DNA Replication, Mutation,

Repair
M 211A MOLECULAR GENETICS
1ST SEMESTER, 2009
NABIL BASHIR
 DNA Replication, Mutation, Repair

a). DNA replication

i). Cell cycle/ semi-conservative replication
ii). Initiation of DNA replication
iii). Discontinuous DNA synthesis
iv). Components of the replication apparatus
b). Mutation
i). Types and rates of mutation
ii). Spontaneous mutations in DNA replication
iii). Lesions caused by mutagens
c). DNA repair
i). Types of lesions that require repair
ii). Mechanisms of repair
Proofreading by DNA polymerase
Mismatch repair
Excision repair
iii). Defects in DNA repair or replication
 Cell Cycle
• The S (for synthesis) phase of the cell cycle
is when chromosomes are replicated. This
requires DNA synthesis and histone
synthesis (the latter to make the proteins
that will package the newly replicated
DNA).
 The mammalian cell cycle

Rapid growth and DNA synthesis and

preparation for histone synthesis
DNA synthesis
S
phase

G0 G1
phase
Quiescent cells
G2
phase Growth and
M preparation for
phase
cell division

Mitosis
Semi-conservative DNA Replication

• DNA replication is said to be "semi-

conservative" because each strand of the
DNA double helix serves as a template for
the synthesis of a new complementary DNA
strand. Hence, the newly replicated
chromosome consists of one old and one
new DNA strand.
DNA replication is semi-conservative

Parental DNA strands

Each of the parental strands serves as a

template for a daughter strand

Daughter DNA strands

Origins Of DNA Replication On Mammalian
Chromosomes

• Each chromosome consists of a very long DNA strand,

the complete length of which needs to be replicated in a
relatively short (a few hours) period of time. This is
accomplished by having multiple origins of DNA
replication on each chromsome, which are spaced every
~150 kb. Replication initiates independently at each
origin and proceeds bidirectionally as the new DNA
strands (red) are synthesized. The resulting "replication
bubbles" then fuse together or merge completing the
synthesis of the daughter chromosomes.
 Origins of DNA replication on mammalian chromosomes
origins of DNA replication (every ~150 kb)

5’ 3’
3’ 5’

bidirectional replication

replication bubble

fusion of bubbles
5’ 3’
3’ 5’
daughter chromosomes
5’ 3’
3’ 5’
 Initiation Of DNA Synthesis At The E. Coli
Origin (Ori)

• The single, circular E. coli chromosome has one

origin of replication (ori). Initiation of replication
begins with the binding of dnaA proteins to the ori
sequence. As these proteins coalesce, the adjacent
DNA is forced to undergo melting into single strands.
This then allows the dnaB and dnaC proteins to bind
the single-stranded DNA and further unwind the
double helix, catalyzed by the dnaB protein which is a
DNA "helicase" or DNA unwinding enzyme.
Initiation of DNA synthesis at the E. coli origin (ori)
origin DNA sequence
5’ 3’
3’ A A A 5’
binding of dnaA proteins

A
A A
DNA melting induced
A A A by the dnaA proteins

dnaA proteins coalesce

dnaB and dnaC proteins bind
to the single-stranded DNA
A
A A
A A A B C
dnaB further unwinds the helix
• As further unwinding occurs, which displaces the dnaA
proteins, the dnaG protein binds. This protein is a
"primase" and synthesizes a short RNA primer of about 5
nucleotides. Because the primase synthesizes an RNA
strand, it is an RNA polymerase. The primer provides a
free 3' OH end to initiate DNA synthesis. As a rule, DNA
polymerases cannot initiate DNA synthesis de novo, but
can only add onto an existing 3' OH. Hence, the need for
the RNA primer. In contrast to DNA polymerases, RNA
polymerases can initiatate synthesis de novo.
 dnaG (primase) binds...

