Gen Lec 20: DNA Mutation + Repair

TEST CONCEPTS: Recognize Mutation given a sequence and explain what the effect would be. Mutation Nomenclature (base 1/ base 2 -> base 1'/base 2'))

Mutation

Heritable change in genetic information

• change from WT

• Source of genetic var. that is necessary in population for evolution to happen

Wild Type

What is observed most often

Mutations Characterized by Size

Chromosomal, Point

Chromosomal mutations (def, e.g.)

large scale genetic alterations that affect structure / #

• e.g. (deletions, inversions duplications, trans)

point mutations

small scale, single/few nucleotides are changed, added, or deleted

• Missense, Nonsense, frameshift, silent

Mutations characterized by change to Nucleotide

Transitions, Transversions

Transition

Purine → Purine / Pyrimidine → Pyrimidine

• More common/less significant change to chem structure

Transversions

Purine ↔ Pyrimidine

• Significant change to chemical structure

Mutations characterized by Affects Mutation has on Protein

Missense, Nonsense, Silent, Frameshift

Missense

point mutation leads to amino acid change in protein

Nonsense

point mutation changes sense codon → nonsense codon

• results in premature stop codon → truncated, usually nonfunctional protein

Sense vs. Nonsense Codon

Sense codes for amino acid; Nonsense Codes for stop

Silent Mutation

Point mutation changes codon, but amino acid stays the same

• happens bc of degeneracy of code

Frameshift

addition/deletion of multiples 1 or 2 but not 3 base pairs

• Shifts reading frame so that amino acid seq downstream of mutation is incorrect

• Often puts nonsense codon in frame so final protein is truncated

Mutations characterized by where Mutations happen

Somatic Mutations, Germline Mutations

Somatic Mutations

Mutations in Body cells

• Not heritable by progeny

• Sometimes result in increased cell division → cancer

• Not all cells in affected individual will have mutation, but daughter cells of a cell with a somatic mutation will have the mutation

Germline Mutations

• in gametes

• Heritable

• All cells in affected individual have mutation

Mutations characterized by how they occur

Spontaneous mutations, Induced mutations

Spontaneous mutations (Definition + causes)

Occur during normal lifetime of a cell

Causes :

1. Errors made during DNA replication

2. Normal biochemistry in cell creates reactive Depurination, Deamination

Errors made during DNA replication

1. Incorrect base insertions likely due to wobble pairing

2. Strand slippage

Incorrect base insertions likely due to wobble pairing

If a replication error due to wobble pairing (e.g., insertion of T instead of C) is not corrected, a mismatch or bulge can form that escapes DNA proofreading

• after a subsequent round of DNA replication → mutation becomes permanently established in genome

wobble pairing

H-bonding between non-Watson/Crick bases

Frameshift mutations due to strand slippage

Strand slippage occurs during DNA replication at repetitive sequences when DNA polymerase “loses count“ and misaligns the strands

• extra bases are added → newly synthesized strand loops out → insertion

• bases are skipped → template strand loops out → deletion

Depurination

Loss of purine base AG

• sugar phosphate backbone stays intact

• Normally repaired (Base excision), but when DNA Pol gets to site w/o base, it doesn’t know what to do and often inserts A

• → subsequent round of DNA rep → permanent change in genome

Deamination (Def and 2 cases)

Loss of amino group from a base

(Can You Anak)

Amino group lost from cytosine → yields uracil → pairs w/ A during replication (C-N=U+A)

• G/C → A/T after 2 rounds of DNA replication (transition)

Amino group lost from Adenine → hypoxanthine → pairs w/ C during replication (A-N = HA+C)

• A/T —> G/C after 2 rounds of DNA replication (transition)

Induced mutations / (mutagen types)

results from exposure to external agent (mutagen)

base analogues, base modifiers, intercalating agents, UV light

AMES Test (Purpose, Method)

Used to identify mutagens

• uses mutant bacterial strain (No DNA repair systems + cannot make Histidine)

1. Expose bacteria to chemical w/ and w/o Liver enzyme (to test if metabolized chemical is a mutagen)

2. Plate bacteria treated w/ chemical, chemical + liver enzyme, and untreated control on minimal media

   1. Growth → His+ mutation (bacteria has been mutated and reverted back to WT)

Base analogues

Chemical that looks like a base

• can be incorporated into DNA during S phase

• ionization will change basepairing properties during subsequent rounds of DNA replication

Base modifiers

add/remove chemical groups from a base so that it will not pair properly when it is template during DNA replication

Intercalating agents

flat planar molecules that intercalate between stacked bases in DNA

• Contorts helical structure → insertions/deletions during DNA replication

UV Light

Cause adjacent pyrimidines to covalently bond together → they will NOT bp with complementary DNA strand during replication

Epistasis

Genotype at 1 locus mask gene expression at another locus

Intergenic Suppression

Individual w/ mutation is double mutant

Mutation 1 → causes muation

Mutation 2 (at another site)→ suppresses effect → WT

Intergenic suppression via suppressor tRNA

Nonsense Mutation 1 Eye Color Gene → Truncated Protein

Suppressor Mutation 2 in tRNA Gene → Mut tRNA has anticodon that can base pair with stop codon → Full length of protein

Intergenic suppression via protein–protein interaction

Enzyme that needs multiple subunits (A/B) to function

• Missense Mut 1 (Subunit A gene) → Subunit A changes and can no longer bind Subunit B

• Suppressor Mut 2 (Subunit B gene) → Subunit B changes and can bind with A

Intragenic Suppresion (def + 2 ecamples)

Second mutation in a SAME gene restores (partially or fully) the phenotype caused by the first mutation

E.g. mutations in same codon, mutations in different codons

Intragenic Suppresion: Both mutations are in a single codon

1st point mut: Leu → Phe

2nd point mut: Phe → Leu

• occurs because of redundancy in genetic code

Intragenic Suppresion: Mutations are in different codons of the same gene (± example)

aa1 and aa2 must interact tfor protein to fold correctly

• 1st point mut: aa1 (-) charge → (+) charge

• 2nd point mut: aa2 (+) charge → (-) charge

• changes restore original proteins structure

DNA Repair Mechanisms

• Require 2 strands of DNA (mutated; nonmutated strand = template)

• Multiple ways to do the same thing

Direct Repair (Def + 2 examples)

Reverse whatever just happened

1. Mutation: Thymine dimers caused by UV light

• Photolyase, activated by light destroys covalent bonds between Ts so they can create H-bonds

2. Base is Methylated

• Transferase enzymes remove alkyl + methyl gr

2 types of excision repair

Nucleotide excision repair, Base excision repair

Nucleotide excision repair

Cells are not actively dividing

1. repair proteins recognize distortion in helix as an error

2. Endonuclease cleave the phosphodiester bond on either side of distortion → remove that piece of DNA

3. Gap is filled by DNA Pol

4. Nick is sealed by DNA

Base Excision Repair

involves initial removal of base

1. DNA Glycolase removes modified base

2. DNA endonuclease nicks DNA and removes sugar

3. DNA polymerase adds new nucleotide

4. Nick is sealed by DNA ligase

Mismatch repair

Corrects mismatches that happen during replication

• new strands (w/ mistake) are distinguished from old strains (no mistake) bc more A’s are methylated on old strand than new strand

1. enzymes look for distortions in DNA

2. section w/ mutation is excised

3. DNA Pol I fills in gap

4. DNA ligase seals nick

Repair Systems that repair DNA mutations in cells that are not actively dividing

Mismatch, Direct, Base, Nucleotide excision

Repair Systems that repair DNA mutations in cells that are actively dividing

Homologous recombination / Nonhomologous end joining