Replication inaccuracy

(Chapt 9, Watson)
Mutation: Key Terms
Mutation: The specific change that occurs in the DNA nucleotide
sequence
-occurs in the coding sequence or the regulatory sequence
Mutagen: a compound that increase the rate of mutation by inducing
changes in DNA sequence, directly/indirectly
Mutagenesis: The introduction of a change in the nucleotide sequence
of DNA
-can occur spontaneously i.e. error in replication
-exposure to mutagen
Mutant: An organism that is altered by a mutation
Mutation
Balance between mutation and genetic variation.
 For survival: DNA sequences must be passed unchanged in the germ-line
Low mutation rates in the soma
 But perfect fidelity →no genetic variation → no diversity
Somatic and Germinal Mutation
Somatic cells – The mutation has to be dominant
 only the mutated cells have the phenotype
 cannot be passed to the next generation

Germ cells (2n)

 dominant → can be seen in the progeny
 recessive →concealed if the progeny is diploid
Cause of mutation:
1. Inaccuracy in DNA replication
2. Chemical damage to the genetic material

Consequence:
1. Change the coding sequence
2. Prevent its use as a template for transcription of replication
Inaccuracy in DNA replication
 Common factors:
1. Tautomerization
The change in the structure during tautomerization causes mispairing of
the bases during replication.
Two types of switch:
 a) transition: a change from a pyrimidine to another pyrimidine or
purine to another purine.
 b) transversion: a change from a pyrimidine to purine or purine to
pyrimidine.
 Tautomerization of bases

Tautomer: structural isomers differing in the location of their hydrogen

atoms and double bonds
Base change substitution: a) transition b) transversion
Consequence of tautomerization:
Point mutation: switches of one base for another, resulting in a
change of the base pair at the next round of DNA replication.
Point mutation will lead to missense or nonsense mutation:
 Missense mutation: change of an amino acid codon to the
codon of another amino acid
 Nonsense mutation: an amino acid codon is changed to a
termination or stop codon.
(continuation on factors for inaccuracy of DNA replication)
2.Insertion and deletion of nucleotides
Many causing factors
Commonly occurs at “hotspots”- high frequency mutation site i.e.
DNA microsatellites – containing sequences of repeats of di-, tri-
or tetranucleotide sequences.
Difficult to replicate accurately
Microsatellites/Simple Sequence Repeats (SSRs)
 Loci consisting of repeating units of 1-4 base pairs in length.
 can be repeated 10-100 times
 common example (CA)n repeat
 Variability of microsatellites due to increase rate of mutation
→slippage during DNA replication
 slippage increases or reduces the number of copies
 thus highly polymorphic in the population
 In research, microsatellites are ideal for
i. Determining paternity
ii. Population genetic studies
iii. Recombination mapping
Leads to frameshift mutation: insertion of one or more (not in
multiples of three) nucleotides in the coding region of a gene.

Example:
Wild type
5’ A T G G T C G C C T A T C G T A 3’ DNA
5’ A U G G U C G C C U A U C G U A 3’ mRNA
NH2-Met-Val-Ala-Tyr-Arg-COOH Polypeptide

Mutation → insertion
5’ A T G G A T C G C C T A T C G T A 3’ DNA
5’ A U G G A U C G C C U A U C G U A 3’ mRNA
NH2-Met-Asp-Arg-Leu-Ser-COOH Polypeptide
Mutation → deletion
5’ A T G G T C G C T A T C G T A 3’ DNA
5’ A U G G U C G C U A U C G U A 3’ mRNA
NH2-Met-Val-Ala-Ileu-Val-COOH Polypeptide
(continuation on factors for inaccuracy of DNA replication)
3. Extensive insertion or deletions and gross rearrangements of
chromosome structures
May due to abnormal recombination process.
Example: Cri-du-Chat
syndrome
-abnormal development of
larynx

Chromosomal mutation: https://mutationssciencie.weebly.com/chromosomal-

mutations.html
A mutation can be permanently incorporated by replication
A mutation may be introducd by misincorparation of a base in the first
round of replication. In the second round of replication, mutation becomes
permanently incorporated in the DNA sequence.
Mismatch Repair System
• Proofreading mechanism during replication is not foolproof. Will be
‘rechecked’ by mismatch repair system.
• Increase the accuracy of DNA synthesis
• Have to find the mismatch and correct it accurately (identify parental
strand)
• Have to be done right after replication since once the second round of
replication take place, the misincorporation can no longer be detected.
• Mutation in this system is correlated to cancer cases
Steps in E. coli mismatch repair system and enzymes that catalyze the repairing activity
1. MutS
 scan DNA
 recognize the mismatch based on the distortion of the backbone
 bind to the mismatch and induce kink
 recruits MutL
2. MutL – activate MutH
3. MutH – cause incision on one of the strands near the site of mismatch
4. UvrD – helicase
- unwind the DNA from the starting of incision to the mismatch
5. Exonuclease – digest the single strand, extend beyond the mismatch
- produce single-strand gap
6.DNA polymerase III – fill in the gap
7. DNA ligase - ligate
How to recognize the parental strand: hemimethylation
Dam methylase methylate A residue on both strands at the site of 5’-
GATC-3’ (once every 256 base) 44
During replication, hemimethylation occur i.e. only one of the strands is
methylated (parental which function as template)
MutH binds to hemimethylation in a latent form.
-activated when in contact with MutL and MutS complex
-starts the incision on the unmethylated strand
Exonuclease
 if the incision is on the 5’-3’, exonuclease VII
 if on 3’-5’, exonuclease VI
Dam methylation at replication fork
a) Replication generates
hemimethylated in E. coli.
b) MutH makes incision in
unmethylated daughter strand.