DNA MUTATION

Lectures -part 1
 What is mutation?
• DNA is a highly stable molecule that replicate with
amazing accuracy, but changes in DNA structure, and
errors in DNA replication do occur that results in mutation
if not corrected
• Term ‘mutation’ refers to an inherited change in
the nucleotide sequence of a short region of a
genome.
• All cells posses DNA repair enzymes that attempt to
minimize the number of mutations that occur.
• They work in two ways:
q Some are pre-replicative and search the DNA for
nucleotides with unusual structures, these nucleotides
being replaced before replication occurs
q Others are post-replicative and check newly synthesized
DNA for errors, correcting any errors that they find
If not corrected, the phenotype as a result
of mutation on a gene can be reflected
 Effects of mutation
• Positive sides: mutations are essential to the continuity of
life, they provide the variation that enables species to
change & adapt to their environment
• Mutations are foundation for evolutionary change
• Mutations provide allelic variations

• Negative sides: New mutations are much more likely to be

harmful rather than beneficial to the individual
• Genes within each species have evolved to work properly-
they have functional promoters, coding sequences,
terminators, etc that allow the genes to be expressed
• Random mutations are more likely to disrupt these
sequences rather than improve their function
• Example: many inherited human diseases result from
mutated genes
 Mutation frequency
• Mutation frequency for a gene is the
number of mutant genes divided by
total number of genes within a
population (human genome size
approx. 3,200,000,000bp)
• If 1 million bacteria were plated & 10
found to be mutant, mutation
frequency would be 1 in 100,000 or 10-5
Types of mutation: Spontaneous Vs
induced mutations
• Spontaneous mutations: Some mutations are
spontaneous errors in replication that evade the
proofreading function of the DNA polymerases that
synthesize new polynucleotides at the replication
fork
• These mutations are called mismatches because they
are positions where the nucleotide that is inserted
into the daughter polynucleotide does not match, by
base-pairing, the nucleotide at the corresponding
position in the template DNA
• If the mismatch is retained in the daughter double
helix then one of the granddaughter molecules
produced during the next round of DNA replication
will carry a permanent double-stranded version of
the mutation.
• Induced mutations:
those that result from
exposure of organisms
to mutagenic agents
such as ionizing
radiations, UV light or
various chemicals that
react with DNA (or
RNA in RNA viruses)
• It’s impossible to prove
that a particular
mutation occurred
spontaneously or a
specific mutation was
induced by a
mutagenic agent
 Errors in replication are a
source of point mutations
• The 3′→5′ exonuclease activity of DNA
polymerase is able to remove an incorrect
nucleotide that evades the nucleotide
selection process and becomes attached to
thye 3’ end of the new polynucleotide. This
is called proofreading
• Each step in the synthesis of a
polynucleotide is a competition between
the polymerase and exonuclease function
of the enzyme
• The polymerase usually winning because it
is more active than the exonuclease, at
least when the 3′-terminal nucleotide is
base-paired to the template. But the
polymerase activity is less efficient if the
terminal nucleotide is not base-paired, the
resulting pause in polymerization allowing
the exonuclease activity to predominate so
that the incorrect nucleotide is removed
 Effect of tautomerism
• Not all of the errors that occur during DNA
synthesis can be blamed on the polymerase
enzymes: sometimes an error occurs even though
the enzyme adds the ‘correct’ nucleotide, the one
that base-pairs with the template
• This is because each nucleotide base can occur as
either of two alternative tautomers, structural
isomers that are in dynamic equilibrium
• After replication, the rare tautomer will inevitably
revert to its more common form, leading to a
mismatch in the double helix
• Tautomers are different forms of the same
molecule that exist in a dynamic equilibrium,
where they can rapidly interconvert. These
forms typically differ in the position of a
hydrogen atom and a double bond. In the
context of nucleotides in DNA, tautomers can
affect base-pairing, potentially leading to
errors during DNA replication.
• Not all errors in DNA synthesis are caused
by polymerase enzymes. Sometimes, even
when the enzyme adds the "correct"
nucleotide, an error can still happen. This
is because each nucleotide can exist in two
forms (tautomers) that are in balance.
After replication, the rare form changes
back to the common one, causing a
mismatch in the DNA.
 Tautomeric shifts
• 3rd way that mutations arise spontaneously involves a
temporary change in base structure: tautomeric shift
• Tautomers- bases, which exist in keto & enol or amino
and imino forms
• These forms can interconvert by a chemical reaction that
involves migration of a hydrogen atom & a switch of a
single bond & an adjacent double bond
• Common, stable form of guanine & thymine is the keto
form; the common form of adenine & cytosine is the amino
form
• At low rate, G & T can interconvert to an enol form; A & C
can change to imino form
• Though the enol & imino forms of these bases are small in
amounts, they still cause mutation & if one of the bases is
in the enol or imino form, hydrogen bonding will promote
TG & CA base pairs
 Tautomeric shifts
• Q: How does a tautomeric shift cause a mutation?
• A: It must occur immediately prior to DNA replication
• When DNA is in a double stranded condition, base pairing
usually holds the bases in their more stable forms
• After strands unwind, a tautomeric shift may occur
• Ex- a thymine base in the template strand has undergone a
tautomeric shift just before replication of complementary
daughter strand
• During replication, daughter strand incorporates a guanine
opposite this thymine, creating a base mismatch
• This mismatch could be repaired by DNA polymerase
proofreading function or by mismatch repair system
• If repair mechanisms fail, next round of DNA replication will
produce a double helix with CG base pair where the correct
base pair should be TA
Example of tautomeric shift
§ A tautomeric shift occurred
in a thymine base just prior
to replication, causing
formation of a TG base pair.
If not repaired, a second
round of replication will
lead to formation of a
permanent CG mutation.

