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DNA Synthesis, Mutation, and Repair

DNA structure:

A -- T
G–C

Antiparallel

Occurs during S phase in eukaryotes

E. coli

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Distinguishing between Models
of DNA Replication
• Three different models of how DNA might
replicate were proposed based on DNA
structure.
– Semi-conservative replication
– Conservative replication
– Dispersive replication

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Alternative Hypotheses for DNA Synthesis

(a) Hypothesis 1: (b) Hypothesis 2: (c) Hypothesis 3:


Semi-conservative Conservative replication Dispersive replication
replication

Intermediate molecule

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Distinguishing Between Models
of DNA Replication
• The Meselsohn and Stahl experiment
determines which
model is correct.
– 15N was fed to growing E. coli cells to mark
DNA, then
cells were switched to 14N.
– DNA replication is semi-conservative: new
DNA has
one 15N strand and one 14N strand.
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• Characteristics of replication in E. coli:
– At the replication fork, DNA polymerase III
builds the
new strands in the 5’-3’ direction.
– New nucleotides are only added to 3’ hydroxyl
groups
of other nucleotides. (creates a problem)
Formation of the leading strand

3'
DNA polymerase III
5'

Newly 5'
synthesized
leading strand 3'

5'
Replication fork

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Formation of lagging strand
3'
5'

Lagging 5'
strands
3'

3' DNA polymerase III


5'

3'
5'

Okazaki 5'
fragments
3'

DNA polymerase III


beginning synthesis
3' of new fragment
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5'
Completely single stranded
3' 5'
Polymerase
G A A T C T G C
inactive

Completely double stranded


3' 5'
G A A T C T G C
Polymerase
inactive
C T T A G A C G
5' 3'
Single strand as template plus 3' end
to start synthesis
3' 5'
G A A T C T G C
Polymerase
active
C T T The new strands are initiated by adding nucleotides
5' OH inserttotext
a short
here RNA primer because there is no DNA on
3' which to build. (Fig. 12.7)
Mutation and DNA Repair
Mechanisms
• Mutations are created by chemicals, radiation, errors
in meiosis and mistakes in DNA replication.
– Mutations can be deleterious, beneficial, or silent.
– Mutations in an individual are usually deleterious, may
cause disease and death.
– Mutations in a population are a source of genetic diversity
that allows evolution to occur.

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Mismatch (about 1/1000 base additions)
A A C T G G C
Wild type
T T G A C C G

A A C T G G C A A C T A G C
MUTANT
3' 5' T T G A T C G T T G A T C G
A A C T G G C
DNA replication DNA replication
T T G A C C G A A C T G G C A A C T G G C
5' 3'
Parental DNA Wild type
T T G A C C G T T G A C C G

First generation
progeny A A C T G G C
Wild type
T T G A C C G

Second generation
progeny
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General Categories of Mutations

Mutation Definition Example Consequence


type G

Insertion Addition of any Original ACC CAT Addition of 1 or 2 bases


number or sequence: ATA GAT GTA disrupts reading frame.
nucleotides due to Usually results in a
ACC CGA
an error in DNA Mutant dysfunctional gene product.
ATA GAT TGT A
synthesis sequence:
A
Deletion Removal of any
Original ACC CAT Deletion of 1 or 2 bases
sequence: ATA GAT GTA disrupts reading frame.
number of
nucleotides due to Usually results in a
ACC ATG dysfunctional gene product.
an error in DNA Mutant ATA GTC TA
synthesis sequence:

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Mutation Definition Example Consequence
type

A Genes A A A A
Gene Addition of a small Produces an extra copy of
B A B B B B one or more genes. If point
duplication chromosome segment
C B C C D C mutations occur in extra
due to an error during
D C D C D DNA, it can produce a new
crossing over at D D
meiosis I. product.
Mutant
Chromosome Change in a Changes gene order along
inversion chromosome segment A A A
B C chromosome. Other types of
when DNA breaks in B C chromosome breaks can
two places, flips, and C B
D D D lead to deletion or addition
rejoins. of chromosome segments.

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DNA DAMAGE

DNA REPAIR MECHANISMS

SHORT-TERM CONSEQUENCES

PHYSIOLOGICAL CELL DEATH ABNORMAL GROWTH &


DYSFUNCTION METABOLISM

Decreased Genomic Defective signalling Impaired protein/


cellular instability pathways gene expression
proliferation

LONG-TERM CONSEQUENCES

Ageing Cancer Disease

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Consequences of mutations: Well-studied
example: DNA point mutation can lead to
a different amino acid sequence.

Start of coding sequence Phenotype

DNA CAC GTG GAC TGA GGA CTC CTC


sequence GTG CAC CTG ACT CCT GAG GAG
Normal
Amino
acid
sequence Histidine Normal red blood cells
Threonine Glutamic
Valine Leucine Proline Glutamic
acid acid

DNA CAC GTG GAC TGA GGA CAC CTC


sequence GTG CAC CTG ACT CCT GTG GAG
Mutant
Amino
acid
sequence Sickled red blood cells
Histidine Threonine Glutamic
Valine Leucine Proline Valine acid

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Overview of DNA repair
pathways

A. Base excision repair


B. Nucleotide excision repair.
C. Mismatch repair
D. Double-strand break repair by
homologous recombination.
E. Double-strand break by end joining

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Base-excision repair
• Deaminated bases are recognized
• Enzymes remove the base

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(NER) Nucleotid Excision Repair
• Enzymes recognize a kink in the DNA
• Nicking
• Removal of the damaged strand
• DNA polymerization
• Ligation

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Double strand break repair.
• Double strand breaks (DSB) are one of the
major products of ionising radiation.
• DSB are are major cytotoxic lesions - even a
single unrepaired DSB can be a lethal event.
• Ataxia telangiectasia (AT) and Nijmegen
breakage syndrome (NBS) are associated with
deficiency in repair of double strand breaks.
• Recent data implicates BRCA1 and BRCA2 in
DSB repair.