A
A A G
A A B C
A
dnaB further unwinds the helix
and displaces dnaA proteins

A ...and synthesizes an RNA primer

A
A G
A B C RNA primer
A
A
 Primasome
• The "primasome" consists of the dnaB,
dnaC, and dnaG proteins.
 Primasome
G dna B (helicase)
B C dna C
dna G (primase)

template strand
5’ 3’
3’ OH 5’
RNA primer
(~5 nucleotides)
• Once the RNA primer has been synthesized, DNA polymerase
can then bind and begin to synthesize DNA. DNA polymerase
catalyzes an attack by the 3' OH on the alpha phosphate of the
dGTP, forming a 3', 5'-phosphodiester bond, and releasing
pyrophosphate, which is then hydrolyzed to two molecules of
inorganic phosphate. All DNA polymerases require a primer
(or a growing DNA chain) with a free 3' OH. The new strand
of DNA grows in a 5' to 3' direction; however, some DNA
polymerases also have a 3' to 5' proofreading activity, which
can remove the 3' terminal nucleotide in case the polymerase
makes a mistake.
 DNA polymerase

5’ 3’
5’
RNA primer

3’
5’
newly synthesized DNA
 Discontinuous Synthesis Of DNA

• Because DNA synthesis always proceeds in a 5' to 3'

direction, and because the two DNA strands are arranged
antiparallel with respect to each other, only one of the two
newly synthesized strands can be made "continuously"
(continuous red line) as the DNA polymerase moves away
from the origin of replication and more DNA template is
exposed. The other strand has to be made
"discontinuously" in short pieces (short red lines). This
latter strand is called the "lagging strand" while the
continuously synthesized strand is called the "leading
strand."
Discontinuous synthesis of DNA

5’ 3’
5’ 3’ 5’ 3’
3’ 5’ 3’ 5’
3’ 5’

Because DNA is always synthesized in a 5’ to 3’ direction,

synthesis of one of the strands...

5’
3’
...has to be discontinuous.

This is the lagging strand.

 Leading And A Lagging Strand

• The small pieces of DNA that comprise the

lagging strand are called "Okazaki
fragments." They are eventually ligated
together forming a continuous DNA strand.
Each replication fork has a leading and a lagging strand

leading strand (synthesized continuously)

replication fork replication fork

5’ 3’
5’ 3’ 5’ 3’
3’ 5’ 3’ 5’
3’ 5’

lagging strand (synthesized discontinuously)

• The leading and lagging strand arrows show the direction

of DNA chain elongation in a 5’ to 3’ direction
• The small DNA pieces on the lagging strand are called
Okazaki fragments (100-1000 bases in length)
• The next series of figures shows the process of DNA
synthesis at the so-called "replication fork." Note
the direction (arrows) of leading strand synthesis and
lagging strand synthesis (both are in a 5' to 3'
direction). As stated previously, DNA polymerase
moves continuously along the leading strand. As the
template DNA unwinds, exposing the single-
stranded template for the lagging strand, primase has
to synthesize an RNA primer to which the DNA
polymerase synthesizing the lagging strand can bind.
RNA primer
direction of leading strand synthesis
3’
5’
replication fork
5’
3’

3’
5’
direction of lagging strand synthesis
 Strand Separation At The Replication Fork Causes
Positive
Supercoiling Of The Downstream Double Helix

• The strand separation process (unwinding the complementary Watson-

Crick DNA strands) causes overwinding ahead of the fork. Any DNA
that is overwound (or underwound) is said to be “supercoiled.”
Overwound DNA is positively supercoiled. The increasing torsional
stress needs to be dissipated in order for the fork to continue to unwind
so that replication can proceed. This is accomplished by DNA
topoisomerases, which cut the DNA strands, unwind them and reseal
the strands. As they do so they introduce negative supercoiling into the
DNA to compensate for the positive supercoiling. Gram-negative
bacteria, such as E. coli, Klebsiella pneumoniae, and Pseudomonas
aeruginosa, can be killed by fluoroquinolone antibiotics, which inhibit
DNA gyrase, a topoisomerase II. Topoisomerase II cuts both strands of
DNA, swivels them and rejoins them. Topoisomerase I cuts only one
strand, and relaxes negative supercoils.
 Strand separation at the replication fork causes positive
supercoiling of the downstream double helix

3’
5’

5’
3’

