BCH 1211/1218

Molecular Biology,
Oncogenesis and Immunology

Week 2

DNA Damage and Repair.

Dr. Nerina Harborne

 Mechanisms of Mutation
A - Replication errors

Damage After Replication:-

B - DNA damage from chemical reactions

C - Ionising Radiation

D - Base Analogs

E - Intercalating agents
 A - Replication Errors
From Alberts et.al,
Molecular Biology of the Cell
Garland Science

DNA Polymerase Proofreads and

Edits the New DNA
 A - Replication Errors
DNA polymerase is extremely accurate in correctly
matching base pairs –
but makes a mistake once in every 105 base-pairs.
Most of these mistakes are corrected by
DNA Polymerase proofreading, reducing the error
rate from 1 in 105 to 1 in 107 base-pairs.
Uncorrected errors result in a Point Mutation.
The error rate is further reduced to 1 in 109
base-pairs by
Post-Replication Mismatch Repair Processes
(which can also correct some DNA damage from
chemical or environmental sources.)
 Replication Error - Mismatch

From Watson et.al,

Molecular Biology of the Gene
pub. Pearson

The mismatch lesion MUST be repaired before a

second round of DNA replication takes place to
prevent it from becoming a permanent mutation.
 Mismatch Repair
For the E. coli model system, the enzymes and other
molecules, and the mechanisms of the mismatch
repair system, are well understood.

Although the Mismatch Repair System in humans and

other eukaryotes is less well characterised, it is
known that they possess many proteins homologous
to those of the E.coli system.

It is also known that mutations causing defects in

these homologous proteins can be linked to cancers.

Mismatch repair in humans is therefore the subject of

much research.
 From Watson et.al,

Mismatch Repair Molecular Biology of the Gene

pub. Pearson

Mismatch repair protein MutS

recognises the damaged DNA
and binds to the mispaired region.
Bound MutS associates with MutL
which then associates with MutH.
MutH is an endonuclease, which
nicks the incorrect strand,
which is then
unwound by DNA helicase,
degraded by an exonuclease,
and repaired by
DNA Polymerase.
How is the incorrect strand
identified?
 Mismatch Repair in E. Coli
From Lehninger Principles of
Biochemistry
WH Freeman & Co.

How is the incorrect

strand identified?

In E. Coli, new and old strands are distinguished by

methylation – the addition of methyl groups to bases.
Methylation of
Adenine in the sequence GATC
(Occurs frequently in the
E. coli genome –
about once every 256bp).
Catalysed by
Deoxyadenosine Methylase –
DAM methylase.
1998 Dr. Michael Blaber
 DAM Methylation
As new DNA is
synthesised, the
template strand will carry
the methylated A bases.
The newly synthesised
strand is not yet
methylated.
DAM methylase From Watson et.al,
Molecular Biology of the Gene

methylates the new pub. Pearson

strand some time

(several minutes) after it
has been replicated.
 E. Coli Methyl Mismatch Repair
If a base pairing
mistake occurs
during replication,
the mismatched
base distorts the
DNA double helix.
Repair mechanisms
will recognise and
repair the damage. © 2013 W. W. Norton & Company.

Repair enzymes recognise which strand to repair by

the methylation status.
 Mismatch Repair in Other Organisms
It is now known that the DAM methylation of newly
synthesised DNA in E. Coli is unusual – not found in
many other bacteria, and not in eukaryotes.
But these organisms do carry out mismatch repair.
It is hypothesised that, in the case of human DNA
replication, identification of the newly synthesised
strand is achieved by association of the human
homolog of MutS (MSH) with the sliding clamp that
had been used by DNA Polymerase during replication.
It is also possible that the nicked DNA of the lagging
strand (prior to ligation by DNA ligase) aids in this
identification.
B - DNA damage from Chemical
Reactions

Hydrolysis - Depurination
Hydrolysis - Deamination
Alkylation - Addition of methyl or ethyl groups
Oxidation
B - DNA damage from Chemical
Reactions

Hydrolysis (Blue arrows)

From Alberts et.al,
Molecular Biology of the Cell
Garland Science

Alkylation (Green arrows)

Oxidation (Red arrows)
 B - DNA damage from Chemical Reactions
Hydrolysis - Depurination
From Alberts et.al,
Molecular Biology of the Cell
Garland Science

The glycosidic bonds that hold the bases to the

DNA phosphate backbone can be hydrolysed,
releasing the base.
Coding information carried by that nucleotide is lost.
This happens more frequently for purine than
pyrimidine bases.
It results in a point mutation.
 B - DNA damage from Chemical Reactions
Hydrolysis - Depurination

Glycosidic bond
hydrolysed.

