GENETICS 380:

DNA Mutation and Repair

Mutations Are Heritable Changes

In The DNA Sequence
The most common (often called normal) sequence is the wild-type
sequence; all changes in the DNA base sequence are referred to as
mutations for the purposes of this unit
Mutations can be helpful, neutral or detrimental to the organisms effort
to thrive in its environment, depending on the demands the
environment makes on the organism
Mutations are necessary for evolution
Mutations are one of the mechanisms whereby organisms acquire new
characteristics

Forward, Reverse And Suppressor Mutations

Reverse mutation, aka reversion, replaces the original (forward)
mutation and restores the wild-type genotype and phenotype
Suppressor mutation is at another site, but it compensates for the
mutation and restore the wildtype phenotype

Suppressor Mutations
Suppressor mutations can hide or suppress the effects of other mutations
The individual is a double mutant, but has a normal phenotype
Some suppressor mutations are intragenicwithin the same genes
coding sequence

Suppressor
Mutations
Some suppressor
mutations are intergenic
in a second genes coding
sequence
Ex. C G nonsense
mutation creates a UAG
STOP codon where there
was a UAC tyrosine codon

Suppressor
Mutations
A mutation changes the
anticodon of a tyrosinecarrying tRNA to AUC,
which can bind to the
STOP codon UAG and
cause the ribosome to put
tyrosine into the
polypeptide
ProblemThe AUC tRNA
is supposed to bring in the
releasing factor that stops
translation at UAG codons
This will cause a failure to
stop translation in other
genes with the same STOP
codon

Germline Versus Somatic Mutations

Germline mutationsPresent in either (or both) the sperm or the egg that
made the individual, therefore present in every cell the individual has

Somatic mutationsArise after fertilization, during cell

replication/division/differentiation/migration, therefore only present in a
subset of the individuals cells

Somatic Mutations Get Passed Down To One

Fourth Of The Descendants Of The Original Cell

One chromosome
2 DNA strands

After mitosis,
one daughter
cell has the
mutation on one
strand

Mutation arises
during DNA
replication in S
phase

After next
S phase
All the
descendants of
this cell have
the mutation

After mitosis

Somatic Mutations Can Be

Passed Down To Offspring

If the mutation exists in the cells that make sperm (spermatogonia) or eggs
(oogonia), the mutation can be packaged into a gamete
From the offsprings perspective, this is a germline mutationit was present
in one of the gametes that made the child, and it will exist in every cell the
child has
From the parents perspective, it is a somatic mutationit occurred after
fertilization and only existed in a subset of the parents cells

Some Mutations Are Conditional

Conditional mutationSome mutations only cause consequences under

certain conditions
Ex. Many people who have a glucose-6-phosphate dehydrogenase
deficiency (an X-linked recessive disease) will experience flare-ups (main
symptom = hemolytic anemia) if they eat fava beans

Some Mutations Involve Large Pieces Of

One Or More Chromosomes
Structural Abnormalities
Deletions and duplications range in size from single nucleotides to pieces
of the chromosome that are large enough to include many genes in them
Inversions and translocations that rearrange large pieces of chromosomes
Numerical Abnormalities
Entire chromosomes can be added or deleted
More on this when we discuss cytogenetics

The Smallest Mutations Are

Single-Nucleotide Point Mutations
Point mutation = substitution, deletion or addition of a single nucleotide
Transition = purine-purine substitution or pyrimidine-pyrimidine substitution
Transversion = purine-pyrimidine substitution or vice versa

Missense mutationsingle-nucleotide substitution causes one amino

acid to replace another
Nonsense mutationsingle-nucleotide substitution creates a STOP codon
at the site of the mutation, and truncates the protein

Point Mutations Have Variable Effects

On The Organisms Phenotype
Not all mutations change the activity of a protein--Those that do not are
often called benign polymorphisms
Some mutations are silent mutationsthey do not change the organisms
phenotype
Synonymous mutationdoes not change the amino acid content of the
protein
Neutral mutationchanges the amino acid content of the protein, but has
no functional consequences