3’
• DNA gyrase is a topoisomerase II, which 5’
breaks and reseals the DNA to introduce negative
supercoils ahead of the fork
• Fluoroquinolone antibiotics target DNA gyrases in many
gram-negative bacteria: ciprofloxacin and levofloxacin (Levaquin)
 Movement Of The Replication
Fork
• As the replication fork moves further to the
left, opening up more DNA, another RNA
primer has to be synthesized.
 Movement of the replication fork

5’
3’ 5’

3’
 Movement Of The Replication Fork

• Each RNA primer (dashed red line) on the

lagging strand then serves as a starting
point for the initiation of DNA synthesis
(Okazaki fragment; solid red line).
Movement of the replication fork

5’ RNA primer
Okazaki fragment
RNA primer
• E. coli DNA polymerase III initiates at the RNA
primer, synthsizing DNA to fill in the gap up to the
next RNA primer. However, it falls off the template
DNA once it reaches the next RNA primer. At this
point, DNA polymerase I takes over. It contains a 5'
to 3' exonuclease activity that can remove the RNA
primer while it simultaneously adds DNA nucleotides
to the 3' end of the Okazaki fragment.
 RNA primer pol III
5’ 5’
3’

DNA polymerase III initiates at the primer and

elongates DNA up to the next RNA primer

5’ 5’
3’

newly synthesized DNA (100-1000 bases) pol I

(Okazaki fragment)
5’
3’

DNA polymerase I inititates at the end of the Okazaki fragment

and further elongates the DNA chain while simultaneously
removing the RNA primer with its 5’ to 3’ exonuclease activity
• However, DNA polymerase I cannot seal the gap
between the two adjacent Okazaki fragments. This
job is carried out by DNA ligase, which catalyzes
the formation of a 3', 5'-phosphodiester bond in an
ATP-dependent reaction. Thus, it takes one RNA
polymerase (primase), two DNA polymerases (III
and I), and DNA ligase to synthesize the lagging
strand. Once initiation occurs on the RNA primer,
the leading strand only requires DNA polymerase III
for its synthesis.
 newly synthesized DNA
(Okazaki fragment)
5’
3’

DNA ligase seals the gap by catalyzing the formation

of a 3’, 5’-phosphodiester bond in an ATP-dependent reaction
5’
3’
 Proteins At The Replication Fork In E. Coli

• This figure shows the proteins required for DNA synthesis at the
replication fork. In addition to the proteins required for leading and
lagging strand synthesis, there are several others that act upstream of
the replication fork. The act of unwinding the DNA double helix puts
torsional stress in the form of positive supercoils in the DNA upstream
of the fork. To overcome this, DNA gyrase, which is a topoisomerase
II, breaks and reseals the DNA in order to introduce negative
supercoils in the DNA, thus overcoming the positive supercoils. The
unwinding itself is carried out by the Rep protein, which is a helicase.
Finally, to keep the unwound strands single-stranded, they bind SSB
(single-strand binding protein).
 Proteins at the replication fork in E. coli

Rep protein (helicase)

3’
pol III
5’

5’
3’ G Primasome DNA ligase
C B
Single-strand
binding protein
(SSB)
pol III

DNA gyrase - this is a topoisomerase II, which

breaks and reseals double-stranded DNA to introduce
negative supercoils ahead of the fork
pol I
 Components Of The Replication Apparatus

• This table lists the proteins required for

replication of the E. coli chromosome, and
their activities.
 Components of the replication apparatus

dnaA binds to origin DNA sequence

Primasome
dnaB helicase (unwinds DNA at origin)
dnaC binds dnaB
dnaG primase (synthesizes RNA primer)
DNA gyrase introduces negative supercoils ahead
of the replication fork
Rep protein helicase (unwinds DNA at fork)
SSB binds to single-stranded DNA
DNA pol III primary replicating polymerase
DNA pol I removes primer and fills gap
DNA ligase seals gap by forming 3’, 5’-phosphodiester bond
 Properties Of DNA Polymerases

