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Mutation in germ line would destroy the

species

Mutation in soma would destroy the

individual.

Maintenance of the correctness of the DNA sequence

is definitely crucial for living organisms. Keeping the
error rate as low as 10-10 is so expensive.

Replication errors
The nature of mutations
Errors Point mutations:

Inaccuracy in DNA replication 1. Transitions (pyrimidine to pyrimidine, purine to
purine)

Chemical damage to the genetic material (environment)
(environment)

Lesions (arose from spontaneous damage)
2. Transversions (pyrimidine to purine, purine to

Damage (caused by chemical agents and radiation
pyrimidine)

To repair an error or damage

 First, Detect the errors

 Second, Mend/repair the errors or lesions in a way

to restore the original DNA sequence.

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Insertions
Deletions
Gross rearrangement of chromosome.
These mutations might be caused by insertion by
transposon or by aberrant action of cellular
recombination processes.
Some replication errors escape proofreading

Expansions of repeated DNA sequences represent a
newly discovered type of human mutation.

Such mutations and their phenotypic consequences
occur when many extra copies are made of three
basepair DNA sequences normally found in low copy
number in or near genes.

Fragile X mental retardation (involves multiple
replications of the nucleotide repeat CGG )

Myotonic Dystrophy (expansion involving excessive
copying of CTG repeat)

Huntington Disease (autosomal dominant, was found to
result from expansion of a triplet repeat (CAG) )
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Rate of spontaneous mutation at any given site
on chromosomal ranges from 10-6 to 10-11 per
round of DNA replication, with some sites
being “hotspot” .
Mutation-prone sequence in human genome are
repeats of simple di-, tri- or tetranucleotide
sequences, known as DNA microsatellites.
These sequences:
(1) are important in human genetics and disease,
(2) hard to be copied accurately and highly
polymorphic in the population.
The 3’-5’ exonuclease activity of replisome only
improves the fidelity of DNA replication by a
factor of 100-fold.
The misincorporated nucleotide needs to be
detected and replaced, otherwise it will cause
mutation.
Generation of Mutation
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Mismatch repair removes errors that DNA damage

escape proofreading
Increase the accuracy of DNA synthesis for 2-3
orders of magnitudes.
Two challenges:
(1) rapidly find the mismatches/mispairs,
(2) Accurately correct the mismatch
Mutations arise not only from errors in replication, but also
from damage to the DNA.
Environment factors – radiation and so-called
mutagens (chemical agents)
Spontaneous damage from action of water
Most common hydrolytic damage is deamination of the base
cytosine, generating the unnatural base uracil in DNA.
Uracil pairs with adenine, and so introduces that
base instead of guanine as directed by cytosine.
Deamination of adenine to hypoxanthine (pairs with cytosine
instead of thymine).

Deamination of guanine to xanthine (pairs with cytosine but

only two hydrogen bonds)

DNA also undergoes depurination by spontaneous hydrolysis

of N-glycosyl linkage, and this produces an abasic site
(deoxyribose lacking a base) in the DNA.

Deamination C→
→U
Hydrolysis creates apurinic deoxyribose

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DNA is damaged by alkylation, oxidation Gamma radiation and X-rays (ionizing radiation) cause double-
and radiation strand breaks and are particularly hazardous (hard to be
repaired).
In alkylation, methyl and ethyl
Ionizing radiation can directly attack the deoxyribose in the
groups are transferred to reactive
DNA backbone, or generating ROS.
sites on the bases and to
phosphates in the DNA backbone.
Mutations are also caused by base analogs and intercalating
agents.
DNA is subjected to attack by

Base analogs: similar enough to the normal bases to be
Reactive Oxygen Species (ROS)
processed by cells and incorporated into DNA during
and UV light. UV radiation causes
replication.
the photochemical fusion of two

But they base pair differently, leading to mistake during
pyrimidines that occupy the
replication.
adjacent positions on the same

The most mutagenic base anolog is 5-bromouracil.
polynucleotide chain. In the case

Intercalating agents: flat molecules that interact with
of two thymines, the fusion is
the normal bases in DNA through hydrogen bonds and base
called thymine dimers, which
stacking.
contains a cyclobutane ring.

