DNA REPLICATION AND
REPAIR
1
SIGNIFICANCE OF DNA REPAIR IN MEDICINE
Example:
DNA repair: enzymes of nucleotide excision repair
Disease: xeroderma pigmentosum
2
Children with xeroderma pigmentosum, showing the multiple skin
tumors characteristic of this disease.
3
DNA REPLICATION AND REPAIR
DNA replication:
1. Essence of DNA replication
2. Replicon and replication fork
3. DNA polymerase
4. Other proteins of replication machinery
5. Mechanisms of DNA replication
6. Replication of ends of eukaryotic chromosomes
7. Proofreading
DNA repair:
8. Mismatch repair
9. Mechanisms of accidental DNA damage
10. Mechanisms of the repair of accidentally damaged DNA
4
1. ESSENCE OF DNA REPLICATION:
DNA replication: the doubling of DNA. Two identical double helixes of
DNA are derived from one original double helix of DNA (entire genetic
information is saved).
Semiconservative character of DNA replication: new strand is
synthesized to each of both original strands on the basis of
complementary pairing (complementary strand).
The original strands function as a template. [FIG.] [FIG.] [FIG.]
5
6
7
8
2. REPLICON AND REPLICATION FORK:
Replicon (a segment of DNA): replication unit with its own replication
origin.
Procaryotic chromosome: one replicon
Eucaryotic chromosome: many (hundreds and thousands) replicons
Replication origin: a specific sequence of DNA (rich in A-T pairs) where
the replications starts. [FIG.]
9
10
Replication fork: the replication continues from the replication origin in
both opposite directions → two replication forks moving apart (the
shape of letter Y). [FIG.]
Procaryotic chromosome: replication fork moves at 1000bp/s.
Eucaryotic chromosome: replication fork moves at 100bp/s.
11
12
Replication fork: the replication continues from the replication origin in
both opposite directions → two replication forks moving apart (the
shape of letter Y). [FIG.]
Procaryotic chromosome: replication fork moves at 1000bp/s.
Eucaryotic chromosome: replication fork moves at 100bp/s.
13
3. DNA POLYMERASE:
DNA polymerase: it catalyzes the formation of phosphodiester bond
between two nucleotides (3´end a 5´end of deoxyriboses) via relevant
phosphate.
Newly added nucleotide of growing DNA strand:
first, complementary pairing with the base of relevant nucleotide of the
template
afterwards, the formation of phosphodiester bond with the previous
nucleotide of growing strand
Nucleotide enters the reaction as nucleoside triphosphate.
Energy released by freeing pyrophosphate (PPi) is used for
polymerization reaction. [FIG.]
14
15
Two important and limiting properties of DNA polymerase:
• It can synthesize new DNA strand only in 5´→3´ direction
(according to the template in 3´→5´ direction)! [FIG.]
• It is unable to start the synthesis of new DNA strand, it can only
extend existing strand of nucleic acid.
DNA polymerases of eukaryotic cell:
• DNA polymerase α
• DNA polymerase δ
and other types (DNA polymerase β)
16
17
Two important and limiting properties of DNA polymerase:
• It can synthesize new DNA strand only in 5´→3´ direction
(according to the template in 3´→5´ direction)! [FIG.]
• It is unable to start the synthesis of new DNA strand, it can only
extend existing strand of nucleic acid.
DNA polymerases of eukaryotic cell:
• DNA polymerase α
• DNA polymerase δ
and other types (DNA polymerase β)
18
4. OTHER PROTEINS OF REPLICATION
MACHINERY:
• Helicase: after the binding to replication origin, it unwinds the
double helix of DNA (energy from ATP is used).
• Single-strand binding protein: molecules of the protein stabilize
single-stranded DNA by binding to it.
• Primase: it starts the replication by the formation of a short RNA
strand (primer).
Primer provides DNA polymerase with 3´ end, DNA polymerase
continues the synthesis of new DNA strand according to the
template. primer in procaryotic cell: 5 bp
primer in eucaryotic cell: 10 bp
• Protein sliding clamp: it keeps DNA polymerase attached to
template strand and it allows DNA polymerase to slide along the
strand.
19
5. MECHANISMS OF DNA REPLICATION:
Synthesis of new strand in 5´→3´on template 3´→5´:
leading strand
synthesis runs continuously (DNA polymerase δ) here
Synthesis of new strand in 3´ →5´ direction on 5´→3´ template:
lagging strand
Synthesis runs discontinously (DNA polymerase α) here
20
Lagging strand:
DNA polymerase „skips“ here forward along the template and then it
synthesizes backwards in proper direction 5´→3´.
The synthesis of new strand is performed piece after piece and these
pieces are referred to as Okazaki fragments (each fragment starts
with its own primer). [FIG.]
Afterwards, RNA primers are removed, missing DNA is synthesized by
relevant DNA polymerase and finally individual fragments are joined by
DNA ligase.
Okazaki fragments of procaryotic cell: about 1000 nucleotides
Okazaki fragments of eucaryotic cell: about 200 nucleotides
[FIG.] [FIG.]
21
22
Lagging strand:
DNA polymerase „skips“ here forward along the template and then it
synthesizes backwards in proper direction 5´→3´.
The synthesis of new strand is performed piece after piece and these
pieces are referred to as Okazaki fragments (each fragment starts
with its own primer). [FIG.]
Afterwards, RNA primers are removed, missing DNA is synthesized by
relevant DNA polymerase and finally individual fragments are joined by
DNA ligase.
Okazaki fragments of procaryotic cell: about 1000 nucleotides
Okazaki fragments of eucaryotic cell: about 200 nucleotides
[FIG.] [FIG.]