• The main DNA polymerases required for DNA replication in E. coli are DNA polymerases I and III.
They both have 5' to 3' polymerizing activity and 3' to 5' proofreading activity. DNA polymerase I
also removes the RNA primer and is also a DNA repair enzyme, and thus requires a 5' to 3'
exonuclease activity.
• In contrast to just three DNA polymerases in E. coli, human cells have at least five DNA
polymerases. The enzymes thought to be responsible for replication of nuclear DNA are DNA
polymerases alpha, delta, and epsilon. Alpha is associated with an RNA primase and it is thought
that these activities are responsible for synthesizing a short RNA-DNA primer. There is some
uncertainty as to the function of alpha in lagging strand synthesis. Since it seems to lack a 3’ to 5’
exonuclease activity, it may not be able to synthesize DNA with high fidelity and thus it may not be
the main lagging strand polymerase. If not, the main polymerase for the lagging strand may be
delta, which is also specifically responsible for synthesis of the leading strand. DNA polymerase
epsilon may function like the bacterial DNA polymerase I, by removing primers and extending the
Okazaki fragment. In humans, there is also a requirement for DNA ligase.
• Proliferating cell nuclear antigen (PCNA) has a role in both replication and repair. It is a
toroidal-shaped (donut) protein which encircles DNA and can slide bidirectionally along the duplex.
One of its functions is to serve as a processivity factor for DNA polymerase delta and epsilon.
PCNA holds the polymerase to the DNA template for rapid and processive DNA synthesis.
Recently, it has been discovered that PCNA also interacts with proteins involved in cell-cycle
progression.
 Properties of DNA polymerases

DNA polymerases of E. coli_

pol I pol II pol III (core)

Polymerization: 5’ to 3’ yes yes yes
Proofreading exonuclease: 3’ to 5’ yes yes yes
Repair exonuclease: 5’ to 3’ yes no no

DNA polymerase III is the main replicating enzyme

DNA polymerase I has a role in replication to fill gaps and excise
primers on the lagging strand, and it is also a repair enzyme
and is used in making recombinant DNA molecules

• all DNA polymerases require a primer with a free 3’ OH group

• all DNA polymerases catalyze chain growth in a 5’ to 3’ direction
• some DNA polymerases have a 3’ to 5’ proofreading activity
 Mutation
• This slide shows the three basic types of
mutational events and their frequencies.
We will be concentrating on "gene
mutations," which are base pair mutations
or small deletions or insertions.
 Mutation

Types and rates of mutation

Type Mechanism Frequency________

Genome chromosome 10-2 per cell division
mutation missegregation
(e.g., aneuploidy)

Chromosome chromosome 6 X 10-4 per cell division

mutation rearrangement
(e.g., translocation)

Gene base pair mutation 10-10 per base pair per

mutation (e.g., point mutation, cell division or
or small deletion or 10-5 - 10-6 per locus per
insertion generation
 Mutation rates* of selected genes

Gene New mutations per 106 gametes

Achondroplasia 6 to 40
Aniridia 2.5 to 5
Duchenne muscular dystrophy 43 to 105
Hemophilia A 32 to 57
Hemophilia B 2 to 3
Neurofibromatosis -1 44 to 100
Polycystic kidney disease 60 to 120
Retinoblastoma 5 to 12

*mutation rates (mutations / locus / generation) can vary

from 10-4 to 10-7 depending on gene size and whether
there are “hot spots” for mutation (the frequency at most
loci is 10-5 to 10-6 ).
 Many Polymorphisms Exist In The Genome

• What is the frequency of new germline mutations?

Consider the following: each sperm contains ~100
new mutations; a normal ejaculate has ~100 million
sperm; 100 X 100 million = 10 billion new mutations;
~1 in 10 sperm carries a new deleterious mutation; at
a rate of production of ~80 million sperm per day, a
male will produce a sperm with a new mutation in the
Duchenne muscular dystrophy gene approximately
every 10 seconds.
 Many polymorphisms exist in the genome

• the number of existing polymorphisms is ~1 per 500 bp

• there are ~5.8 million differences per haploid genome
• polymorphisms were caused by mutations over time
• polymorphisms called single nucleotide polymorphisms
(or SNPs) are being catalogued by the Human
Genome Project as an ongoing project
 Types Of Base Pair Mutations

