lab 2

gene mutagenesis

creation/generation of mutations in genes

types of mutations

• nucleotide

  • substitution (X → Y)

  • insertion (XY → XAY)

  • deletion (ABC → AC)

  • frameshift

    • caused by loss of nucleotide in group not divisible of 3

• large-scale chromosomal rearrangements

  • duplication

  • deletion

  • inversion

    • may be epigenetically modified (ex: now next to heterochromatin)

  • translocation

genome instability

result of competing DNA modification and DNA repair pathways

DNA modification rates

• 2000-10000 hydrolytic depurinations every day/cell

• ~1 cytosine deamination every 5 days/cell

• ~1 event of guanidine oxidation (→ 8-oxo-guanine) every 5 days/cell

• ~600 events of methylation of adenine (→ 3-meA) per day/cell

DNA repair pathways

• base excision repair

• nucleotide excision repair

• mismatch repair

• homologous recombination

• non-homologous end-joining

evolution of mutation rate

• viruses, bacteria, archaea: mutation rate inversely correlates with genome size

• archaea, bacteria, eukaryotes: mutation rate scales with genome size

  • longer DNA replication times → more time for errors to occur

• SARS-CoV-2: evolution of virus variants trackable using point mutations

sources of mutations

• most incorporated occur during DNA replication

• replicative DNA polymerases have very high fidelity → make mistake every ~10⁴ nucleotides incorporated

• activity of proofreading exonucleases → additional increase in fidelity 100x

• mismatch repair systems → additional increase ~1000x

• in total: probability of incorporating a single mistake of 10^-9-10^-10 per nucleotide in human cells

mutagenesis from UV/sunlight exposure

• direct DNA damage

• cyclobutane thymine dimer formation

• up to 50-100 reactions/second might occur in skin cell during exposure

• lesions repaired by photolyase (microorganisms, plants, animals other than humans) and nucleotide excision repair

mutagenesis from ionizing radiation

• direct DNA damage creating double-stranded breaks

• used to treat cancer

• presence of DNA damage induces apoptosis

mutagenesis from benzo(a)pyrene

• polycycling hydrocarbon (PCH) and potent carcinogen

• exposure from burning of organic material, tobacco smoke, grilled meats, asphalt

• must be processed by liver to become carcinogenic

• causes mismatch/point mutations (adduct)

mutagenesis from aflatoxin B₁ (Aspergillus flavus)

• fungal toxin and potent carcinogen

• exposure from contaminated plant products or meat/dairy products of animals that ate contaminated feed

• must be processed by liver to become carcinogenic

• causes mismatch/point mutations (adduct)

Ames test

tests ability of substances to revert mutations in a strain of bacteria (mutagenicity)

steps of Ames test

1. rat liver (containing liver enzymes) + Salmonella strain (auxotrophic mutant requiring histidine for growth)

2. experimental plate

   1. + possible mutagen

   2. plate on media with minimal histidine

   3. incubate

   4. high number of revertants (his- → his+) suggests mutagen causes mutations

3. control plate

   1. plate on media with minimal histidine

   2. incubate

   3. natural revertants

random mutagenesis methods in lab

• mutagenic bacterial strains

  • specially engineered or selected microorganisms with increased susceptibility to DNA changes

• mutagens

  • EMS/MMS

  • highly toxic

• error-prone PCR

  • manipulating PCR reaction to become lower fidelity

typical PCR reaction components

• template DNA

• primers (short complementary single strand DNA fragments)

• dNTPs (deoxynucleoside triphosphates)

• buffer (containing Mg²⁺), ddH₂O

• thermostable DNA polymerase (Taq)

steps of typical PCR reaction

1. denaturation (98°C)

   • separate DNA strands

2. annealing (48-72°C)

   • allow primers to base pair to complementary DNA template

3. extension (68-72°C)

   • polymerase extends primer to form nascent DNA strand

Taq polymerase

• derived from hyperthermophile Thermus aquaticus in Yellowstone mushroom pool

• heat-stable

• does not have proofreading ability

  • polymerases with proofreading have 1-2 orders of magnitude greater fidelity → good for preserving original sequence amplified

proofreading exonuclease

1. wrong base is incorporated and detected immediately

2. transcript is pulled back and new strand is directed to exonuclease active site

3. mispaired base is removed

4. transcript is returned to polymerase domain

5. correct base is incorporated

methods of modulating mutation rate of PCR

• using error-prone DNA polymerase

• altering ratio of dNTPs so there is an imbalance

• adding mutagenic nucleotide analogues

• increase [Mg²⁺] and/or replacing Mg²⁺ with Mn²⁺

effect of altering ratio of dNTPs in PCR

changing ratio favours mutations (scarcity of correct dNTPs)

effect of using nucleotide analogues in PCR

• ambiguous nucleotides → can pair with multiple other nucleotides

• 8-oxo-guanine → C or A

• dPTP → G or A

effect of increasing Mg²⁺ or replacing Mg²⁺ with Mn²⁺ in PCR

• Mg²⁺ helps coordinate growing end of growing chain with incoming dNTPs and in discrimination of nucleotides

• excess of Mg²⁺ interferes with polymerase’s ability to position Mg²⁺ correctly → blocks ability to sense errors

• presence of Mn²⁺ permits incorrect incorporation by increasing rate of dNTP incorporation and time that an incorrect dNTP remains at catalytic site

consequences of single nucleotide replacements

• premature stop codons

• silent mutations

• conservative amino acid changes

• amino acid changes with impact on protein function

DpnI restriction endonuclease digestion

• recognizes methylated DNA at GATC site → cleaves

• does not recognize PCR products since they are not methylated (only cleaves plasmid DNA generated in E. coli)

agarose gel electrophoresis

• movement of negatively-charged DNA in an electrical field

• size-dependent retention in agarose gel matrix

  • small fragments migrate faster

• visualization of bands using DNA-intercalating dye (mixed into gel)