Transcript Eukaryotic and Prokaryotic Cells

DNA Damage and
Repair
Why do we care?


Genetic diseases
Cancer
Cellular Responses to DNA Damage
Reversal of DNA Damage
 Enzymatic photoreactivation
 Ligation of DNA strands
 Repair of photoproduct
Tolerance of DNA Damage
 Replicative bypass of template damage with gap
formation and recombination (gap repair)
Excision of DNA Damage
 Base excision repair
 Nucleotide excision repair
 Mismatch repair
Mutagens and Carcinogens
Essentially all mutagens are carcinogens
Most carcinogens are mutagens
Somatic vs. germ line mutations
Somatic mutations can lead to cancer
Germ line mutations can lead to birth defects
Most mutations cause neither
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Some fall in non-coding DNA
Others are silent
Types of mutations
Types of substitutions
Missense

Results in an amino acid substitution
Nonsense

Results in a stop codon (TAG, TAA, TGA)
Same sense

No effect (silent mutation)
Slippage
Types of mutations
Multisite
Point mutations
Multisite mutations
Cause gross chromosome abnormalities
Involve large regions of DNA
Arise during meiosis
Types of multisite mutations
Inversions:
ACBDEF
Duplications ABCDEEF
Deletions:
ABCDF
Insertions:
ABCDSEF
Substitutions: ATCDEF
Point mutations
Involve only one or a few nucleotides
Arise during DNA replication
Require two errors

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An error during DNA replication
Failure to correct that error
Types of point mutations
Substitutions: GATC
CATC
Insertion:
GATC
GGATC
Deletion:
GATC
GTC
Duplication:
GATC
GAGATC
Inversion:
GATC
GTAC
What is the first defense against
mutations?
3’ to 5’ exonuclease activity of the
polymerases
Natural causes of mutations
Base tautomerization
UV damage
Spontaneous deamination
Adenine tautomer
Generation of
a mutation by
the adenine
tautomer
- About every
1 in 104 bases
UV damage
Spontaneous deamination
Three of the four bases have exocyclic
amino groups
Adenosine produces hypoxanthine
Guanine produces xanthine
Cytosine produces uracil
Hypoxanthine
Deamination of cytosine
produces uracil
If replication occurs a
mutation will result
Removal of
uracil from
DNA
Why do cells use thymine
rather than uracil?
Answer
The reason cells use thymine in their DNA

Is to allow recognition of uracil formed from cytosine
But what about RNA?

RNA is short lived and in many copies.
Chemical mutagens
Chemicals that accelerate the deamination
reaction
Base analogues
Alkylating agents
Intercalation agents
Base analogues
5-bromouracil
Goes in as T
Can base pair with A but also G to a
smaller degree
Generation of a
mutation by
5-bromouracil
Intercalation
Flat aromatic compounds
Acridine dyes
Ethidium bromide
Cause frame-shifting
Repair mechanisms
We are exposed to mutagens all the time

you would expect repair mechanisms to
exist
A number of different repair mechanisms
do exist
Repair Mechanisms
In mismatch repair
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Incorrect base is identified
On short section of a newly synthesized DNA
Removed, and replaced
by DNA synthesis directed by the correct template.
In excision repair

bulky lesions in DNA
exposure to UV light
removed by specialized nuclease systems
DNA polymerase fills gap
DNA ligase joins the free ends.
DNA Mismatch Repair
Intro to DNA Mismatch Repair
Mismatch Repair Genes

recognition and repair of certain types of DNA
damage or replication errors
Function to help preserve the fidelity of the
genome