• This slide illustrates the four basic types of base pair

mutations. Two of them result in the conversion of
one base pair to another (base pair substitution). The
others result in removal (deletion) or addition
(insertion) of one or more base pairs. (Note that a
transition mutation results when a pyrimidine on one
strand is converted to another pyrimidine on the same
strand. The complementary strand would see a
conversion from one purine to the other purine.)
Types of base pair mutations
normal sequence
CATTCACCTGTACCA
GTAAGTGGACATGGT
transition (T-A to C-G) transversion (T-A to G-C)
CATCCACCTGTACCA CATGCACCTGTACCA
GTAGGTGGACATGGT GTACGTGGACATGGT
base pair substitutions
transition: pyrimidine to pyrimidine
transversion: pyrimidine to purine

deletion insertion
CATCACCTGTACCA CATGTCACCTGTACCA
GTAGTGGACATGGT GTACAGTGGACATGGT
deletions and insertions can involve one
or more base pairs
 Spontaneous Mutations Can Be Caused By
Tautomers

• There are many different causes of mutations.

Spontaneous mutations (those that result from no
external cause) can occur simply by rearrangement of
bonds and by the repositioning of hydrogens in the
purine and pyrimidine bases. The common forms of
adenine and cytosine are the amino forms, which can
rearrange to the imino forms. The repositioning of
hydrogens changes their base pairing, hydrogen
bonding chemistry.
Spontaneous mutations can be caused by tautomers
Tautomeric forms of the DNA bases

Adenine

Cytosine

AMINO IMINO
 Tautomeric Forms Of The DNA Bases

• The common forms of guanine and thymine

are the keto forms, which can rearrange to
the enol forms.
 Tautomeric forms of the DNA bases

Guanine

Thymine

KETO ENOL
 Mutation Caused By Tautomer Of Cytosine

• This figure illustrates how a conversion from the amino to the imino
form of cytosine changes the locations of hydrogen donor and acceptor
groups, such that the imino form of cytosine base pairs with adenine
instead of guanine. As shown in the next figure, this will ultimately
lead to a transition mutation. As for the other tautomers, the imino
form of adenine "looks sort of like" a guanine and should be able to
form two hydrogen bonds with cytosine; the enol form of guanine
looks like an adenine and should be able to form three hydrogen bonds
with thymine; and the enol form of thymine looks like a cytosine and
should be able to form three hydrogen bonds with guanine. In all cases,
the wrong nucleotide will be inserted into the growing DNA chain.
 Mutation caused by tautomer of cytosine

Cytosine

Normal tautomeric form Guanine

Cytosine

Rare imino tautomeric form Adenine

• cytosine mispairs with adenine resulting in a transition mutation
 Mutation Is Perpetuated By Replication

• The conversion of a C-G base pair to a T-A base pair

takes two steps. If the tautomeric form of cytosine is
present during DNA replication, an adenosine will be
inserted into the daughter DNA strand (instead of the
normal guanosine). During the next round of DNA
replication, the adenosine then serves as a template
for the insertion of a thymidine in the new DNA
strand, resulting in a transition mutation (the
conversion of a C-G to a T-A).
 Mutation is perpetuated by replication

C G C G
• replication of C-G should give daughter strands each with C-G

C G C A
• tautomer formation C during replication will result in mispairing
and insertion of an improper A in one of the daughter strands

C A T A
• which could result in a C-G to T-A transition mutation in the next
round of replication, or if improperly repaired
 Chemical Mutagens

• Mutation can also occur by the action of

chemical mutagens. This figure shows how
oxidative deamination of cytosine converts it to
uracil, and how the oxidative deamination of
adenine converts it to hypoxanthine, both
processes altering the hydrogen bonding
specificities of these bases.
 Chemical mutagens

Deamination by nitrous acid

 Attack By Oxygen Free Radicals
Leading To Oxidative Damage

• The deoxyribose ring is also susceptible to

damage, which can be cleaved by oxygen
free radicals and break the phosphodiester
backbone of DNA.
 Attack by oxygen free radicals O
leading to oxidative damage
N
NH