Point Mutations Have Variable Effects

On The Organisms Phenotype
Those that alter protein activity may increase or decrease it, by a small
or large amount
If the amino acid substitution, deletion or addition changes the proteins
3D shape or ability to move as it works, it will disrupt the proteins
function
The degree to which it alters the proteins ability to bend and move
determines how severely it affects protein activity

Amino Acids Differ In Size, Polarity,

Bendability And Electrical Charge
Arginine, histidine and
lysine are positively charged
Glutamic acid and aspartic
acid are negatively charged

Some of the neutral ones are polar, some

are nonpolar

A Proteins 3D Shape Is Determined By

Interactions Among Amino Acids

Some Amino Acid Substitutions Will Disrupt The

Protein's Function; Others Will Not
Alanine replaces glycine
will probably be tolerated

Arginine replaces glycine

will probably not be tolerated

Small
uncharged
Small
uncharged

Small
uncharged

Large
positive charge

Some Mutations Are Synonymous

The genetic code is partially redundant (often referred to as degenerate)
Some base substitutions do not change the amino acid content of the
protein; these are called synonymous mutations

Synonymous And Intronic Mutations May

Change The Pattern Of RNA Splicing
Synonymous mutations may change the splicing of the mRNA (intronic
mutations may also)
Recall that almost all human introns begin with GT and end with AG
If a mutation creates a new splice site (ex. a C T mutation changes a GC
that lies in an exon to a GT), the spliceosome will splice the pre-mRNA
abnormally
This will either add intronic nucleotides to the mRNA or delete exonic
nucleotides from the mRNA
Either way, this changes the instructions the ribosome gets, and it will
incorporate the wrong amino acids into the polypeptidethis almost
always abolishes the activity of the protein

Insertions And Deletions May Shift The

Ribosomes Reading Frame
The ribosome reads the mRNA in 3-base codons
An insertion or deletion in the mRNA involving a multiple of three
nucleotides will not shift the ribosomes reading framethe amino acid
sequence will be normal before and after the mutation
Ex. Deletion of a codon results in loss of one amino acid
Normal sequence TAC TGG CTA GCT CCU
tyr trp leu ala pro
3 bp deletion

TAC TGG GCT CCU

tyr trp ala pro

The amino acid sequence is normal before and after the deletion

Insertions And Deletions May Shift The

Ribosomes Reading Frame
If the insertion or deletion does not involve a multiple of three bases,
the ribosomes reading frame gets shifted
The ribosome will incorporate the wrong amino acids, until it hits a
STOP codon (usually fairly soon)
Normal sequence TAC TGG CAT TCT CCT
tyr trp his ser pro
1 bp deletion

TAC TGG ATT CTC CT

tyr trp ile leu leu

All the amino acids after the deletion are abnormal

Mutations In The Regulatory Sequences Affect

The Rate Of Protein Production
Coding sequence mutations can change the activity of each molecule
of the protein
Mutations in the promoter and other regulatory sequences affect the
rate at which the gene makes its protein, i.e. the number of protein
molecules in the body

Trinucleotide Repeats Can Expand

And Disrupt A Genes Activity
There are some places in the genome where three bases get repeated
(trinucleotide repeats), with different people often having a different
number of repeated units in the string
Trinucleotide repeats are found in both coding regions and regulatory
sequences
These trinucleotide repeats can expand during meiosis
The larger the repeated string gets, the more it disrupts gene function
Expansions are most common, but sometimes the repeat can contract
Whether it expands or contracts often depends on the parent of origin
more likely to contract during spermatogenesis than oogenesis

Trinucleotide
Repeats Can
Cause Strand
Slippage
In this case, the newly
synthesized strand forms a
hairpin loop
This causes DNA polymerase
to replicate that portion of the
template strand again
When the newly synthesized
strand is used as a template
itself, both strands end up
having the expanded repeat

