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L14 Mutation


Forward Genetics depends on phenotypic variation as an index of
genetic variation: its inheritance or transmission-genetic pattern; how
transmission is related to chromosome variation; how character and
phenotype differences depend on metabolic pathways involving several
genes that may or may not be functional; how they replicate but not the
source(s) of mutation .

Scale of mutation: this can range from a change in a single base (substitution),
through several bases (insertiondeletion), to a chromosome fragment.



Mutation rate is low What really governs mutation rate is the efficiency of
repair mechanisms. Regardless, the rate is never 0, some base changes (1) go
un-repaired, or, (2) repair mechanisms introduce a mutant variant.

If one of these (1 or 2) occurs in a germ cell (sperm,eggs) which happens to
segregate and produce an embryo, it will be passed on to descendent
generations.

E coli has 5 DNA polymerases





DNA pol III
replicase with 3-5 proofreading

DNA pol I -


major repair -5-3 exonuclease activity
enables it to start replication at a nick (+ 3 - 5
proofreading)

DNA pol II -


minor, SOS repair

DNA pol IV, V

- SOS repair


Mutations are:
(1) potentially permanent (mutations in somatic cells), and



(2) possibly inherited changes in DNA sequence (mutations in germ cells).








(A) spontaneous
- occurring in the absence of known mutagens

or


(B) induced mutations - require an known agent that increases the
mutation rate significantly above the spontaneous rate.

Spontaneous mutations

Rare tautomers

Spontaneous mutations



Tautomeric or keto-enol,
amino - imino shifts
suggested by Watson &
Crick (1953), involve:

(1) the migration of a Hbond ,

(2) the switch of adjacent
single and double bonds



Depurination -covalent
bond between sugar and
purine is less stable than
sugar-pyrimidine.

= rare single base loss

Note this tautomeric shift must happen during the process, just
before/as DNA replicates.

An apurinic site - any complementary base could be substituted
during replication.

Spontaneous (single strand) mutations in germ


cells may be inherited, if a mutant gamete
forms a viable zygote that survives to
reproduce.

Faithful, but not perfect



spontaneous mutations during replication

Copy errors in replication could cause heritable changes to a DNA
sequence.

There are 3 error - checking steps in DNA replication:
(2) proof reading (3) mismatch repair

(1) base selection

(1) Base selection if the correct base is not chosen, the polymerase site in the
holoenzyme will not be activated.

(2) Proof reading: 3-5 exonuclease activity (DNA pol III)

(3) Mismatch repair after replication, the wrong nucleotide will not generally
pair correctly, it causes a bulge in the helix, mismatch repair enzymes (Mut S, L
and MutH) scan for bulges, if found soon after replication, non-methylated
strands are repaired based on its methylated complement. See fig 15-26

If both strands are methylated, the mutation may be inherited (pp 536-538).

Eukaryotes & prokaryotes: A replication fork that is blocked by


damage to several bases signals the cell death pathway,

But, it may be repaired before replication by:








(1) Nucleotide Excision Repair (NER) involving a complex of
proteins that:



-Recognition of a distorted double helix
UVr A dimer, B

-Cuts on both sides of the damaged strand
UVr B, C

-Removes the damaged segment
UVr D

-DNA polymerase I fills, and DNA ligase links the new segment
to the strand.



(2) During replication E coli- SOS repair use DNA polymerase II,
IV, V (eukaryotes - translesion repair). These are low - fidelity
(error prone or high mutation) systems involving recombinationlike repair, that puts an undamaged strand near the damaged one ,
excise damaged copy undamaged.

A homology-dependent base-excision
repair in post-replicated DNA

Recognition Each glycosylase recognizes a specific
type of altered base (oxidized, deaminated etc)

(a) Cut Glycosylase cleaves the glycosidic bond, (basesugar), leaving unattached bases

(b)AP endonuclease(s) (APurinic, APyrimidinic), nicks
the damaged strand

(c) Removal deoxyRibophosphodiesterase (dRpase)
removes neighbor bases

(d) Restore DNA polymerase I and DNA ligase.


