Lecture Notes on Mutation and DNA Repair

Nature of Mutation

• Luria-Delbrück Experiment (1943) (Fluctuation Test):
  • Dealt with understanding the nature of mutations in bacteria. Presented two possibilities:
    • Possibility 1: Mutations occur independently of natural selection and are already present in the population.
    • Possibility 2: Mutations are directed by the selection pressure.

Luria-Delbrück Experiment

• Experiment Setup:
  • A giant flask containing bacteria was sampled to assess the population's variation.
  • The bacteria were tested for resistance to streptomycin.
  • A culture was divided into 50 tubes, and each was plated on a streptomycin-containing medium.
• Observations:
  • There was fluctuation in the number of resistant bacterial colonies between different plates.
  • Bacteria select for traits already existing in the population.
• Results interpretation:
  • In the Same Culture (Control), the mean number of T1-Resistant Bacteria colonies was 16.7 with a variance of 15.0.
  • In Different Cultures, the mean number of T1-Resistant Bacteria was 26.2, and the variance was significantly higher at 2178.0.
  • Fluctuation was observed, with some cultures showing many resistant bacteria and others few.
  • Small-scale mutations can lead to significant variations in resistance.

Spontaneous Mutation Rates in Humans

• Study Details:
  • Genomes of 78 parent/offspring sets (219 individuals) were studied by comparing single-nucleotide polymorphisms (SNPs).
  • A newborn baby’s genome contains an average of 60 new mutations compared to parents.
  • The number of new mutations is related to the father’s age at conception.
  • Identified 4933 new SNP mutations, with 73 located in exons.
• Family Structures in the Study:
  • 57 simple trios (mother, father, and child).
  • 5 three-generation families.
  • 6 sibling pairs.
• De Novo Mutations (DNMs):
  • Most new mutations come from the paternal side.
• Age of Father and DNMs:
  • The number of de novo mutations increases with the father's age at conception.
• Parental Age and DNMs:
  • Females do not contribute as many de novo mutations compared to males.

Tautomerization

• Tautomerization is a phenomenon involving the rearrangement of a molecule, specifically the movement of a proton and a double bond.
• Tautomers Examples
  • keto-enol tautomerism: conversion between keto and enol forms by shifting a proton and a double bond.

Base Pairing and Tautomeric Shifts

• Standard Base Pairing:
  • Adenine (A) pairs with Thymine (T).
  • Cytosine (C) pairs with Guanine (G).
• Anomalous Base Pairing:
  • Tautomeric shifts can lead to incorrect base pairings.
    • Thymine (enol form) pairing with Guanine.
    • Guanine (keto form) pairing with Thymine.
    • Cytosine (imino form) pairing with Adenine.
    • Adenine (amino form) pairing with Cytosine.
• Replication and Tautomeric Shifts:
  • During DNA replication, tautomeric shifts can cause mispairing.
    • A tautomeric shift in Thymine can cause it to pair with Guanine instead of Adenine.
    • This mispairing can lead to a transition mutation in subsequent replication rounds.

Deamination

• Deamination Process:
  • Deamination involves the removal of an amino group from a base.
    • Cytosine is converted to Uracil.
    • Adenine is converted to Hypoxanthine.
• Consequences:
  • Introduces point mutations.
  • Uracil in DNA is an issue.

5-Bromouracil (5-BU)

• Analogue of Thymine:
  • 5-BU is an analogue of thymine and can be incorporated into DNA.
• Inducing Mutations:
  • Feeding cells the enol form of 5-BU can cause mispairing, leading to mutations.
    • Encourages anomalous base pairing without the need for X-rays.
  • Can result in Guanine pairing with Thymine.

EMS and ENU

• Function:
  • EMS (Ethyl MethaneSulfonate) and ENU (N-Nitroso-N-ethylurea) convert normal bases to strange bases.
• Mechanism:
  • These compounds modify existing bases, causing anomalous carrying properties.
    • For example, EMS modifies Guanine by adding a methyl group.
      • This leads to anomalous base pairing.

Ames Test

• Purpose:
  • The Ames test determines if a compound causes mutations and is therefore mutagenic.
• Procedure:
  • A compound is either treated or untreated on a plate.
  • Each tiny spot on the plate represents a colony.
  • The background level of revertants is noted.
• Revertants:
  • The test is based on reverse mutations that occur within the mutant gene.
• Interpretation:
  • More colonies after treatment indicate that the compound is mutagenic.
  • If disk is just to concentrate the compound in one spot.
  • The mutagenic property can be inferred to indicate carcinogenic potential, but it is not direct evidence.
  • If there isn't natural revertants you can't do this.
• Controls
  • negative basal control represents the background number of mutations, or revertants.