23
24
25
6. REPLICATION OF ENDS OF EUCARYOTIC
CHROMOSOMES:
A problem of synthesizing the lagging strand at the end of
chromosomes (telomere): it solves telomerase
Telomerase: it adds short repeats of a DNA sequence to the 3´end
it uses a RNA template that is part of the enzyme
Repetitive DNA sequence then acts as a template to complete
replication of the end of lagging strand. [FIG.]
26
27
7. PROOFREADING:
Proofreading: correcting activity of DNA polymerase on new DNA
strand in 3´→5´ direction while it synthesizes new strand in 5´→3´
direction.
Functioning of DNA polymerase before binding a new nucleotide:
• It verifies whether previously bound nucleotide has the base
complementary to template
• If yes, it continues by binding a new nucleotide
• If no, it removes previous wrong nucleotide and, instead of this
nucleotide, corresponding nucleotide is bound [FIG.]
28
29
Proofreading activity of DNA polymerase explains why DNA
polymerase has only 5´→3´ polymerase activity and proofreading in
3´→5´ direction.
Proofreading in 5´→3´ direction (hypothetical polymerization in 3´→5´
direction) is not possible from the chemical point of view. [FIG.]
30
31
8. MISMATCH REPAIR:
Mismatch repair: it corrects wrongly paired bases of newly
synthesized DNA strand (it corrects mistakes of replication machinery).
[FIG.]
Proteins, involved in mismatch repair, recognize pairing which is not
complementary (mismatch) due to the deformation of DNA double helix.
Afterwards, they remove wrong segment of new DNA strand and
synthesize this segment again. [FIG.]
Replication machinery: 1 error/107 nucleotides
Mismatch repair: correction of 99% errors of replication machinery
→
Overall accuracy of DNA replication: 1 error/109 nucleotides
32
33
8. MISMATCH REPAIR:
Mismatch repair: it corrects wrongly paired bases of newly
synthesized DNA strand (it corrects mistakes of replication machinery).
[FIG.]
Proteins, involved in mismatch repair, recognize pairing which is not
complementary (mismatch) due to the deformation of DNA double helix.
Afterwards, they remove wrong segment of new DNA strand and
synthesize this segment again. [FIG.]
Replication machinery: 1 error/107 nucleotides
Mismatch repair: correction of 99% errors of replication machinery
→
Overall accuracy of DNA replication: 1 error/109 nucleotides
34
35
8. MISMATCH REPAIR:
Mismatch repair: it corrects wrongly paired bases of newly
synthesized DNA strand (it corrects mistakes of replication machinery).
[FIG.]
Proteins, involved in mismatch repair, recognize pairing which is not
complementary (mismatch) due to the deformation of DNA double helix.
Afterwards, they remove wrong segment of new DNA strand and
synthesize this segment again. [FIG.]
Replication machinery: 1 error/107 nucleotides
Mismatch repair: correction of 99% errors of replication machinery
→
Overall accuracy of DNA replication: 1 error/109 nucleotides
36
9. MECHANISMS OF ACCIDENTAL DNA DAMAGE:
• Depurination: release of guanine or adenine from DNA
(spontaneous)
• Deamination: conversion of cytosine to uracil (spontaneous)
[FIG.] [FIG.]
• Formation of pyrimidine (thymine) dimers: caused by UV
irradiation [FIG.]
37
38
Figure 6-25 Chemical modifications of nucleotides, if left unrepaired, produce mutations. (A) Deamination of
cytosine, if uncorrected, results in the substitution of one base for another when the DNA is replicated. As shown
in Figure 6-23B, deamination of cytosine produces uracil. Uracil differs from cytosine in its base-pairing properties
and preferentially base-pairs with adenine. The DNA replication machinery therefore inserts an adenine when it
encounters a uracil on the template strand. (B) Depurination, if uncorrected, can lead to the loss of a nucleotide
pair. When the replication machinery encounters a missing purine on the template strand, it can skip to the next
complete nucleotide, as shown, thus producing a daughter DNA molecule that is missing one nucleotide pair. In
other cases (not shown), the replication machinery places an incorrect nucleotide across from the missing base,
again resulting in a mutation.
39
9. MECHANISMS OF ACCIDENTAL DNA DAMAGE:
• Depurination: release of guanine or adenine from DNA
(spontaneous)
• Deamination: conversion of cytosine to uracil (spontaneous) [FIG.]
• Formation of pyrimidine (thymine) dimers: caused by UV
irradiation [FIG.]
40
41
10. MECHANISMS OF THE REPAIR OF
ACCIDENTALLY DAMAGED DNA :
General steps of the repair of damaged DNA:
• recognition of the damage of DNA strand → excision of the
damaged DNA by specific nucleases
• synthesis of removed DNA according to complementary strand by
repair DNA polymerases
• rejoining newly synthesized DNA segment with repaired DNA strand
by DNA ligase (ligation)
• Base excision repair
• Nucleotide excision repair [FIG.]
The stability of DNA and thus also the stability of genetic information
depends on mechanisms of DNA repair.
42
43
10. MECHANISMS OF THE REPAIR OF
ACCIDENTALLY DAMAGED DNA :
General steps of the repair of damaged DNA:
• recognition of the damage of DNA strand → excision of the
damaged DNA by specific nucleases
• synthesis of removed DNA according to complementary strand by
repair DNA polymerases
• rejoining newly synthesized DNA segment with repaired DNA strand
by DNA ligase (ligation)
• Base excision repair
• Nucleotide excision repair [FIG.]
The stability of DNA and thus also the stability of genetic information
depends on mechanisms of DNA repair.
44
LITERATURE:
• Alberts B. et al.: Essential Cell Biology. Garland Science. New York
and London, pp. 197217, 2010
45