through successive cycles of cell division
Mismatch repair
Occurs just after replication
Improves accuracy 102 - 103 fold
Must distinguish the parent from the
daughter strand
History of MMR
System first discovered in bacteria
Partially homologous system in yeast
Marked homology between yeast and higher
order organisms
Human MMR genes first described 1993.
DNA Mismatches
Damage to nucleotides in ds-DNA
Misincorporation of nucleotide
Missed or added nucleotides
Acquired DNA Damage
M
-C-A-G-T-
Demethylation
-T-A-G-T-
Nucleotide Misincorporation
-C-A-G-C-T-
CT substitution
-G-T-C-C-A-
-C-A-G-C-T-C-A-G-C-T-
-G-T-C-C-A-
-G-T-T-C-A-
-C-A-G-C-T-G-T-C-C-Acorrectly copied
Added Nucleotides
-C-A-G-C-Tnucleotide added
-G-T-C-C-A-
-C-A-G-C-T-C-A-G-C-T-
-G-T-C C-AA
-G-T-C-C-A-
-C-A-G-C-T-G-T-C-C-Acorrectly copied
Mismatch Repair Genes
Recognition and repair of mismatches
Other functions
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Repair of branched DNA structures
Prevent recombination of divergent sequences
Direct non-MMR proteins in nucleotide excision
and other forms of DNA repair
MSH4 & MSH5 involved (with MLH1) in meiotic
crossover
Human Mismatch Repair
Genes
MLH1
PMS1
PMS2
MSH2
MSH3
MSH6
(3p21)
(2q31-33)
(7p22)
(2p16)
(5q3)
(2p16) (=GT Binding Protein)
Mismatch Repair Function
MMR Proteins combine as heterodimers
Recognise and bind mismatches
ATP consumption
Recruit other proteins
Separate, destroy and resynthesise new DNA strand
Mechanism works for up to 20 base pairs
MSH Protein Complexes
MutS (MSH2-MSH6)
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GT mispairs and short (1 base pair) loops/deletions
MutS (MSH2-MSH3)
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Larger mispair loops and deletions
Some overlap in function
MSH2 loss is greater cancer risk
MLH Protein Complexes
MutL (MLH1-PMS2)
MutL (MLH1-PMS1)
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No established function
Can bind other MMR proteins, MSH heterodimers and
replication factors
As for MSH2, overlap means loss of MLH1 confers
the greater cancer risk
Other MMR Proteins
DNA ligase
Replication protein A
Replication factor C
Proliferating Cell Nuclear Antigen
Exonucleases
DNA polymerase 
Defective Mismatch Repair
Defects in MMR Genes and Function
Microsatellite Instability
Cancer development
Defects in MMR Genes
Control sequences
Premature stop codon
Point mutations
Insertions/Deletions
 Nonexpression
 Truncated protein
 Altered sequence
 Frameshift effects
Somatic loss of second allele
Microsatellite Instability
Simple nucleotide repeat sequences
Length should be stable at any one locus
Poly-A and poly-CA repeat sequences particularly prone to
mismatch errors
Alterations in length are a sign of deficient mismatch repair
Also called RER (Replication ERror)
Microsatellite Instability
-C-A-C-A-C-A-C-A-G-T-G-T-G-T-G-T-
shortened repeat
-C-A-C-A-C-ACA skipped
-C-A-C-A-C-A-
-G-T-G-T-G-T
-G-T-G-T G-T-
G-T
heteroduplex
results
-C-A-C-A-C-A-C-A
-G-T-G-T-G-T-G-T
MI Positive Tumours
90% of HNPCC colorectal cancers
20% of sporadic colorectal cancers
30% of sporadic uterine cancers
Cancer Development
Activation of Oncogenes
Inactivation of Tumour Suppressor Genes
Repeat sequences are common in both of
these classes of gene
Susceptible to mutation
Cancer Genes
Cell Cycle Control
Cellular differentiation
Cell Death
Other Genes
Summary
Inherited mutations of MMR genes lead to high relative
and absolute risk of cancer
Colorectal and endometrial cancers
Cancer at early age
High risk of further cancers
Identification of families and individuals

Amsterdam criteria
Amsterdam criteria
One patient should be a first degree relative of
the other two
At least two successive generations should be
affected
At least one tumor should be diagnosed <50
years of age
Tumors should be verified by histopathology
Let’s get back to molecular cell
genetics!
How to distinguish the mother and daughter
strands
Strands are methylated at GATC sequences on A
The parental strand is methylated the daughter
strand is not yet methylated
Mismatch repair of single-base mispairs
What happens when mismatch
repair fails in humans?
Missing enzymes homologous to MutS and MutL
Patients usually die by age 30
Disease: hereditary nonpolyposis colorectal
cancer (HPCC)
1 in 200 people affected
Excision repair in
eukaryotes
Excision Repair in Eukaryotes
Mechanism responsible for most of the
DNA repair in eukaryotes
Damaged or incorrect bases are excised
from the DNA and replaced with the
correct nucleotide
5-step pathway
Base excision repair
Base excision involves removal of DNA damage
such as:
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Deaminated bases
Alkylated or oxidized bases
Bases with open rings
Apurinic/apyrimidinic sites
Pathway removes the types of DNA damage that
occur spontaneously in all living cells
Steps in excision repair
1. Damage recognition
2. Incision of the lesion containing the DNA
strand on both sides of the lesion
3. Excision of the damaged nucleotide(s)
4. Synthesis of new DNA by a DNA polymerase
using the complementary strand as template
5. Ligation
Direct repair (Nucleotide excision repair)
Pyrimidine dimers
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Repaired only in the presence of light
Requires a DNA photolyase enzyme
M6Guanine
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Requires a methyl transferase
Chemically modified bases, such as thyminethymine dimers, are corrected by nucleotide
excision repair
DNA damage and repair and the role
in carcinogenesis
A DNA sequence can be changed by copying errors
introduced by DNA polymerase during replication and by
environmental agents such as chemical mutagens or
radiation
If uncorrected, such changes may interfere with the ability of
the cell to function
DNA damage can be repaired by several mechanisms
All carcinogens cause changes in the DNA sequence and
thus DNA damage and repair are important aspects in the
development of cancer
Prokaryotic and eukaryotic DNA-repair systems are
analogous
Proofreading by DNA polymerase
corrects copying errors
Schematic model of the proofreading
function of DNA polymerase
Chemical carcinogens react with DNA directly
or after activation, and the carcinogenic effect
of a chemical correlates with its mutagenicity
Mutations in the DNA
repair machinery
Almost all mutations in genes encoding
repair proteins are lethal
There are a few rare exceptions to this and
the best characterized is Xeroderma
pigmentosa
Xeroderma pigmentosa
Rare skin disease in humans
Patients are very sensitive to sunlight
Usually they develop skin cancer and die
before the age of 30
Frequent neurological abnormalities
Distribution of Xeroderma pigmentosa
1 in 100,000 in Europe and the US
1 in 40,000 in Japan
What causes Xeroderma
pigmentosa?
Autosomal recessive trait
Fibroblasts from patients are unable to excise
pyrimidine dimers
In one case, cells were missing the
endonuclease activity
Mutations in seven other genes will cause the
same effect
Mutations and Cancer
What is the evidence that many cancers arise from
mutations -- usually multiple mutations?
Cancers are common in animals with
damaged DNA repair machinery
Most cancers arise as clones of a single cell
Mutagens are carcinogenic
Most carcinogens are also mutagenic