Faithful DNA replication

Species health - individual

survival - species
Mutations
in somatic cells in germline cells
DNA MUTATIONS
Evolution
and established
after 2 cell 1 stable change
REPAIR divisions per average protein
in 200 000 years

Cell changes:
cell death / disease new species
/ survival & division
mostly deleterious for essential for longterm
the affected individual survival of life

Somatic cells Mutations occur continuously WHAT CAUSES MUTATIONS?

must be protected at a low rate - difficult to estimate
to safeguard individuals Rate / cell cycle (in tissue culture)
= 1 mutation / 109 b.p.
(7x109 b.p./ human genome)
SPONTANEOUS
Extensive damage occurs continuously.
continuously
Faulty ‘etidoryal’ proofreading during replication.

CHEMICAL
Chemicals from the environment (mutagens, carcinogens)
(some activation by our own metabolism)
DNA repair Repair mechanism Modification of bases (alkylation)
mechanisms unavailable / faulty
Insertion between bases

UV, IONIZING RADIATION

Mutation established Cross linking of base pairs
Accurate Ring opening
DNA replication after 2 DNA replications
(cell divisions) DNA strand breaks

DNA Change Cause Repair DNA Change Cause

Missing base Depurination Base Missing base Depurination (104/day/cell)

Excision
Incorrect base Spontaneous deamination Repair Incorrect base Spontaneous deamination
Alkylating agents Alkylating agents
Tautomeric shifts Tautomeric shifts
Ionizing radiation Ionizing radiation
Replication Mismatch Mismatch
Repair Replication Mismatch
Thymine dimers UV radiation Thymine dimers UV radiation
Nucleotide
Deletion/Insertion Intercalating agents Excision Deletion / insertion Intercalating agents
Repair
Strand breaks Ionizing radiation Strand breaks Ionizing radiation
Chemicals Photolyase Chemicals

1
 Missing base: DEPURINATION (104 / cell / day) Incorrect base: SPONTANEOUS DEAMINATION
(102 / cell / day)
Spontaneously and through heat/acid e.g. C deaminates to U

NH2 O O
CH3
N Deamination NH NH

N O N O N O
Spontaneous - base is lost
depurination - sugar-phosphate sugar sugar sugar
backbone is left
- if not repaired,
strand cannot Cytosine Uracil Thymine
replicate

Deamination of C: G-C → A-T transition mutation Base excision repair

(102/cell/day)
Uracil DNA 1. Recognition-removal of base
Generation Normal base →U
Deamination C→ glycosylase
Base-specific glycosylases
remove incorrect base.
1 G C GU Note: Repair of depurinated
base pairs starts here.
AP endo-nuclease &
Phosphodiesterase 2. Excision
2 GC GC GC AU Sugar-phosphate backbone
is excised.

3 AT AU
Needs to be repaired Polymerase I
Ligase 3. Resynthesis - DNA Pol I
here, before the
mutation passes to the 4. Ligation - Ligase
next generation

Incorrect base: ALKYLATING AGENTS

DNA Change Cause
Alkalytion: adds methyl or ethyl groups
Missing base Depurination (104/day/cell) to O & N of bases & phosphates of the DNA strand

Incorrect base Spontaneous deamination

Effects:
Alkylating agents
Tautomeric shifts (1) disrupts normal base pairing - strands distort
Ionizing radiation Distort DNA: (2) DNA strands break or form inter-strand bridges
Replication Mismatch Uvr-ABC (3) Replication = faulty
Nucleotide
Thymine dimers UV radiation Excision
Repair Source: environmental carcinogens / mutagens, eg
Deletion / insertion Intercalating agents
Methylnitrosourea (MNU)
Strand breaks Ionizing radiation Ethylmethanesulfonate (EMS)
→ Bulky lesions
Chemicals Chemotherapeutic agents (e.g. Cisplatin)

2
 Thymine dimers: UV RADIATION
2 adjacent
thymine residues

6-4 photoproduct
dimerization occurs
with T or C (i.e.
pyrimidines)
UV
light

T-T dimer is pulled together,

UV energy is
A’s are not hydrogen bonded
absorbed by T Replication stops at this point
In normal cells, 50% is repaired in < 24 hours Cyclobutane thymine dimer 6-4 photoproduct