Fragile X Syndrome Is Due To

An Expanding CGG Repeat In
The X Chromosome (Xq28)
Chromosomes from cells that are grown in
culture will show fragile sites, where they look
stretched out as if the chromosome was
about to break
There are common fragile sites all over the
genome, in everyones chromosomesthese
are not associated with disease
There are also rare fragile sites that are
associated with diseasesex. Fragile X
mental retardation syndromefragile site
depicted here

Fragile X Syndrome Is Due To An Expanding

CGG Repeat In The X Chromosome (Xq28)
The Fragile X Syndrome CGG repeat is located in the 5' untranslated
region of the FMR1 gene
Unaffected individuals have 5-44 CGGs in the string
45-54 repeats is a grey areasometimes these can expand
Those with 55+ CGGs often see the repeat expand during meiosis
When there are greater than 200 repeats, the CGG repeat and the FMR1
gene promoter get methylated, which silences the gene

Fragile X Syndrome Is Due To An Expanding

CGG Repeat In The X Chromosome (Xq28)

The FraX trinucleotide repeat can also contract during meiosis

The repeat is more likely to contract during Dads spermatogenesis than
Moms oogenesis
There are asymptomatic male carriers who pass a repeat down to their
daughters that is smaller than the repeat they have in their genome
The daughters may not be affected, but will pass on an expanded repeat
to some of their children; the repeat will be big enough to affect the
children

Fragile X Syndrome

Trinucleotide Repeat Expansion Causes

Anticipation At The Clinical Level

The larger a repeat gets, the more severely it disrupts gene activity
When the repeat expands through meiosis, subsequent generations are affected
earlier and more severely than previous generations
At the clinical level, this is known as genetic anticipation

Anticipation In Three Generations

With Myotonic Dystrophy

Transposable Elements Can Affect Gene Activity

Transposable elements (aka transposons) can move from one place to

another within the same cells DNA
They can cut and paste, (nonreplicative transposition) and leave their
original place to insert elsewhere, or they can make a copy of themselves
and copy and paste (replicative transposition) themselves into another
location

Approx. 45% of the human genome has come from transposons

All Organisms Genomes

Contain Transposable Elements
Most transposable elements have inverted terminal repeats at their
ends (shown in green in the figure below)
ex 5-ACAGTTCAGCTGAACTGT-3
3-TGTCAAGTCGACTTGACA-5

This enables them to form loops that help them break out

Inverted Repeats Help Transposable Elements

Break Out Of Their Places

The Enzyme Transposase Makes

Staggered Cuts In The DNA
This creates short single-stranded overhangs that are complementary
to each other
The transposon inserts between the ends of the cut

Transposable
Elements
Generate Direct
Flanking
Repeats When
They Insert
The inverted terminal
repeat is part of the
transposon
The direct flanking
repeat is not part of the
transposonit is
created as a result of
the transposition
process

Transposable Elements Generate Direct Flanking

Repeats When They Insert

If you were searching through a genome for transposons, you would see
the terminal inverted repeat flanked by the direct repeat

Retrotransposons Use Reverse Transcription

Transposable Elements Can Disrupt Genes

Transposable
Elements Can
Disrupt Genes
In this case, the transposable
element is inserted in the genes
regulatory sequences
This shuts the gene down,
causing the grapes to stop
producing the anthocyanin
pigments that make them look
black, making white grapes
Later, a portion of the
transposable element leaves,
partially restoring gene function
and allowing the grapes to
produce enough anthocyanins to
look red

Transposable Elements Can Jump Back

Out And Restore Gene Function
In corn, there is a transposable element that contains an Ac element and
a Ds element
The Ac element produces transposase, while the Ds element does not
By producing transposase, the Ac element can cause the Ds element to
transpose into the pigment-making genes sequence, disrupting pigment
formation and causing the kernels to be yellow

