 x 109 base pairs) were replicated from a
single origin at the same rate  3 weeks (30,000 minutes).
33
The genomes of mammalian cells are typically replicated within a few hours,
necessitating the use of thousands of replication origins.
Human genome has about 30,000 origins of replication

Replication origins in eukaryotic chromosomes
34
Telomeres and Telomerase:
Maintaining the Ends of Chromosomes
Because DNA polymerases extend primers only in the 5' to 3' direction, they
are unable to copy the extreme 5' ends of linear DNA molecules.
Special mechanisms are required to replicate the terminal sequences of the

linear chromosomes of eukaryotic cells.
These sequences (telomeres) consist of tandem repeats of simple-
sequence DNA.
Telomerase is able to catalyze the synthesis of telomeres in the absence of a

DNA template.
35
Telomeres and Telomerase:
Maintaining the Ends of Chromosomes
Telomerase
• is a reverse transcriptase
• is one of a class of DNA polymerases
• synthesizes DNA from an RNA template
• carries its own template RNA
Template RNA is complementary to the telomere repeat sequences.
36
Action of telomerase
37
DNA Repair
Various chemical changes occur in DNA either spontaneously or as a result of
exposure to chemicals or radiation.
Spontaneous damage to DNA Examples of DNA damage induced by
radiation and chemicals
38
DNA Repair
Such damage to DNA can block replication or transcription, and can result in a
high frequency of mutations.
Cells have therefore had to evolve mechanisms to repair damaged DNA.
DNA repair can be divided into two classes:

1) direct reversal of the chemical reaction responsible for DNA damage
2) removal of the damaged bases followed by their replacement with newly
synthesized DNA.
39
Direct Reversal of DNA Damage
UV light is one of the major sources of damage to DNA.
The major type of damage induced by UV light is the formation of

pyrimidine dimer.
Pyrimidine dimer  adjacent pyrimidines on the same strand of DNA

are joined by the formation of a cyclobutane ring resulting from
saturation of the double bonds between carbons 5 and 6.
40
Direct Reversal of DNA Damage
The formation of such dimers distorts the structure of the DNA chain
and blocks transcription or replication past the site of damage
41
Excision Repair
Excision repair is a more general means of repairing a wide variety of chemical alterations
to DNA.
In excision repair, the damaged DNA is recognized and removed.
The resulting gap is then filled in by synthesis of a new DNA strand, using the undamaged
complementary strand as a template.
Excision repair:
1- base-excision
2- nucleotide-excision
3- mismatch repair
42
Excision Repair
Base-excision repair: single damaged

bases are recognized and removed from
the DNA molecule.
43
Excision Repair
Uracil can be formed in DNA by the deamination of cytosine
alters the normal pattern of complementary base pairing and thus represents a
mutagenic event
The excision of uracil in DNA is catalyzed by DNA glycosylase.
DNA glycosylase is an enzyme that cleaves the bond linking the base (uracil) to
the deoxyribose of the DNA backbone.
44
Excision Repair
Nucleotide-excision repair of thymine dimers
Nucleotide-excision repair:
The damaged bases (a thymine dimer)
are removed as part of an
oligonucleotide containing the lesion.
45
Excision Repair
In E. coli, nucleotide-excision repair is catalyzed by the products of three

genes:
1- uvrA: recognizes damaged DNA and recruits UvrB and UvrC to the site
of the lesion
2- uvrB: cleave on the 3' and 5' sides of the damaged

site, respectively, thus excising an
3- uvrC: oligonucleotide consisting of 12 or 13 bases
46
Nucleotide-excision repair
in mammalian cells
47
Excision Repair
an alternative form of nucleotide-excision repair, called

transcription-coupled repair.
Transcription-coupled repair is specifically dedicated to

repairing damage within actively transcribed genes.
48
Excision Repair
Mismatch repair system  recognizes mismatched bases

that are incorporated during DNA replication.
If a mismatch is found, the enzymes of repair system are

able to identify and excise the mismatched base specifically
from the newly replicated DNA strand, allowing the error to
be corrected and the original sequence restored.
49
Excision Repair
In E. Coli, DNA of this bacterium is modified by the

methylation of adenine residues within the sequence GATC to
form 6-methyladenine.
Since methylation occurs after replication, newly synthesized