• many different oxidative modifications occur NH N NH2

• by smoking, etc.
• 8-oxyG causes G to T transversions guanine
O
H
N
NH
O
NH N NH2
8-oxyguanine (8-oxyG)

• the MTH1 protein degrades 8-oxy-dGTP preventing misincorporation

• mutation of the MTH1 gene causes increased tumor formation in mice
 Ames test for mutagen detection

• named for Bruce Ames

• reversion of histidine mutations by test compounds
• His- Salmonella typhimurium cannot grow without histidine
• if test compound is mutagenic, reversion to His+ may occur
• reversion is correlated with carcinogenicity
 Thymine Dimer Formation By UV Light

• Sunlight is particularly damaging to DNA.

This figure shows the formation of a
thymine dimer, catalyzed by UV light. The
thymine dimer bridges two adjacent
thymine residues on the same DNA strand
Thymine dimer formation by UV light
 DNA Lesions

• As shown here, DNA is prone to many

different kinds of damaging reactions, any
of which can alter DNA function and cause
mutations if not repaired.
 Summary of DNA lesions
Missing base Acid and heat depurination (~104 purines
per day per cell in humans)

Altered base Ionizing radiation; alkylating agents

Incorrect base Spontaneous deaminations
cytosine to uracil
adenine to hypoxanthine

Deletion-insertion Intercalating reagents (acridines)

Dimer formation UV irradiation

Strand breaks Ionizing radiation; chemicals (bleomycin)

Interstrand cross-links Psoralen derivatives; mitomycin C

Tautomer formation Spontaneous and transient
 Mechanisms of Repair

• Mutations that occur during DNA replication are repaired when

possible by proofreading by the DNA polymerases

• Mutations that are not repaired by proofreading are repaired

by mismatch (post-replication) repair followed by
excision repair

• Mutations that occur spontaneously any time are repaired by

excision repair (base excision or nucleotide excision)
 Mismatch (Post-replication) Repair
(Reduces DNA Replication Errors 1,000-fold)

• For DNA to be repaired properly following the misincorporation of a

nucleotide into the newly synthesized DNA strand, the replication machinery
must have a means by which to distinguish between the "old" (template) strand
shown in black and the "new" (daughter) strand shown in red. After DNA
replication takes place, the newly synthesized DNA is methylated on certain
adenine bases. This, however, does not occur right away - there is a "window
of time" in which the newly synthesized DNA (in red) is not methylated.
Thus, if a mutation occurs in the new strand, the repair machinery can tell
which is the template strand (presumable the correct strand) and the new strand
(containing the mutation). It will then repair the mismatch by excision repair.
Once some time has passed, the new strand will also become methylated; at
that point it will not be possible to distinguish the correct nucleotide from the
incorrect nucleotide at the site of a base pair mismatch. Defects in mismatch
repair are a cause of hereditary nonpolyposis colon cancer (Thompson &
Thompson, Case 13). There are six mismatch repair genes that can be affected
in HNPCC.
 Mismatch (post-replication) repair
(reduces DNA replication errors 1,000-fold)

• the parental DNA strands are

methylated on certain CH3
adenine bases
CH3
• mutations on the newly
replicated strand are
5’ identified by scanning
3’ for mismatches prior to
CH3
methylation of the newly
replicated DNA

• the mutations are repaired

by excision repair mechanisms
• after repair, the newly CH3
replicated strand is methylated
 Excision Repair