Transposable Elements Can Jump Back

Out And Restore Gene Function

Transposable Elements Can Jump Back

Out And Restore Gene Function
The Ds element can transpose back out again, thereby restoring the
function of the pigment gene and allowing the cell to produce purple
pigment again
This happens one cell at a time; as that cell continues to undergo mitosis
in the developing kernel, the cells it gives rise to also produce purple
pigment; this causes purple speckles in the kernel
The larger speckles represent a group of cells that have descended from
a cell in which the Ds element transposed out of the pigment gene early
in the development of the kernel, when there were still many rounds of
cell replication and division to go before the kernel was completely
developed
Smaller speckles represent cells that have descended from a cell in
which the Ds element transposed out of the pigment gene at a later time

Transposable Elements Can Jump Back

Out And Restore Gene Function

Transposable Elements Can Also Activate Genes

Inactive gene

PRO

PRO

Transposable element has sequence

that is capable of acting as a promoter

Activated gene
RNA

Transposable Elements Can Cause

Chromosome Rearrangements That
Disrupt The Activity Of Genes

Transposable Elements Can Cause

Chromosome Rearrangements That
Disrupt The Activity Of Genes

Transposable Elements Can Cause

Chromosome Rearrangements

Regulating The Activity Of Transposons

The transposable elements produces the enzyme transposase, which
is what enables transposition
In many cases, the cell methylates the DNA in the region of the
transposon, which inhibits production of transposase
In some cases, the cell uses interfering RNAs to interrupt transposase
production

Spontaneous And Induced Mutations

Spontaneous Mutations
Errors during DNA replication
Hydrolytic and other reactions can cause base changes in DNA after
replication
Induced Mutations
Environmental agents such as UV light or radon
Chemical exposures
Metabolic byproducts such as the superoxide ion O2Agents that induce mutations are called mutagens

A Genes Mutation Rate Is Influenced By Factors That

Vary By Gene And Species
Spontaneous DNA mutation rate (varies from gene to gene)
Exposure to dietary, environmental and lifestyle factors
Capacity to repair mutations--DNA polymerase has 3-5
exonuclease activity so it can remove an incorrect base
Some mutations convey advantages in some environments
Stressful environment may increase mutation rate

Bases Exist In
Protonated
And
Nonprotonated
Forms
Shifting the proton
changes the number
and position of
hydrogen bonds the
base makes
This causes the base
to pair with a different
base than it usually
pairs with

Incorporation
Errors
Arise During
DNA
Replication
The ability to pair with
an atypical base
because of shifts in
the arrangement of
hydrogen bonds is
sometimes referred to
as wobble or non
Watson-Crick
basepairing

Incorporation Errors Lead To Replication Errors

During the next cell cycle, the strands with the mismatched bases serve
as templates for DNA replication
Replication proceeds properly, resulting in one of the two new chromatids
having the wrong base on both strands of its DNA

Depurination: Hydrolysis Removes The Purine

Ring From Adenines And Guanines
The N-glycosidic bond that holds the base to the sugar is cleavedthe
base is removed but the sugar/phosphate remains

Usually corrected quickly, but sometimes an A gets incorporated across from

the depurinated site during the next round of replicationthis results in a
mutation, because the base that belongs there is either a T or a C

Deamination Converts Between Pyrimidines

Pyrimidines (C, U and T) can be deaminated by hydrolysis of the NH 2
group; the reaction doesnt require an enzyme
Some Cs exist in methylated form; they get deaminated to yield Ts
Unmethylated Cs get deaminated to yield Us

Deamination Followed By Replication

A
T
G
C

T
A
C
G

1ST round
A T replication A T

T A
G C
U G

Deamination
converts C to U

T A
G C
U A

A
T
G
C

T
A
C
G

2nd round
replication

A
T
G
U

T
A
C
A

A
T
G
T

T
A
C
A

Many Chemical Mutagens Change The

Number Of Hydrogen Bonds The Base Makes

5-Bromouracil Replaces A Thymine

But Bonds With A Guanine

Reactive Oxygen Species Also

Change Base Pairing
Reactive oxygen species (ex. superoxide ion O2-) oxidize guanine to 8oxoguanine, which pairs with A, rather than C, as G should
Reactive oxygen species (ex. superoxide ion O2- ) are created by
normal metabolism