Deletion/insertion: INTERCALATING AGENTS Deletion/insertion: INTERCALATING AGENTS

Stretching changes the Intercalating agents: flat, multi-ring aromatic molecules

frame needed by DNA For example:
Polymerase during
replication.
Extra nucleotides are Benz[a]anthracene
added during replication from cigarette smoke
& charred meat
The DNA reading frame for Aflatoxin:
RNA synthesis is changed: activated by our
liver enzymes from
amino acid changes mouldy
altered protein peanuts
Ethidium Bromide
(DNA stain used
Original DNA stretched by in Laboratory)
DNA intercalated ligands O

Thymine dimer Uvr ABC Nucleotide excision repair

5’ 3’ 1. Recognition- Repair system = NUCLEOTIDE EXCISION REPAIR
A A DNA bending
B
UvrA, UVrB For DNA distorting lesions in prokaryotes, e.g. E.coli:
ATP
Uvr ABC nucleotide excision repair
2. Excision-Excinuclease e.g. alkyl adducts, UV-induced dimers of bases,
B C UvrA dissociates intercalating agents)
UvrB~UvrC
UvrD helicase
unwinds fragment In eukaryotic cells, e.g. humans,
17 proteins cooperate in nucleotide excision repair.
3. Resynthesis In the disease Xeroderma Pigmentosum some of these
Polymerase I or II 17 components are mutated & non-functional.
4. Ligation Investigation of XP patients helped to identify some
Ligase of the components (named: XPA, XPB, XPC…XPG )

3
DNA Change Cause Repair Photolyase - Photoreactivation

Photolyase contains two

Missing base Depurination Base
Excision chromophores
Incorrect base Spontaneous deamination Repair (can absorb light of a
Alkylating agents characteristic wavelength)
Tautomeric shifts
Ionizing radiation
Replication Mismatch Mismatch
Repair
Thymine dimers UV radiation
Nucleotide
Deletion/Insertion Intercalating agents Excision
Repair 1. THF (tetrahydrofolate) derivative harvests light
Strand breaks Ionizing radiation energy & transfers it to 2nd chromophore
Chemicals Photolyase 2. FADH – transfers electrons to pyrimidine dimers
to break the dimerising bonds

Mismatch DNA repair

DNA Change Cause
Despite proofreading during replication
Missing base Depurination (104/day/cell) SOME mismatch replication errors occur

Incorrect base Spontaneous deamination The Mismatch DNA Repair system repairs mismatches
Alkylating agents in newly replicated DNA strands.
Tautomeric shifts
Ionizing radiation
Parent strand T
Replication Mismatch
Thymine dimers UV radiation Correct match A
Deletion / insertion Intercalating agents Repaired by proofreading C
Strand breaks Ionizing radiation
→ Bulky lesions
Chemicals Repaired by DNA Mismatch G
Repair system

Mismatch DNA repair Mismatch DNA repair

3. MutH = an endonuclease
• recognizes AMe
• nicks the opposite strand.

CH3
2. MutL
T~G mismatch in newly synthesized DNA. attaches& links MutL
MutH & MutS
Which strand must be repaired?

Parental GATC = methylation of adenine 1. MutS

Repair before the new strand is methylated. scans DNA &
binds to
mismatched
bases

4
 Mismatch DNA repair Mismatch DNA repair
Then: bacterial eukaryotic
(1) a Helicase (UvrD) MutS hMSH2
Mutations in these proteins
unwinds DNA from MutL hMLH1, specifically permit random
nick →past the mismatch. hPMS1, new mutations.
(2) an Exonuclease - hPMS2
cuts away single bases
from nick. MutH
endonuclease recognizes AMe mutations not known
(3) SSB - single strand
(as yet?)
binding proteins. Helicase
Exonuclease
Then DNA polymerase III Significant other functions -
SSB
• fills the site mutations would be lethal
Polymerase III
• Ligase seals the gap. Ligase