L14 Biol261PmutationF2013

8/9/2019 L14 Biol261PmutationF2013

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L14 Mutation

Forward Geneticsdepends on phenotypic variation as an index of

genetic variation: its inheritance or transmission-genetic pattern; howtransmission is related to chromosome variation; how character andphenotype differences depend on metabolic pathways involving severalgenes that may or may not be functional; how they replicate but not thesource(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 lowWhat really governs mutation rate is the efficiency ofrepair mechanisms. Regardless, the rate is never 0, some base changes(1) goun-repaired, or, (2) repair mechanisms introduce a mutantvariant.

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

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E coli has 5 DNA polymerases

DNA pol III - replicase with 3

-5

proofreadingDNA pol I - major repair -5

-3

exonuclease activity

enables it to start replication at a nick (+ 3

- 5

proofreading)DNA pol II - minor, SOS repairDNA pol IV, V - SOS repair


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

(2)possibly inheritedchanges 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.

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Spontaneous mutations

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

(1)the migration of a H-bond ,

(2)

the switch of adjacentsingle and double bonds

Depurination -covalent

bond between sugar andpurine is less stable thansugar-pyrimidine.= rare single base loss

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Rare tautomers

Spontaneous mutations


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Spontaneous (single strand) mutations in germcells may be inherited, if a mutant gameteforms a viable zygote that survives toreproduce.

5Note this tautomeric shift must happen during the process, justbefore/as DNA replicates.

An apurinic site - any complementary base could be substituted

during replication.


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Faithful, but not perfect

spontaneous mutations during replication

Copy errors in replication could cause heritable changes to a DNAsequence.

There are 3 error - checking steps in DNA replication: (1) base selection(2) proof reading (3) mismatch repair

(1) Base selectionif the correct base is not chosen, the polymerase site in theholoenzyme will not be activated.

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

(3)Mismatch repair after replication, the wrong nucleotide will not generallypair 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-methylatedstrands are repaired based on its methylated complement. See fig 15-26

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

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Eukaryotes & prokaryotes: A replication fork that is blocked bydamage to several bases signals the cell death pathway,But, it may be repaired before replication by:

(1) Nucleotide Excision Repair (NER) involving a complex ofproteins 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 segmentto the strand.

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

excise damaged copy undamaged.


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A homology-dependent base-excisionrepair in post-replicated DNA

Recognition Eachglycosylaserecognizes a specifictype of altered base(oxidized, deaminated etc)

(a) CutGlycosylase cleavestheglycosidic bond, (base-sugar), leaving unattached bases

(b)AP endonuclease(s) (APurinic,APyrimidinic), nicksthe damaged strand

(c) Removal deoxyRibophosphodiesterase(dRpase)

removes neighbor bases

(d) Restore DNA polymerase I andDNA ligase.

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Cause:Measured as % death of F2

(hemizygous) malescarrying irradiated , P, Xchromosomes throughmarked , heterozygous F1females

Inducedmutationsthrough the actionof mutagens.

High energy likeX rays causedouble strand

breaksamongvarious othermutations

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Double Strand Break Repair ?

(1)NonHomologous, EndJoining(NHEJ)mechanismsinvolve dedicatedproteins repairing non-replicatingchromosomesinG0 or 1(pp540)

(2)Synthesis-Dependent StrandAnnealing(SDSA) mechanismuses a sisterchromatidas a template (homologousrecombination repair) G2.

(3)

Meiosis-mechanisms:repair is basedon homologues as well as sisterchromatids. (15.5)

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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 samechemical 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 basefrom a different category

Base analogues(pp 526) cause transitions or transversions in thefirst 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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Rare, but frequentcompared to the enolform of thymine.

5 bromouracil is an analogue ofthymine

It forces a tautomericshift


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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 deletionof nucleotides,-insertion or loss of one or two nucleotides

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Point mutations :Synonymous mutations are changes orsubstitutions in a nucleotide sequencethat do not cause a change in

amino acid sequence.

DNA: 3

TAC GCT CCT CTT GGT GCT

Protein Met- Arg- Gly- Glu- Pro- Arg-

Mutant

DNA: 3

TAC GCT CCT CTT GGT GCG

Protein Met- Arg- Gly- Glu- Pro- Arg-

Synonymous mutations have no effect on the protein function

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Point mutations:Missense mutations change a codon, and itmutates to a different amino acid, it is potentially non-silent, it may

be chemically similar, it may be chemically different , thus having anon neutral effect on a character expression. The effect of the amino

acidsubstitutionmay be conservative (similar chemical properties) or

non conservative.

Wild type

DNA: 3

TAC GCT CCT CTT GGT GCT CTA

Protein Met- Arg- Gly- Glu- Pro- Arg- Asp-

Mutant

DNA: TAC GCT CCT CTT GGT CCT CTA

Protein 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 aSTOP codon

Sense mutationschange a protein coding or

a stop codon to a START codon.

WT

DNA: 3

TAC GCT CCT CTT GGT GCT CTA


Mutant

DNA: TAC GCT ACT CTT GGT GCT CTA

Protein Met- Arg

Non sense mutations produce truncated proteins and are nearly

always deleterious except those at the extreme carboxyl end.

STOP

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Nonsense mutations change a codon to a

STOP codon.

Sense mutationschange a stop codon to aprotein coding codon.

Same - sense mutationor silent mutation asynonomous 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 deletionof nucleotides,

-insertion or loss of one or two nucleotides

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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

WT

DNA: 5

ATG CGA CCTGAA GGT GCT CTA


Mutant

DNA: ATG CGA CCTCGA AGG

Protein Met- Arg- Gly- Arg - Arg.

Frame shift mutations are deleterious

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C


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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.

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 orwithout one of hundreds of amino acids.Any addition or

deletion notdividable by 3will cause a frame shift.

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Forward Genetic Analysis is only possible with genetic

differences: mutants, variants, polymorphisms etc..

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 mutantsare 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 chromosomestructure. See fig 16.19

1.Gene Duplications2.Deletions

3.Inversions4.Translocations

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Human

Normal

Karyotype

Karyotype

from

cancer cell.Note extra copies

lost copies

translocations


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Duplications

A B C D E F G H I J

A B C C D E F G H I J

A B C D E C D E F G H I J

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 , ).

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Same gene, different functions, probablyevolved through an inactive duplication

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1.Duplications

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 C D E F G H I J

A B C E F G H I J

Deletions are usually deleterious they are the lossof genes. They may exist in a heterozygous state. But, (1)imbalance in gene

number can be deleterious. (2) they can uncoverdeleterious recessive

allelesthat may not otherwise be expressed (3) probably due to slippage duringreplication, mispairing in meiosis breakage, deletion & duplication.

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1. Duplications

2. Deletions

3. Sequence inversions of many bases to several genes.

4.

Translocations

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A B C D E F G H I J

A B C F E D G H I J

Inversions either include the centromere or not

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

A B C G F E D H I J

Pericentric inversion no recombinant offspring, includes thecentromere (normal & inverted)

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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 genesin the region of the inversion ?

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1. Duplications

2.

Deletions

3. Inversions

4.

Translocations of chromosome segments

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Reciprocaltranslocations-

break and reciprocal fusion

of non-homologous

chromosomes.

Meiosis I:

Alternate segregation - bothpairs have a complete gene

set - fine

Adjacent segregation pattern

produces inviable gametes

Thus translocation

heterozygotes are semisterile

(fig. 16.30)

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

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L14 Biol261PmutationF2013

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