Base lost.

Information lost.
A deletion
point mutation
results.

From Alberts et.al,

Molecular Biology of the Cell
Garland Science
 B - DNA damage from Chemical Reactions
Hydrolysis - Deamination

From Alberts et.al,

Molecular Biology of the Cell
Garland Science

Cytosine can undergo hydrolytic deamination to Uracil

So, on replication,
a GC base-pair becomes an AT base-pair,
a point mutation.
Methylcytosine residues are deaminated at a higher
rate than cytosine, so form mutation 'hotspots'.
 B - DNA damage from Chemical Reactions
Hydrolysis - Deamination
Deaminated C
becomes U –
pairs with A.

U (in DNA) will be

replaced with T.

C-G base pair has

become A-T base
pair -

A substitution
From Alberts et.al,
Molecular Biology of the Cell
Garland Science
point mutation.
 B - DNA damage from Chemical Reactions
Alkylation From Alberts et.al,
Molecular Biology of the Cell
Garland Science

Alkyl groups (e.g. methyl groups -CH3) can be added

to many positions on DNA. (Green arrows)
DNA methylation has important biological functions,
but uncontrolled methylation can cause DNA damage.
Alkylation can result in 'misreading' of bases.
 B - DNA damage from Chemical Reactions
Oxidation
Oxidation can be From Watson et.al,
Molecular Biology of
the Gene
be carried out pub. Pearson

by reactive oxygen
molecules
(free radicals)
produced within
the cell by
ionizing radiation
or chemicals.

The modified bases can be mis-read by the DNA

replication machinery, resulting in point mutations.
 B - DNA damage from Chemical Reactions
Oxidation
For example, From Watson et.al,
Molecular Biology of
the Gene
pub. Pearson

Oxidation of
Guanine results
in oxoG
(7,8-dihydro-
8-oxoguanine).

This can base-pair with A as well as C –

so can cause G:C to T:A transversion mutation, one of
the most common mutations found in human cancers.
 C - Ionising Radiation
Gamma-Radiation and X-Rays
Produce Free Oxygen Radicals which react with DNA
causing Chemical Damage (as above).
Can also break the DNA Phosphate backbone,
causing Double Stranded breaks in the DNA:-
can result in DNA Rearrangement mutations.
 C - Ionising Radiation
UV Light (260 nm wavelength)
is strongly absorbed by DNA bases.
Can cause Thymine Dimer to form between 2
adjacent T residues.

Cannot form
base-pairs,
so DNA
Replication
HALTS at
this damage.

From Watson et.al,

Molecular Biology of the Gene
pub. Pearson
 D – Base Analogs
Structurally similar to DNA
bases,
but functionally different.
Can be incorporated into DNA,
but cannot
base-pair correctly.
e.g. 5-Bromouracil –
analog of Thymine,
but can be paired with
Guanine.
Thus, can cause http://acade
mic.brooklyn

Point Mutations
.cuny.edu/
 E – Intercalating agents
Flat, ring-shaped
molecules, can
'slip between' the
DNA bases
Distort the shape
of the double
helix
e.g. Ethidium
Bromide
Can cause
Insertion or
Deletion
mutations. http://what-when-how.com/molecular-
biology/ethidium-bromide-molecular-biology/
 E – Intercalating agents
Ethidium Bromide
When intercalated with
DNA, fluoresces under
UV light.
Widely used in
laboratories to visualise
DNA on gels etc.
It is a potent carcinogen,
so care must be taken
with its use and disposal.

Copyright © 2013 Bio-Rad Laboratories, Inc.

 Major DNA Repair Pathways
DNA polymerase proofreading
Mismatch Repair System

Excision Repair Base Excision Repair

Systems:- Nucleotide Excision Repair

Direct Reversal of Photoreactivation

damage:-
Recombinational Double-Strand Break
Repair:- Repair

Translesion DNA Synthesis

 DNA Repair
Prokaryote or Eukaryote?

Most known about prokaryote (E. coli)

“model systems”.

But – much also applies to eukaryote systems.

So – the following principles apply to all cell types,

but the details apply to prokaryotes.
 Base Excision Repair
Glycosylase enzymes (specific
for different types of DNA base
damage) “check” the DNA.

When they find a damaged base,

they remove it by cleaving the
glycosidic bond connecting the
base to the sugar-phospate
backbone.