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Repair of Double-Strand Breaks
• Double-strand breaks (ds breaks) are repaired
by at two different mechanisms.
• One mechanism takes advantage of proteins
that promote homologous recombination to use
information from the homologous or sister
chromosome for precise repair of breaks (HR).
• The other mechanism permits joining of ends
even if there is only small sequence similarity
between them. It is called non-homologous end
joining (NHEJ).

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Double-strand break repair by
homologous recombination
• The mechanism of DSB repair by HR is
intensively studied, many models have been
proposed.
• Proteins encoded by genes in the RAD52
epistasis group of S. cerevisiae (or their
homologs in other eukaryotic organisms) are
important for this process.
• These are RAD51, RAD52, RAD54, RAD55,
RAD57 and RAD59.
• The RAD51 protein is a key member of this
group – it is a RecA homolog and forms a
complex with ss DNA.
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SDSA - repair by conservative DNA synthesis is
accomplished in the following
way:
• A DSB is introduced into one of two homologous chromosomes.
• The 5' ends are then cut by an exonuclease to expose the 3' ends in
single-stranded form.
• With the help of RecA/Rad 51-like proteins, the 3' ends locate
complementary regions in the homologous or sister chromosome –
process of "strand invasion."
• In the next step 3' ends serve as primers for new DNA synthesis and
homologous or sister chromosome serve as template.
• Upon synthesis, the new strands are unwound from template and
anneal with each other.
• Any overhangs are removed by a flap endonuclease
• Gaps are filled in by a polymerase.
• Nicks are sealed by a ligase.
• The chromosome is repaired, but contains information from the
homologous chromosome.

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Single-strand annealing (SSA)
• DSB repair by SSA begins in similarly.
• A break is introduced and the 5' ends are cut.
• But, when the ends are cut this exposes regions
of complementary sequence in 3' strands -
repeated sequences flanking the DSB.
• Annealing of homologous sequences
• Flaps are removed by a FEN1-like
endonucleases
• DNA ligase seals the nicks
• DSB is repaired-but there was a deletion of DNA
between the two repeated sequences.
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Double-strand break repair by non-homologous
end joining (NHEJ)

Initial processing of breaks:


The 3 yeast gene products - Rad50, Mre11 and Xrs2,
interact with each other and are important for initial
processing of DSBs regardless of whether the breaks
are to be repaired by homologous recombination or by
NHEJ.
In their absence, NHEJ is reduced about 50-fold, but HR is
only delayed.
Mammalian homologs of these yeast proteins have now
been identified. The human RAD50 and MRE11 proteins
closely resemble their yeast counterparts, but the human
NBS1 protein is only distantly related to yeast Xrs2.

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• DNA Repair Mechanisms during replication
– DNA polymerase I proofreads and corrects point mutations
during replication.
– Other excision repair systems scan newly formed DNA and
correct remaining mutations.
– Repair enzymes identify the correct template strand by its
methyl groups.
– Defects in repair system enzymes are implicated in a variety
of cancers.

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Mismatch repair
• Occurs just after replication
• Improves accuracy 102 -103 fold
• Must distinguish the parent from the
daughter strand

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Mismatch repair model. MutS
MutL ssb
MutU
GATC G GATC GATC GATC

CTAG CTAG CTAG CTAG


T

GATC GATC
MutH
CTAG CTAG

GATC G GATC
GATC GATC

CTAG CTAG
GATC C
Synthesis
Mismatch repaired
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Mismatched 3' 5'
A
bases. T G T C C T C G C

A C A G G
5' G
OH 3'

But, how does the “system” know which is the correct


sequence and which is the mutant sequence?

Polymerase III can 3' 5'


repair mismatches.
T G T C C A T C G C

A C A G G
5'

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METHYLATION-DIRECTED MISMATCHED BASE REPAIR Answer

1. Where a mismatch occurs, the


correct base is located on the
methylated strand: the incorrect base
occurs on the unmethylated strand.
Mismatch

2. Enzymes detect mismatch and nick


unmethylated strand.

3. DNA polymerase I excises


nucleotides on unmethylated strand.

4. DNA polymerase I fills in


gap in 5' 3' direction.

5. DNA ligase links new and


old nucleotides.
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Repaired Mismatch
Interesting and common: UV-induced thymine dimers caused DNA to kink

P O H P
N O H
CH2 CH2
O N Thymine O O N
N O
DNA strand H UV light
CH3
with adjacent H CH3
Thymine
thymine P O H P Kink O H dimer
bases N N
CH 2 O N Thymine O CH2 O N O

H CH3 H CH3
P P

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General repair mechanisms needed
• EITHER Reverse damage (e.g. PHOTOREACTIVATION)
• OR excise DNA and patch repair the region

Photoreactivation: Discovered in Actinomycetes in 1949


UV - DNA Damage - Cell Death
UV - Bright visible light - survival !

3 Steps:
• Photolyase (encoded by phrA and phrB genes in E. coli)
recognises distortion at dimer.
• Light activates photolyase
• Dimer cleaved
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Excision repair........
UvrABC Pol1
5’ 3’
3’ 5’

OR
5’ 3’ 5’ 3’
3’ 5’ 3’ 5’

5’ 3’ 5’ 3’
3’ 5’ 3’ 5’

5’ 3’
3’ 5’

5’ 3’
3’ 5’
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