• There are two types of excision repair: base excision repair (left) and nucleotide
excision repair (right). While they differ in their initial steps (top), they are
similar in the latter steps (bottom).
• If cytosine is deaminated forming uracil, the U can be recognized as being
an improper base in DNA by the enzyme, uracil DNA glycosylase. This
enzyme cleaves the uracil base from the phosphodiester backbone, and the space
is opened up by repair nucleases that remove a number of nucleotides from one
strand (the other strand has to be left intact to serve as the template for DNA
repair). The repair polymerase, DNA polymerase beta, then fills in the gap and
DNA ligase seals the last phosphodiester bond. The double strandedness of
DNA makes possible both DNA replication and DNA repair, because the
template strand always contains the information for the synthesis of a
complementary strand.
• Nucleotide excision repair occurs when the DNA lesion is larger, for
example when there is a thymine dimer. In this case, a special repair
excinuclease removes about 30 nucleotides, including the lesion. The DNA is
then resynthesized and ligated together as with base excision repair.
 Excision repair
deamination
ATGCUGCATTGA
TACGGCGTAACT
uracil DNA glycosylase thymine dimer
ATGC GCATTGA ATGCUGCATTGATAG
TACGGCGTAACT TACGGCGTAACTATC
repair nucleases excinuclease
AT GCATTGA AT (~30 nucleotides) AG
TACGGCGTAACT TACGGCGTAACTATC
DNA polymerase β DNA polymerase β
ATGCCGCATTGA ATGCCGCATTGATAG
TACGGCGTAACT TACGGCGTAACTATC
DNA ligase DNA ligase
ATGCCGCATTGA ATGCCGCATTGATAG
TACGGCGTAACT TACGGCGTAACTATC
Base excision repair Nucleotide excision repair
 Deamination Of Cytosine Can Be Repaired

• Deaminated cytosine is fairly straightforward to repair because uracil is

recognized as being "foreign" in the DNA molecule. However, many
cytosines are reversibly methylated at CG sites (or CpG sites to
emphasize that the C and G are adjacent nucleotides on the same DNA
strand). It is believed that this methylation functions to regulate gene
expression because 5-methylcytosine (5mC) residues are often
clustered near the promoters of genes in so-called "CpG islands." The
problem that arises from these methylations is that subsequent
deamination of a 5mC results in the production of thymine, which is
not foreign to DNA. Thus, while a base pair mismatch is seen in the
DNA by the repair machinery, it does not know which of the two
strands to repair (50% of the time it will make the right choice and
50% of the time it will make the wrong choice). As such, 5'-mCG-3'
sites are "hot-spots" for mutation.
 Deamination of cytosine can be repaired

cytosine uracil

Deamination of 5-methylcytosine cannot be repaired

5’-methyl- thymine
cytosine

More than 30% of all single base changes that have been detected
as a cause of genetic disease have occurred at 5’-mCpG-3’ sites
 Correlation Between DNA Repair
Activity And
The Life Span Of The Organism

• There is a direct correlation between DNA repair

enzymatic activity and the life span of
organisms, suggesting that DNA repair activity
slows down cellular senescence and that cellular
senescence is caused by mutations in DNA.
Defects in DNA repair or replication can lead to
a number of abnormalities. See Baynes &
Dominiczak, pg. 601.
Correlation between DNA repair
activity in fibroblast cells from
various mammalian species and
the life span of the organism

100
human
elephant

cow
Life span

10

hamster
rat
mouse
shrew
1
DNA repair activity
 Defects In DNA Repair Or Replication

• As shown here, there are a number of defects of

DNA replication and repair that are associated
with a predisposition to cancer and other
disorders. This highlights the importance of high
fidelity DNA replication and for the presence of
DNA repair mechanisms for normal cell function
and longevity. See Baynes & Dominiczak, pg.
315.
Defects in DNA repair or replication
All are associated with a high frequency of chromosome
and gene (base pair) mutations; most are also associated with a
predisposition to cancer, particularly leukemias
• Xeroderma pigmentosum
• caused by mutations in genes involved in nucleotide excision repair
• associated with a >1000-fold increase of sunlight-induced
skin cancer and with other types of cancer such as melanoma
• Ataxia telangiectasia
• caused by gene that detects DNA damage
• increased risk of X-ray
• associated with increased breast cancer in carriers
• Fanconi anemia
• caused by a gene involved in DNA repair
• increased risk of X-ray and sensitivity to sunlight
• Bloom syndrome
• caused by mutations in a a DNA helicase gene
• increased risk of X-ray
• sensitivity to sunlight
• Cockayne syndrome
• caused by a defect in transcription-linked DNA repair
• sensitivity to sunlight
• Werner’s syndrome
• caused by mutations in a DNA helicase gene
• premature aging