Ames Test Results and Controls

• Comparing to Zero:
  • Comparing results to zero helps determine if there is an increased mutation rate.
• Positive Control:
  • A known mutagen is used as a positive control to ensure the assay worked.
• Bacterial Strains:
  • Different bacterial strains (e.g., TA97, TA98, TA100, TA102) are used.
  • Cannot compare across strains because they have different backgrounds.
• Histidine Biosynthesis Pathway
  • Curois property:- they cannot grow without histomine.
  • The salmonella strains are auxotrophic for histidine but can do natural revertants.
  • Meaning that if you take that bacteria and throw them on a plate a small number will grow due to a point mutation in the histidine biosynthesis pathway.
  • the reversion back mutates it
• Liver Extract (S9):
  • Liver extract is used to determine if processing by the liver fixes toxicity or makes it worse.

Interpreting Ames Test Data

• Different Strains of Salmonella:
  • TA97, TA98, TA100, and TA102 are different strains of Salmonella used in the Ames test.
  • These strains are auxotrophic for histidine, meaning they cannot grow without it.
  • They can undergo natural reversion, allowing a small number of colonies to grow on a plate without histidine.
• Measuring Mutations:
  • The assay measures the reversion of a mutation in the histidine biosynthesis pathway.
  • An increased mutation rate indicates the compound is mutagenic.
• Liver Extract (S9):
  • Liver extract is used to assess whether the liver's processing of the compound alters its toxicity.
• Positive Control:
  • Uses a known mutagenic to see if the assay worked.
• Considerations:
  • Cannot compare results across different strains due to their different backgrounds.
  • Looking down the first column, TA 97, TA 98… these are different Strains of salmonella.
• using the broken "thing", fixed as the assay way to measure something.

Strains and Treatment

• Real Data Analysis:
  • The data shows if a compound is mutagenic or not.
  • The '0 addition control' represents the background level of mutations.
• Strain Differences:
  • Different strains are used because mutations behave differently in each.
  • TA100, TA97a, TA98, and TA102 are different strains with different types of mutations.
• Types of Mutations:
  • Insertions
  • Deletions
  • Point mutations (single nucleotide polymorphisms)
• Liver Extract Effect:
  • The liver extract can either make a compound more or less toxic.
  • It can take something toxic and make it non-toxic.
• The values in parenthesis represent the mutagenic index [MI, red color, * p < 0.05 (ANOVA)] or signs of mutagenicty [green color,**p<0.05 (ANOVA)].

Benzo(a)pyrene and Liver Extract

• Concentration and Revertant Colonies:
  • The bar graph data comparing the number of revertant colonies at different concentrations of Benzo(a)pyrene ((μg/mL)).
  • Should use multiple strains because they behave differently.

Human Disorders Caused by Single-Gene Mutations

• Types of Mutations:
  • Missense
  • Nonsense
  • Insertion
  • Deletion
  • Trinucleotide repeat expansions
• Examples of Disorders:
  • Achondroplasia: Glycine to arginine at position 380 of FGF R3 gene (Missense mutation).
  • Marfan syndrome: Tyrosine to Stop codon at position 2113 of fibrillin-1 gene (Nonsense mutation).
  • Familial hypercholesterolemia: Various short insertions throughout the LDL R gene (Insertion).
  • Cystic fibrosis: Three-base-pair deletion of phenylalanine codon at position 508 of CFTR gene (Deletion).
  • Huntington disease: >40 repeats of (CAG) sequence in the coding region of Huntingtin gene (Trinucleotide repeat expansions).

Types of Mutations and Their Effects

• Missense Mutation:
  • Involves changing an amino acid.
  • Example: Glycine → Arginine.
• Nonsense Mutation:
  • Results in an early stop codon.
  • Positional effect matters: If it's close to the actual stop codon, the phenotype is usually a weak loss of function. If it's close to the start, the phenotype is a strong loss of function or null.
• Frameshift Mutation:
  • Considered terrible regardless of position due to knocking the protein out of frame.
• Insertions and Deletions:
  • Have different effects depending on the size.
  • Inserting a single base when the index is 3 results in a frameshift mutation.
• Triplet Repeats:
  • Cannot be fixed and are post-reproductive.
  • More repeats increase the likelihood of the protein not functioning correctly.
  • More repeats means more chance of the protein not functioning properly.