Cause:


Induced
mutations
through the action
of mutagens.



High energy like
X rays cause
double strand
breaks among
various other
mutations


Measured as % death of F2
(hemizygous) males
carrying irradiated , P, X
chromosomes through
marked , heterozygous F1
females



Double Strand Break Repair ?



(1) Non Homologous, End Joining (NHEJ)
mechanisms involve dedicated
proteins repairing non-replicating
chromosomes in G0 or 1 (pp540)

(2) Synthesis-Dependent Strand Annealing
(SDSA) mechanism uses a sister
chromatid as a template (homologous
recombination repair) G2.

(3) Meiosis-mechanisms: repair is based
on homologues as well as sister
chromatids. (15.5)

Mutations and their molecular basis (15.3)





High energy radiation cause transitions and transversions.


Transitions - replacement of a base by the other base in the same


chemical category (purine/purine A-G, pyrimidine/ pyrimidine (C-T). e.g.
tautomeric shift (pp 520)

Transversions - replacement of a base of one category with a base


from a different category









Base analogues (pp 526) cause transitions or transversions in the
first replication. E.g. 5 bromouracil



Intercalating agents (pp 528) cause indels -










insertions and deletions.



?conservative chemically similar neutral mutation

?non-conservative - chemically different


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5 bromouracil is an analogue of

thymine

It forces a tautomeric

shift

Rare, but frequent


compared to the enol
form of thymine.

Intercalating agents mimic base pairs and insert


themselves between them (intercalate) usually
causing an indel mutation - an insertion or
deletion of one or more bases

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Point Mutations - summary Fig 15-2


1. Point mutations base substitutions,
- synonymous or silent
- missense mutations,
- nonsense mutations
- sense mutations
2. Point mutations -Frame shift mutation -indel mutants
- insertion or deletion of nucleotides,
-insertion or loss of one or two nucleotides

Point mutations : Synonymous mutations are changes or

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substitutions in a nucleotide sequence that do not cause a change in
amino acid sequence.

DNA: 3TAC GCT CCT CTT GGT GCT


Protein Met- Arg- Gly- Glu- Pro- ArgMutant
DNA: 3TAC GCT CCT CTT GGT GCG
Protein Met- Arg- Gly- Glu- Pro- ArgSynonymous mutations have no effect on the protein function

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Point mutations: Missense mutations change a codon, and it

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mutates to a different amino acid, it is potentially non-silent, it may


be chemically similar, it may be chemically different , thus having a
non neutral effect on a character expression. The effect of the amino
acid substitution may be conservative (similar chemical properties) or
non conservative.
Protein Met- Arg- Gly- Glu- Pro- Arg- AspWild type
DNA: 3 TAC GCT CCT CTT GGT GCT CTA
Mutant
DNA:
Protein

TAC GCT CCT CTT GGT CCT CTA


Met- Arg- Gly- Glu- Pro- GLY- Asp-

Some point mutations are very deleterious those that change


critical amino acids in a protein.

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Point mutations: Nonsense mutations change a codon to a 19



STOP codon
Sense mutations - change a protein coding or
a stop codon to a START codon.
WT
DNA: 3TAC GCT CCT CTT GGT GCT CTA
Protein Met- Arg- Gly- Glu- Pro- Arg- AspMutant
DNA: TAC GCT ACT CTT GGT GCT CTA
Protein

Met- Arg STOP

Non sense mutations produce truncated proteins and are nearly


always deleterious except those at the extreme carboxyl end.

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Nonsense mutations change a codon to a


STOP codon.


Sense mutations change a stop codon to a
protein coding codon.



Same - sense mutation or silent mutation a
synonomous mutation

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Mutation,
1. Point mutations substitutions,
- synonymous
- missense mutations,
- nonsense mutations
- sense mutations
2. Point mutations -Frame shift mutation - indels
- insertion or deletion of nucleotides,
-insertion or loss of one or two nucleotides

Frame shift mutation - the addition or loss of 1 or 2


nucleotides:
Frame shift mutations change all the amino acid sequence
after the mutation and often introduce stop codons
C

WT
DNA: 5ATG CGA CCT GAA GGT GCT CTA
Protein Met- Arg- Gly- Glu- Pro- Arg- AspMutant
DNA:
Protein

ATG CGA CCT CGA AGG


Met- Arg- Gly- Arg - Arg .