§ A tautomeric
shift:temporary situation;
during 2nd round of
replication, thymine base
that shifted prior to first
round of DNA replication is
likely to have shifted back
to its normal form. So,
during 2nd round of
replication, adenine base is
found opposite this thymine
 Molecular basis of
mutagenesis: Point mutations
• A gene mutation can involve a base
substitution in the sequence within a
gene or a removal or addition of one
or more base pairs
• Point mutation base substitution-
a change in a single base pair within
the DNA
• Point mutations can also be insertion &
deletion
• Example:
Examples
 Molecular basis of mutagenesis:
Possible effects (consequences) of
point mutation
•
 Nonsense mutation
• Involves a change from a normal codon to a stop codon
• This terminates translation of the polypeptide earlier
than expected thus producing a truncated polypeptide
• When a nonsense mutation occurs in a bacterial operon,
it inhibits the expression of downstream genes
(Downstream of a gene refers to the region 3' to the
transcription termination site of the reference gene.)
• This phenomenon is called ‘polarity’ (in genetics, in
which a nonsense mutation in one gene affects the
translation of a downstream gene in an operon)
• Polar mutation-a mutation in an operon that affects
expression of another gene that’s found downstream of
an operon
 Frame shift mutations
• 3) Frame shift mutation: Involve addition or deletion
of a number of nucleotides that is not divisible by three
• Because the codons are read in multiples of 3, this shifts
the reading frame
• Translation of the mRNA then results in a completely
different amino acid sequence downstream from the
mutation
• 4) Neutral mutation: when a missense mutation has no
detectable effect on protein function it’s referred to as
neutral mutation
• Why? A missense mutation is less likely to alter function as
it involves a change of a single amino acid within
polypeptides. Silent mutations are also included as neutral
mutation
Effects of mutation
 Replication slippage
• Replication slippage is probably also responsible for the trinucleotide
repeat expansion diseases that have been discovered in humans in
recent
• Each of these neurodegenerative diseases is caused by a relatively
short series of trinucleotide repeats becoming elongated to two or
more times its normal length
• For example, the human HD gene contains the sequence 5′-CAG-3′
repeated between 6 and 35 times in tandem, coding for a series of
glutamines in the protein product. In Huntington's disease this
repeat expands to a copy number of 36–121, increasing the length of
the polyglutamine tract and resulting in a dysfunctional protein
• Several other human diseases are also caused by expansions of
polyglutamine codons
• Some diseases associated with mental retardation result from
trinucleotide expansions in the leader region of a gene, giving a
fragile site, a position where the chromosome is likely to break
• Expansions involving intron and trailer regions are also known