Diagram of a

replication fork in
E. coli showing
the major
components of
the replication
apparatus. rNMP
ribonucleoside
monophosphates.

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 DNA Polymerases
 DNA polymerases are the enzymes that catalyze
the attachment of nucleotides to make new DNA

 In E. coli there are five proteins with polymerase

activity
 DNA pol I, II, III, IV and V

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 DNA POLYMERASE I

• Prokaryotic Family A polymerases include

the DNA polymerase I (Pol I) enzyme
• This repair polymerase is involved in excision
repair with 3'-5' and 5'-3' exonuclease activity and
processing of Okazaki fragments generated during
lagging strand synthesis.
• Pol I is the most abundant polymerase, yet lacking
in cells.
• Adds ~15-20 nucleotides per second, poor
processivity.
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 DNA polymerase II

• Family B polymerase, is a polB gene product also known

as DinA.
• Pol II has 3'-5' exonuclease activity and participates in
DNA repair, replication restart to bypass lesions, and its
cell presence can jump from ~30-50 copies per cell to
~200-300 during SOS induction.
• Pol II is also thought to be a backup to Pol III as it can
interact with holoenzyme proteins and assume a high
level of processivity.
• The main role of Pol II is thought to be the ability to direct
polymerase activity at the replication fork and helped
stalled Pol III bypass terminal mismatches.

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 DNA polymerase III

• belongs to Family C polymerases.

• It consists of three assemblies: the pol III core, the beta
sliding clamp processivity factor and the clamp-loading
complex.
• The core consists of three subunits - α, the polymerase
activity hub, ɛ, exonucleolytic proofreader, and θ, which may
act as a stabilizer for ɛ.
• The holoenzyme contains two cores, one for each strand, the
lagging and leading.
• The beta sliding clamp processivity factor is also present in
duplicate, one for each core, to create a clamp that encloses
DNA allowing for high processivity. The third assembly is a
seven-subunit (τ2γδδ′χψ) clamp loader complex.
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DNA polymerase III

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 Bacterial DNA polymerases may vary in their
subunit composition
 However, they have the same type of catalytic subunit
Structure resembles a
human right hand
Template DNA thread
through the palm;
Thumb and fingers
wrapped around the DNA

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 DNA Polymerase III is a
Processive Enzyme
 DNA polymerase III remains attached to the
template as it is synthesizing the daughter strand

 This processive feature is due to several different

subunits in the DNA pol III holoenzyme
 b subunit is in the shape of a ring
 It is termed the clamp protein
 g subunit is needed for b to initially clamp onto the DNA
 It is termed the clamp-loader protein
 d, d’ and y subunits are needed for the optimal function of
the a and b subunits
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 DNA Polymerase III is a
Processive Enzyme
 The effect of processivity is quite remarkable

 In the absence of the b subunit

 DNA pol III falls off the DNA template after a few
dozen nucleotides have been polymerized
 Its rate is ~ 20 nucleotides per second

 In the presence of the b subunit

 DNA pol III stays on the DNA template long enough to
polymerize up to 50,000 nucleotides
 Its rate is ~ 750 nucleotides per second
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 DNA polymerase IV

• An error-prone DNA polymerase involved in non-targeted mutagenesis.

• Pol IV is a Family Y polymerase expressed by the dinB gene that is
switched on via SOS induction caused by stalled polymerases at the
replication fork.
• During SOS induction, Pol IV production is increased tenfold and one of
the functions during this time is to interfere with Pol III holoenzyme
processivity.
• This creates a checkpoint, stops replication, and allows time to repair DNA
lesions via the appropriate repair pathway.
• Pol IV is to perform translation synthesis at the stalled replication fork like,
for example, bypassing N2-deoxyguanine adducts at a faster rate than
transversing undamaged DNA.
• Cells lacking dinB gene have a higher rate of mutagenesis caused by
DNA damaging agents.
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 DNA polymerase V
• A Y-family DNA polymerase that is involved in SOS
response and translession synthesis
• Transcription of Pol V via the umuDC genes is highly
regulated to produce only Pol V when damaged DNA is
present in the cell generating an SOS response.
• Stalled polymerases causes RecA to bind to the ssDNA,
which causes the LexA protein to auto-digest.
• LexA then loses is ability to repress the transcription of the
umuDC operon.
• The same RecA-ssDNA nucleoprotein post-translationally
modifies the UmuD protein into UmuD' protein.
• UmuD and UmuD' form a heterodimer that interacts with
UmuC, which in turn activates umuC's polymerase catalytic
activity on damaged DNA.
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DNA polymerases cannot
initiate DNA synthesis