This creates an “AP” site

(apurinic or apyrimidinic)

From Alberts et.al,

Molecular Biology of the Cell
Garland Science
 Base Excision Repair
AP Endonuclease and
Phosphodiesterase
enzymes recognise the AP site
and cleave the sugar-phosphate
backbone on either side, leaving
a gap.

This gap is filled by

DNA Polymerase (specialised for
DNA damage repair), and the
nick in the sugar-phosphate
backbone is sealed by
DNA Ligase.
From Alberts et.al,
Molecular Biology of the Cell
Garland Science
 Base Excision Repair – Base Flipping
Glycosylase
enzymes find
damaged bases by
“running along” the
DNA helix, and
“flipping out” each
base.
If it is damaged in a
way that matches
the specific
From Alberts et.al,
damage
Molecular Biology of the Cell
Garland Science recognised by that
enzyme, the base
is removed.
 Nucleotide
Excision Repair
Nucleotide Excision
Repair enzymes
recognise distortions in
the DNA double helix.
They cleave the DNA
backbone on either side
of the damage.
DNA Helicase unwinds
the damaged section.
The gap is filled by
DNA Polymerase and
From Alberts et.al,
Molecular Biology of the Cell
DNA Ligase.
Garland Science
Nucleotide Excision Repair
Nucleotide
Excision Repair
in E. coli, using
UvrA, B and C
enzymes.
The equivalent
repair pathway in
humans uses XP
enzymes.
From Watson et.al,
Mutations in XP
Molecular Biology of the Gene
pub. Pearson enzyme genes
result in the genetic disorder
xeroderma pigmentosum.
 Photoreactivation
Direct reversal of damage to DNA
Photolyase enzyme - Uses energy from visible light
Breaks covalent bonds
linking pyrimidine dimers
This DNA repair can
only happen in the Light
Humans do not have this
From Watson et.al,
repair pathway
Molecular Biology of the Gene
pub. Pearson
 Double-Strand Break Repair
Double strand breaks are very damaging to the
chromosome
Can occur as a result of ionising radiation
Can also occur as a result of exposure to
clastogenic agents (chemicals which cause double-
stranded DNA breaks)
such as the anti-cancer drug bleiomycin
 Double-Strand Break Repair
Cannot be repaired using information from the
other DNA strand (as both are broken)

Are repaired by

Non-Homologous End Joining

or
Homologous Recombination
Double-Strand Break Repair
Non-Homologous End
Joining

Information is lost from

the DNA

However, this may not be

important if the
information is not vital for
cell function.
Double-Strand Break Repair
Non-Homologous End
Joining

is also important in the

'normal' cell mechanisms
involved in adaptive
immunity.

DNA is broken and rejoined

to “shuffle” the elements
needed to produce a wide
range of different antibodies
and T-cell receptors.
From Watson et.al,
Molecular Biology of the Gene
pub. Pearson
Double-Strand Break Repair Homologous
Recombination
information is
restored from an
undamaged copy
of the chromosome.
Can only occur
when chromosomes
are paired during
mitosis
Uses the same
mechanisms which
also lead to genetic
recombination.
 Translesion DNA Synthesis (TLS)
“Failsafe” Measure to deal with unrepaired DNA
damage
Prone to errors (therefore likely to introduce
mutations)
But – allows DNA replication to proceed, so saving the
cell from incomplete replication (VERY damaging)
 Translesion DNA Synthesis (TLS)
“Failsafe” Measure to deal with unrepaired DNA
damage
Prone to errors (therefore likely to introduce
mutations)
But – allows DNA replication to proceed, so saving the
cell from incomplete replication (VERY damaging)

Carried out by specialized DNA polymerases

These need a DNA template, but incorporate bases
without base-pairing.
Thus, DNA synthesis can be continued over a region
of damaged template.
Translesion DNA Synthesis (TLS)
From Watson et.al,
Molecular Biology of the Gene
pub. Pearson
DNA Pol III is unable to
replicate DNA across a
DNA lesion
(e.g. a thymine dimer).
It is replaced by a
specialised TLS
Polymerase which adds
bases to fill the gap.
DNA Pol III is therefore
able to continue DNA
synthesis, but there is a
point mutation in the
DNA sequence.
 SOS Response

Translesion DNA polymerases are not always

present in the cell,
but are synthesised as part of the
SOS response pathway –
a damage-induced series of changes in the
proteins transcribed within the cell.
The same pathway produces proteins needed for
Double-Strand Break Repair.