DNA strands are not methylated and thus can be specifically
recognized by the mismatch repair enzymes.
50
Excision Repair
Mismatch repair in E. coli
51
Excision Repair
Eukaryotes have a similar mismatch repair system.
Eukaryotic cells do not have a homolog of MutH and the strand-specificity of

mismatch repair is not determined by DNA methylation.
Eukaryotic homologs of MutS and MutL bind to the mismatched base .
52
Translesion DNA Synthesis
The replication of damaged DNA by specialized polymerases,

called translesion DNA synthesis,
Translesion DNA synthesis provides a mechanism by which

the cell can bypass DNA damage at the replication fork,
which can then be corrected after replication is complete.
53
Translesion DNA Synthesis
Translesion DNA synthesis
54
Recombinational Repair
Recombinational repair
Recombination is a process by which

pieces of DNA are broken and recombined
to produce new combinations of alleles.
Recombinational repair, relies

on replacement of the damaged
DNA by recombination with an
undamaged molecule.
55
Recombinational Repair
Repair of double strand breaks
56
Recombination between Homologous
DNA Sequences
Recombination is an important mechanism for repairing damaged DNA.
Recombination is key to the generation of genetic diversity.
Genetic differences between individuals provide the essential starting

material of natural selection, which allows species to evolve and adapt to
changing environmental conditions.
57
Recombination between Homologous
DNA Sequences
Recombination is involved in rearrangements of specific DNA

sequences that alter the expression and function of some genes
during development and differentiation.
Homologous recombination involves the exchange of information

between DNA molecules that share sequence homology over
hundreds of bases.
58
Models of Homologous Recombination
Recombination results from the breakage and rejoining of two

parental DNA molecules, leading to reassortment of the genetic
information of the two parental chromosomes.
59
Homologous recombination by
complementary base pairing
60
61
Enzymes Involved in Homologous Recombination
Recombination requires specific enzymes, in addition to

proteins:
• DNA polymerase
• Ligase
• single-stranded DNA-binding proteins
62
Different types of Nucleotide Polymerases
1) DNA polymerase
Uses a DNA template to synthesize a DNA strand
2) RNA polymerase
Uses a DNA template to synthesize an RNA strand
(= transcription)
3) Reverse transcriptase
Uses an RNA template to synthesize a DNA strand
Found in many viruses
Telomerase is a specialized reverse transcriptase

63

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6 - DNA Replication

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Introduction to Biology

Each time a cell divides

DNA replication is a semiconservative process.

DNA polymerase  the central enzyme

• The DNA replication model is known as the

Polymerase I is principally involved in repair of damaged DNA.

Both prokaryotic and eukaryotic cells contain multiple different

DNA polymerases play distinct roles in DNA replication and

The structure of a DNA replication fork.

Since the two strands of double-helical DNA run in opposite (antiparallel)

One strand of DNA is synthesized in a continuous manner.

small pieces of newly synthesized DNA

Okazaki fragments are joined by the action of DNA ligase,

The continuously synthesized strand

the leading strand

Short fragments of RNA serve as primers for DNA replication

Initiation of Okazaki fragments with RNA primers 13

In contrast to DNA synthesis, the synthesis of RNA can initiate de novo,

Okazaki fragments are then synthesized via extension of these RNA

To form a continuous lagging strand of DNA, the RNA primers must

Removal of RNA primers and joining of Okazaki fragments

In addition to its DNA polymerase activity, polymerase I acts as an

The action of polymerase I as a 5' to 3' exonuclease removes

In eukaryotic cells, RNA primers are removed by the combined action

Roles of DNA polymerases in E. coli and mammalian cells

DNA polymerase γ is localized to mitochondria.

Polymerases + primase + proteins

at the replication fork

• clamp-loading proteins replication factor C [RFC]

• sliding-clamp proteins  proliferating cell nuclear antigen [PCNA]

Helicases catalyze the unwinding of

Single-stranded DNA-binding proteins

Unchecked, this rotation would cause circular DNA molecules to become

Topoisomerases, enzymes that catalyze the reversible breakage and rejoining of

There are two types of these enzymes:

• Type I topoisomerases break just one strand of DNA

• Type II topoisomerases introduce simultaneous breaks in both strands.

Although eukaryotic chromosomes are composed of

• The histones bound to the parental chromatin are divided between

• New histones are then added to reassemble nucleosomes by

• The accuracy of DNA replication is critical to cell reproduction.

• DNA polymerase increases the fidelity of replication.

2. has proofreading activity.

• have an exonuclease activity that can

1- polymerase III This 3' to 5' exonuclease operates in the

Proofreading by DNA polymerase

Origin replication serves as binding sites for proteins .

Replication always begins at a unique site on the bacterial chromosome.

Origin of replication in E. coli

Single origins are sufficient to direct the replication of bacterial.

Viral genomes and multiple origins are needed to replicate the

Human genome has about 30,000 origins of replication

Special mechanisms are required to replicate the terminal sequences of the

Telomerase is able to catalyze the synthesis of telomeres in the absence of a

Template RNA is complementary to the telomere repeat sequences.

Cells have therefore had to evolve mechanisms to repair damaged DNA.

DNA repair can be divided into two classes:

UV light is one of the major sources of damage to DNA.

The major type of damage induced by UV light is the formation of

Pyrimidine dimer  adjacent pyrimidines on the same strand of DNA

In excision repair, the damaged DNA is recognized and removed.

Base-excision repair: single damaged

Uracil can be formed in DNA by the deamination of cytosine