Problem is overcome by
the RNA primers
synthesized by primase

Problem is overcome by
synthesizing the 3’ to 5’
strands in small fragments

DNA polymerases can

attach nucleotides only in
the 5’ to 3’ direction

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DNA Damage and Repair
1. Defects in repair cause disease
2. Common types of DNA damage
3. DNA repair pathways
Direct enzymatic repair
Base excision repair
Mismatch repair
Nucleotide excision repair
Double-strand break repair
Non-homologous end joining
Homologous recombination

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 Type of Mutations(I)
I. Point mutation:
A. Base substitution
Change in DNA
Transition: One purine replaced by a different
purine;or one pyrimidine replaced by a
diferent pyrimidine
A G T C
Transversion: A purine replaced by a pyrimidine
or vice versa
A T C G
 Type of Mutations (II)
Change in protein
1. Silent mutation: altered codon codes for the
same a.a. GAG (Glu) --->GAA (Glu)
2. Neutral mutation: altered codon codes for
functional similar a.a. GAG--->GAC (Asp)

3. Missense mutation: altered codon codes for

different dissimilar a.a. GAG ---> AAG (Lys)
4. Nonsense mutation: altered codon becomes a
stop codon GAG ---> UAG (stop)
 Type of Mutations (III)
B. Frameshift mutation: addition or deletion of one
base-pair result in a shift of reading frame and alter
amino acid sequence

1. Wild type: ATG ACC AGG TC

Met Thr Arg

2. Base addition: ATG ACA CAG GTC

Met Thr Gln Val

3. Base deletion: ATG ACA GGT C

Met Thr Gly
 Type of Mutations (IV)
II. Insertion

III. Deletion

IV. Translocation

V. Inversion
 Sources of mutation
• Natural polymerase error
• Endogenous DNA damage
oxidative damage
depurination
• Exogenous DNA damage
radiation
chemical adducts
• “Error-prone” DNA repair

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 Cellular protection from DNA
damage
• Natural errors: polymerase base selection,
proofreading, mismatch repair
• Endogenous/exogenous DNA damage: base excision
repair, nucleotide excision repair, (recombination,
polymerase bypass)
• Recombination and polymerase bypass do not remove
damage but remove its block to replication.
Polymerase bypass is itself often mutagenic.

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 Induced Mutations
(a) Physical agents that damage DNA:
--- Ionizing radiation: OH, O2-, H2O2, damage
base and sugar residues.
--- UV radiation: Cyclobutane pyrimidine dimers,
Thymidine dimers (T-T) dimer

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(b) Chemical agents that damage DNA:
---Alkylating agents: Alkylating agents are
electrophilic compounds with affinity for
nucleophilic centers in organic macromolecules.
These include a wide variety of chemicals, many of
which are proven or suspected carcinogens (such as
nitrous acid, hydroxylamine, and ethylmethane
sulfonate, EMS), Adding alkyl group to hydrogen-
bonding oxygen of G or T, resulting in G-T
mispairing

G-C ---> G*T --->A-T

T-A --->T*-G ---> CG 25
 Base-analogue Agents
A base analogue is a substance other
than a standard nucleic acid base
that can be incorporated into a DNA
molecule by the normal process of
polymerization. Such a substance
must be able to pair with the base on
the complementary strand being
copies, or the 3'->5' editing function
will remove it. For example, 5-
bromouracil is an analogue of
thymine and might cause an A-T to
G-C transition mutation.
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Base Analogue

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 Intercalating Agents:
Intercalating agents: Substances whose dimensions are
roughly the same as those of a purine-pyrimidine pair.
In aqueous solutions, these substances form stacked
arrays, and are also able to stack with a base-pair by
insertion between two base-pairs. This may result in
frameshift mutation.