Intercalating
Agents Insert
Themselves
Between
Nucleotides
Intercalating agents increase
the distance between
neighboring basepairs,
confusing the DNA
polymerase and causing
insertions and deletions in
the DNA
Includes some common DNAvisualizing agents, ex. acridine
orange, ethidium bromide

Ultraviolet (UV) And Ionizing

Radiation Break The DNA Strands
Both UV radiation
(from the sun) and
ionizing radiation
(from X rays or radon
in homes) cause your
cells to form highly
reactive free radicals
These free radicals
can cause breaks in
the DNA
The attempt to fix the
damage may result in
mutations arising

The Suns UV Rays Cause Pyrimidine Dimers

Bonds form between neighboring Cs or Ts in the same DNA strand

This brings the two neighboring nucleotides closer together, leaving
the bases unable to bond with the bases from the other DNA strand
ACTTGC
| | | | | |
T G AAC G

UV rays

A C T= T G C
| |
| |
T GA AC G

This prevents DNA replication, which prevents the cell from going
through the cell cycle, causing the cell to go into apoptosis
This is why UV light kills bacteria and is a good water sterilizer
Eukaryotes have a special DNA polymerase eta that puts AA across from
the pyrimidine dimer and restores the DNA
If the dimer is not a T-T dimer, this will lead to mutation after replication

Base Mismatch Repair

DNA polymerase III proofreads itself, but still makes an error every approx.
10,000-100,000 nucleotides (104 105)
After the repair systems fix what they can, the frequency of errant
nucleotides is only approx. 1 in 1 billion (109) nucleotides
Mismatched bases and loops such as those that lead to deletions and
duplications form bubbles in the DNA double helix, which are recognized by
the repair systems
At the bubble, the cell must recognize which of the two strands is the one
that needs to be taken out
After replication, the As in GATCs get methylated, but theres a delay, so
immediately after replication, the newly synthesized DNA is unmethylated,
while the template DNA is methylated

Methylation Differentiates The

Old DNA Strand From The New One

Direct Repair
Some mutations, ex. pyrimidine dimers, can be repaired directly by
chemical reactions that restore the original base without removing anything

Ex. photolyase uses light energy to break the bonds in pyrimidine dimers,
which enables the bases to make their proper bonds with their
complementary bases again

If guanine gets methylated to O6-methyl guanine, the enzyme

06-methylguanine DNA methyltransferase removes the methyl group

Deaminations Are Repaired By Base Excision

Deaminations Are Repaired By Base Excision

Deaminations Are Repaired By Base Excision

Deaminations Are Repaired By Base Excision

Base Excision Repair Is Error-Prone

In eukaryotes, DNA polymerase beta, which has no proofreading

ability, fills in the gap
DNA polymerase betas error rate is high enough to leave approx. 10
new mutations uncorrected per day

Sometimes the error leaves DNA ligase unable to seal the gap
The AP endonuclease will return and excise the nucleotide, allowing
DNA polymerase beta another chance to put the right nucleotide in

Nucleotide
Excision Repair
Sometimes the cell
removes a short string of
nucleotides
Same steps as base excision:
1. Recognize problem
2. Remove bad nucleotide(s)
3. Replace with proper
nucleotide(s)
4. Seal with DNA ligase

endonuclease

DNA Damage
Bypass

Replication stalls briefly

at the gap, then resumes
after a short time on the
other side of the gap

DNA Damage
Bypass
The corresponding portion of the
other DNA strand is excised and
inserted into the gap
This fixes the original gap but
leaves a new gap

DNA polymerase fills in the gap

that was created by the excision
The newly synthesized strand is
available as a template