Base analogs

Some damages, such as thymine dimer, nick or breaks in

the DNA backbone, create impairments to replication
or transcription

Some damages creates altered bases that has no
effect on replication but cause mispairing, which in turn can
Intercalating agents Can cause deletions
be converted to mutation.
and additions in
genes, thus shifting
the coding sequence
out of range.

Recombination repair/

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Mismatch repair
To repair mismatched bases, the system
has to know which base is the correct
one.
In E. coli, this is achieved by a special
methylase called the "Dam methylase",
which can methylate all adenines that
occur within (5')GATC sequences.
Immediately after DNA replication, the
template strand has been methylated,
but the newly synthesized strand is not
methylated yet. Thus, the template
strand and the new strand can be
distinguished.

MutS, also known as the "mismatch recognition" enzyme, is
essential for the DNA mismatch repair biological pathway.

It recognizes base-base mismatches and small nucleotide
insertion/deletion mispairs generated during DNA synthesis
or damage caused by various agents.

MutS scans the DNA, recognizing the mismatch from the
distortion they cause in the DNA backbone

MutS embraces the mismatch-containing DNA, inducing a
pronounced kink in the DNA and a conformational change
in MutS itself
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In E.coli, MutS/MutL and
MutH are essential for DNA
mismatch repair.
Eukaryotic cells also repair
mismatches and do so
using homologs to MutS
(MSH) and MutL (MLH).
The underlying
mechanisms are not the
same and not well
understood.

The repairing process begins with the protein MutS which
binds to mismatched base pairs.

Then
Then,, MutL is recruited to the complex and activates MutH
which binds to GATC sequences.

Activation of MutH cleaves the unmethylated strand at the
GATC site.

Subsequently
Subsequently,, the segment from the cleavage site to the
mismatch is removed by exonuclease (with assistance from
helicase and SSB proteins).

If the cleavage occurs on the 3' side of the mismatch, this
step is carried out by exonuclease I (which degrades a single
strand only in the 3' to 5' direction).

If the cleavage occurs on the 5' side of the mismatch,
exonuclease VII or RecJ is used to degrade the single
stranded DNA. The gap is filled by DNA polymerase III and
DNA ligase.
ligase.
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Different exonucleases are used to remove ssDNA

between the nick created by MutH and the
mismatch.
If GATC is located on 5’ of mutation
If GATC is located on 3’ of mutation
Base excision
• DNA's bases may be mutated
by deamination or alkylation.
• The position of the modified
(damaged) base is called the
"abasic site" or "AP site".
• In E.coli, the DNA glycosylase
can recognize the AP site and
remove its base.
• Then, the AP endonuclease
removes the AP site and
neighboring nucleotides.
• The gap is filled by DNA
polymerase I and DNA ligase.

Nucleotide excision

In E. coli, proteins UvrA, UvrB,

and UvrC are involved in
removing the damaged
nucleotides (e.g., the dimer
induced by UV light).

Photoreactivation
The gap is then filled by DNA
polymerase I and DNA ligase.
Methyltransferase action

In yeast, the proteins similar to

Uvr's are named RADxx ("RAD"
stands for "radiation"), such as
RAD3, RAD10. etc.

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Photoreactivation Methyltransferase

Photoreactivation directly reverses the formation of pyrimidine

dimers that result from UV irradiation. Direct removal of the methyl group from the methylated O6-
methylguanine. The methyl group is transferred to the protein
The enzyme DNA photolyase captures energy from light and uses itself, inactivating the protein. Costly!!
it to break the covalent bonds linking the adjacent pyrimidines,
pyrimidines,
and mending the DNA directly.

Translesion DNA synthesis enables

replication to proceed across DNA damage
 Error-prone repair***
 Occurs when the above repairs are not efficient
enough so that a replicating polymerase
Translesion encounters a lesion
 Translesion synthesis is also called a fail-safe or
polymerase and last resort mechanism.

process 1. Translesion synthesis is catalyzed by a specialized

class of DNA polymerases that synthesize DNA
directly across the damage site.
2. Translesion polymerase is produced by cell in
response to the DNA damage
3. Translesion polymerases are expressed as part of
the SOS response pathway.

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Translesion DNA
synthesis

Upon encountering a
lesion in the template
during replication, DNA
polymerase III with its
sliding clamp dissociates
from the DNA, and is
replaced with translesion
polymerase, which extends
DNA synthesis across the
thymine dimer.

Translesion polymerase is
then replaced by DNA
polymerase III.