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Model of intercalating agent
induced mutagenesis

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 Metabolite Mutagens
Chemicals that are metabolized to electrophilic
reagents: Aflatoxins, benzo[a]pyrene
A mutagen is a physical or chemical agent that
causes mutations to occurs.
Mutagenesis is the process of producing a mutation.
Mutant refers to an organism or a gene that is
different from the normal or wild type.

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(a) Mismatches:
Occurs during DNA synthesis
(i.e. replication, repair, or
recombination)

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 (b) Tautomeric shifts
Nucleotides spontaneously under go a transient
rearrangement of bonding, e.g. a shift from NH2 (amino
form) to NH (imino form) or C=O (keto) to C-OH (enol).
Therefore, if any base in a template strand exists in its
rare tautomeric form during DNA replication,
misincorporation in the daughter strand can result.

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Base Pairing of Imino A-C

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 (c) Deamination
Three of the four bases normally present in DNA
(cytosine, adenine, and guanine) contain amino
group (NH2). The loss of the amino group
(deamination) can occur spontaneously and result in
the conversion of the affected bases to uracil,
hypoxanthine, and xanthine, respectively.

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 (d) Loss of bases
Depurination and depyrimidination:
The loss of purines or pyrimidines
from DNA usually occurs at acidic
pH; however, it can also happen in
physiological pH (~10,000 purine
per day in mammalian cell; ~500
pyrimidine/day). This will results in
breaking the 3‘ phosphodiester bond
called b-elimination.

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 DNA Repair Mechanisms

( 1 ) Repair by direct reversal: The simplest

mechanism. e.g. UV induced T-T dimer is
recognized by photolyase and is cleaved into
intact thymine (light dependent). This is called
photoactivation
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 Excision Repair
(2) Excision Repair:
The most ubiquitous
repair mechanism,
which can deal with
a large variety of
structural defects in
DNA.
Base excision repair (BER)
• Major pathway for repair of modified bases, uracil
misincorporation, oxidative damage
• Various DNA glycosylases recognize lesion and
remove base at glycosidic bond, thereby producing
an “abasic” or AP (apurinic/ apyrimidinic) site by
base “flipping out”
• One of several AP endonucleases incises
phosphodiesterase backbone adjacent to AP site
• AP nucleotide removed by exonuclease/dRPase and
patch refilled by DNA synthesis and ligation
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 Mismatch repair (MMR)
• Despite extraordinary fidelity of DNA synthesis, errors do
persist
• Such errors can be detected and repaired by the post-replication
mismatch repair system
• Prokaryotes and eukaryotes use a similar mechanism with
common structural features
• Defects in MMR elevate spontaneous mutation rates 10-1000x
• Defects in MMR underlie human predisposition to colon and
other cancers (“HNPCC”)
• MMR also processes mispairs that result from heteroduplex
DNA formed during genetic recombination: act to exclude
“homeologous” recombination
 Basis of MMR recognition
• MutS dimer (in yeast, Msh2/Msh3 or Msh2/Msh6
heterodimer)
• By DNA binding experiments in vitro and DNA
heteroduplex repair experiments in vivo: MMR
can recognize all base substitutions except C:C
and short frameshift loops <4 bp
• Transition mispairs G:T and A:C and one base
loops are particularly well-recognized (these are
also the most common polymerase errors)
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 Nucleotide excision repair (NER)
• Recognizes bulky lesions that block DNA
replication (i. e. lesions produced by carcinogens)-
-example, UV pyrimidine photodimers
• Common distortion in helix
• Incision on both sides of lesion
• Short patch of DNA excised, repaired by
repolymerization and ligation
• In E. coli, mediated by UvrABCD
• Many more proteins involved in eukaryotes
• Can be coupled to transcription (TCR,
“transcription coupled repair”)
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The SOS response
The SOS response: The SOS response system is
only active in response to some signal such as a
blocked of replication fork. In E. Coli, recA and
lexA govern the expression of a number of other
genes involved in DNA repair. This is an error-
prone DNA repair mechanism and result in higher
than normal mutagenesis.
SOS DNA Repair
1. DNA damage
2. RecA converted to RecA*
3. RecA* facilitated LexA self-cleavage
4. Increased synthesis of SOS proteins
5. Error prone repair induced
6. DNA damage repaired
7. RecA* returned to RecA
8. LexA no longer self-cleaved
9. LexA repressed SOS genes
10. LexA repress lexA gene expression
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