Mutations in the HBB Gene That Cause β-Thalassemia

• 5' Upstream Region:
  • 22 known mutations occur between -101 and -25 upstream from the transcription start site.
  • Example: A T → A transition in the TATA sequence at -30 results in decreased gene transcription and severe disease.
• mRNA CAP Site:
  • 1 known mutation: A → C transversion at +1 position leads to decreased levels of mRNA.
• 5' Untranslated Region:
  • 3 known mutations at +20, +22, and +33 cause decreases in transcription and translation and mild disease.
• ATG Translation Initiation Codon:
  • 7 known mutations alter the mRNA AUG sequence, resulting in no translation and severe disease.
• Exons 1, 2, and 3 (Coding Regions):
  • 36 known mutations including missense and nonsense mutations, and mutations that create abnormal mRNA splice sites.
  • Disease severity varies from mild to extreme.
• Introns 1 and 2:
  • 38 known mutations
  • Single base-pair transitions and transversions that reduce or abolish mRNA splicing and create abnormal splice sites that affect mRNA stability.
  • Most cause severe disease.
• Polyadenylation Site:
  • 6 known mutations.
  • Single base-pair changes in the AATAAA sequence reduce the efficiency of mRNA cleavage and polyadenylation, yielding long mRNAs or unstable mRNAs.
  • Disease is mild.
• Throughout and Surrounding the HBB Gene:
  • (>100) known
  • Short insertions, deletions, and duplications that alter coding sequences, create frameshift stop codons, and alter mRNA splicing.

Thymine Dimers

• Formation:
  • Thymine dimers are created by forming covalent bonds between adjacent thymine bases on a single strand of DNA.
• Replication Issues:
  • Cells struggle to replicate past thymine dimers.
  • DNA polymerase may insert random bases due to the inability to replicate the dimer.

Postreplication Repair

• Process Overview:
  • DNA is unwound before replication.
  • Replication skips over the lesion, leaving a gap.
  • The undamaged complementary region of the parental strand is recombined.
  • The new gap is filled by DNA polymerase and DNA ligase.
• Outcomes:
  • Best-case scenario involves leaving a hole that can be fixed.
  • Potential issues include single-stranded DNA breaks.
  • The process may involve stealing information from the other strand.
  • Once excised you can fill the gap.

Base Excision Repair

• Steps:
  • Recognition of an incorrect base (e.g., uracil) in the DNA duplex.
  • Uracil DNA glycosylase recognizes and excises the incorrect base (Uracil).
  • AP endonuclease recognizes the lesion and nicks the DNA strand.
  • DNA polymerase and DNA ligase fill the gap.
• Considerations:
  • Uracil has to be recognized.
  • Leaving a gap can cause DNA to break.

Nucleotide Excision Repair

• Process:
  • Nuclease excises the lesion in the DNA strand.
  • DNA polymerase I fills the gap using the free 3' end.
  • DNA ligase seals the gap, restoring normal pairing.
• Mechanism:
  • The whole thing is cut out because a distortion in the helix because of damage on a larger scale (series of thymine dimers).
  • involves uvr gene products

Repair of Double-Stranded DNA Breaks

• Process Overview:
  • A double-stranded break is detected, and 5'-ends are digested.
  • 3'-end invades the homologous region of a sister chromatid after the S phase.
  • DNA synthesis occurs across the damaged region using the sister chromatid as a template.
  • Heteroduplex is resolved, and gaps are filled by DNA synthesis and ligation.
• Key Principles:
  • Uses the principle that sister chromatids or homologous chromosomes are identical.
    • bd this is invading a sister chromatid it has to be AFTER s phase
  • Functions as a primer using sequence on homologous sister chromatid using complementary base pairing
• Potential Issues:
  • The process can induce translocation, where DNA moves to an incorrect location.
  • Multiple DNA breaks can lead to DNA segments being inverted, breaking the chromosome.

Lac Operon

• Need to Memorize the lac operon from CH 15., what does the gene make, what does it do Fig 15-8 + don't need to know structural genes.

Transposable Elements (TEs)

• Definition:
  • Transposable elements (TEs)—“Jumping genes” are DNA sequences that move within and between chromosomes.
• Function:
  • Insert themselves into various locations within the genome.
• Occurrence:
  • Found in all organisms; precise function still unknown.