Frame shift mutations are deleterious

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A 3 base pair addition or deletion mutation
will add or delete one amino acid. In this case the effect
depends on the chemical similarity of the protein with or
without one of hundreds of amino acids. Any addition or
deletion not dividable by 3 will cause a frame shift.

Frame shift mutations can be corrected


by a second mutation up or downstream and
nearby. The negative effect increases with the
size of the frame-shifted sequence.
All amino acids encoded between the two mutations
will be changed. Sometimes short stretches of changed
amino acids will not inactivate a protein.

Frame shift correction by 2nd indel mutation:


WT
DNA: 5ATG CGA CCT GAA GGT GCT CTA
Protein Met- Arg- Gly- Glu- Pro- Ala- LeuC addition

Mutant
DNA:
ATG CGA CCT CGA AGG..
Protein
Met- Arg- Gly- Arg - Arg.
2nd

G deletion

Mutation
DNA:
ATG CGA CCT CGA AGT GCT CTA
Protein

Met- Arg- Gly- Arg - Arg Ala- Leu-

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Forward Genetic Analysis is only possible with genetic


differences: mutants, variants, polymorphisms etc..

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The most useful mutations for research are conditional


(lethal) mutations:
(a) Auxotrophic mutants - unable to synthesize essential
metabolites- e.g Beadle & Tathum
(b) Temperature sensitive mutations- grow or express at one
temperature .
(c) Suppressor-sensitive mutants are viable when a second
genetic factor a suppressor is present , inviable in the
absence of the suppressor

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Large Sequence Alterations


Large -scale changes in chromosome


structure. See fig 16.19

1. Gene Duplications

2. Deletions

3. Inversions

4. Translocations

Human
Normal
Karyotype

Karyotype
from
cancer cell.
Note extra copies
lost copies
translocations

Duplications
A B

A B

C C

A B

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E
D

F
E

G
F

E C D

H
G
E F

I
H

J
I

J
H

Duplications could create an imbalance in the number


of genes.
Evolutionarily, it is clear that duplications give rise to
extra copies that can evolve to produce novel genes
(paralogs Haemoglobin , ).

Same gene, different functions, probably


evolved through an inactive duplication

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1.Duplications
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2. Deletions of genes or a large portion of its sequence
3. Inversions
4. Translocations
Many mutations occur in meiosis

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Deletions:
A B

A B

Deletions are usually deleterious they are the loss


of genes. They may exist in a heterozygous state. But, (1)imbalance in gene
number can be deleterious. (2) they can uncover deleterious recessive
alleles that may not otherwise be expressed (3) probably due to slippage during
replication, mispairing in meiosis breakage, deletion & duplication.

1. Duplications
2. Deletions
3. Sequence inversions of many bases to several genes.
4. Translocations

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Inversions either include the centromere or not


A B

Paracentric inversion:- no recombinant offspring (normal & inverted)

A B

F E

Pericentric inversion no recombinant offspring, includes the


centromere (normal & inverted)

A B

G F E

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In both, recombinants are not viable

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Inversion heterozygotes are never recovered


If inversions lead to reduction of numbers of
surviving offspring from crossovers in the inversion
region , what effect do they have on map distance of genes
in the region of the inversion ?

1. Duplications
2. Deletions
3. Inversions
4. Translocations of chromosome segments

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Reciprocal translocationsbreak and reciprocal fusion
of non-homologous
chromosomes.
Meiosis I:
Alternate segregation - both
pairs have a complete gene
set - fine
Adjacent segregation pattern
produces inviable gametes
Thus translocation
heterozygotes are semisterile
(fig. 16.30)

Figure 4-14 Molecular Biology of the Cell ( Garland Science 2008)