Repairing Double-Stranded Breaks

When there is a double-stranded break, there are three ways in which it
can be resolved:
Nonhomologous end joiningThe ends simply join back together
This results in there being a deletion in the chromosome
There are two specific mechanisms for double-strand gap repair
In both cases, the homologous chromosome is used as a template to fill in
the gap on one of the broken strands
The gap in the other strand may be filled using either the newly repaired
strand or the homologous chromosome as the template

The Newly
Synthesized
Strand
May Be Used To
Fill The Gap In
The Other
Strand
The light blue strand uses a
red strand as a template to
fill its gap

The light blue strand then

acts as a template to fill the
gap in the dark blue strand

The Homolog May Be Used To

Fill The Gap In The Other Strand

The Homolog May Be Used To

Fill The Gap In The Other Strand

Good Practice Questions From Chapter 18

Comprehension Questions1-7, 9, 10, 12, 15, 16
Application Questions34, 36, 42

Answers Not Given By The Textbook

Comprehension Questions
1. Transition = purine to purine, or pyrimidine to pyrimidine; transversion =
purine to pyrimidine or pyrimidine to purine.
2. The size of the repeat increases from one generation to the next, as
does the effect the repeat has on gene function. This causes subsequent
generations to be more severely affected, and at younger ages, than
previous generations.
3. Missense = one amino acid substituted for another; nonsense creates a
STOP codon at the site of the mutation; Silent doesnt change the amino
acid sequence of the protein; neutral changes the amino acid sequence
but not the activity of the protein

Answers Not Given By The Textbook

Comprehension Questions
4. A second mutation in the codon could restore the original amino acid; a
frame-shifting insertion or deletion could be followed by a deletion or
insertion (respectively) that restores the reading frame
5. Strand slippage; unequal crossovers due to repeated sequences
causing misalignment of chromosomes
6. They get incorporated into the DNA, then pair during DNA replication
with the wrong nucleotide.
7. Alkylating agents add methyl or ethyl groups; nitrous acid deaminates
cytososine; hydroxylamine add an OH group to cytosine. All cause the
base to pair with a base other than the one it is supposed to pair with

Answers Not Given By The Textbook

Comprehension Questions
9. Terminal inverted repeats and the transposase or reverse transcriptase
gene as part of the transposon. Flanking direct repeats around it after it
has inserted into a new location.
10. Transcription produces RNA, then reverse transcription creates a DNA
copy of the sequence, which then translocates into a new spot in the
genome.
12. The Ac element produces transposase. This causes the Ds element to
insert itself into the pigment-producing gene, preventing pigment
production and causing the kernels to be yellow instead of purple. Later,
the Ac element can cause the Ds element to transpose back out of the
gene in certain cells, allowing the gene to resume making pigment in those
cells only. Replication and division create more pigment-producing cells
from those that have lost the Ds element. The earlier in the development of
the kernel the transposon jumps out, the more opportunity for replication
and division to produce descendants of that cell, leading to larger
pigmented spots around that cell.

Answers Not Given By The Textbook

Comprehension Questions
15. Mismatch repairMismatched bases on the new (unmethylated)
strand are removed and replaced by DNA polymerase.
Direct repairOriginal base and bonds are restored by enzymes such as
photolyase or O6-methylguanine DNA methyltransferase.
Base excision repairglycosylase removes the damaged base, AP
endonuclease removes the rest of the nucleotide, DNA polymerase
replaces the nucleotide, DNA ligase knits it all together
Nucleotide excision repairSystem recognizes the mismatch, nucleases
and helicases remove a portion of the new (unmethylated) strand, DNA
polymerase replaces the nucleotides, DNA ligase knits it all together

Answers Not Given By The Textbook

Comprehension Questions
16. Homologous recombination requires a sister chromatid and a complex
sequence of invasion, displacement and synthesis of new DNA.
Nonhomologous end joining simply joins the broken ends.
Application Questions
34. The white eyes are due to a transposable element that interrupts the
function of the gene that makes the red pigment. Red spots represent the
cells that have descended from cells that have had the transposon
transpose back out, restoring pigment production.