Lecture Notes on Mutations and DNA Repair Mechanisms

Mutations

  • Goals of Lecture:
    • Different types of mutations
    • DNA repair mechanisms

Supplementary Resources

  • Chelsea Marks: Supplementary TA Zoom meetings
    • Mondays, Tuesdays, & Fridays, 1:15-2:30 PM
    • Zoom link: https://ccny.zoom.us/j/6746950051?pwd=llT8XQk2A6Zkzd8L Q3bCo4pdBgzbbi.1&omn=87606418947
  • Datius Mai: Tutor
    • Tuesdays/Thursdays, 2-3:30 PM, room MR403
  • Andrew Blake: Workshop leader
    • Wednesdays, 3-4:30 PM; Thursdays, 11 AM-1 PM, room MR403
  • Note: Course standing should be based on the instructor's grade breakdown, not Brightspace.

Mutation Induction

  • X-rays Induce Mutations
    • Example using Bar-eye females in Drosophila
    • Muller found matings where only Bar eye males were observed, indicating X-rays can cause lethal mutations.

Mutagens and DNA Alteration

  • How Mutagens Alter DNA: Chemical Action of Mutagens
    • All endogenous mutagens are also found as environmental mutagens
    • Deaminating agents

Environmental Mutagens

  • Environmental Agents That Can Act as Mutagens:
    • Increase levels of reactive oxidative species (ROS)
      • Sources: Inflammation, chronic stress, tumors, irradiated food, chlorinated dioxins, furans, biphenyls (forest fires/incinerators, byproduct of herbicides, bleach, cigarette smoke).
    • Double stranded breaks
      • Sources: X-rays, UV boxes (tanning machines), radiation treatment.

Chemical Mutagens

  • Intercalating agents: Inserts between bases
  • Replace a base: Base analogs - chemical structure almost identical to normal base

Impact of Unrepaired Mutations

  • Germ line mutations
    • Occur in gametes or in gamete precursor cells
    • Mutation does not affect parent
    • Mutation transmitted to next generation
    • Provides raw material for natural selection
  • Somatic mutations
    • Occur in non-germ cells
    • Not transmitted to next generation of individuals
    • Can affect survival of an individual
    • Can lead to cancer

DNA Repair Importance

  • Effect of mutation depends on whether mutation in somatic or germline cells.
  • DNA repair must occur before replication, or the mutation is permanent.

DNA Repair Mechanisms: High Accuracy

  • Proofreading during DNA replication
    • Mispaired base is recognized and excised by 3'-to-5' exonuclease activity of DNA polymerase
    • Improves fidelity of replication 100-fold
  • Reversal of DNA base alterations
    • Alkyltransferase – removes alkyl groups
    • Photolyase – splits covalent bond of thymine dimers in presence of light (only in prokaryotes).
  • Homology-dependent repair of damaged bases or nucleotides
    • Base excision repair
    • Nucleotide excision repair
  • Correction of DNA replication errors
    • Methyl-directed mismatch repair

Base Excision Repair

  1. Deaminated cytosine DNA (looks like uracil)
  2. Glycosylase cleaves deaminated cytosine from sugar, leaving an AP site
  3. AP endonuclease nicks backbone at the AP site.
  4. DNA exonucleases remove nucleotides near the nick, creating a gap
  5. DNA polymerase I/b synthesizes new DNA to fill in the gap.
  6. DNA ligase seals the nick.
  • Different glycosylases cleave specific damaged bases (e.g., deaminated, apurinated, mismatched, & alkylated bases) and SSBs.
  • At least 11 glycosylases in mammals.
  • AP=apurinic/apyrimidinic deaminated cytosine released

Nucleotide Excision Repair (NER)

  1. Exposure to UV light.
  2. Thymine dimer forms.
  3. UvrA in complex with UvrB recognizes DNA distortion region.
  4. Once lesion found, UvrA dissociates.
  5. UvrB now forms complex with UvrC. UvrC has two endonuclease pockets to nick 5’ and 3’ to thymine dimer.
  6. Damaged single-stranded fragment is released from DNA by UvrD (helicase).
  7. DNA polymerase I/k/d/e fills in the gap with new DNA and ligase re-seals the repaired strand.

Methyl-Directed Mismatch Repair

  • In Bacteria

    • Parental DNA strand marked by adenine methylase
      • Methyl group added to A in GATC sequence
      • Newly-replicated DNA is not yet methylated
    • MutS recognizes mismatches
    • With MutL recruits & activates MutH, which has endonuclease activity
    • MutH nicks the unmethylated strand opposite the methylated GATC
    • Gap made in unmethylated (new) strand by DNA exonucleases.
    • Gap filled in by DNA polymerase III using the methylated (old) strand as template; fragments ligated.
    • New strand gets methylated.

  • In Eukaryotes

    • DNA repair enzymes: MutSa/MutSb (several isoforms of both) and MutLa /MutLb in complex with other proteins.
    • MutL proteins have endonuclease activity.
    • MutH recognizes parental vs newly synthesized strand.
    • Process similar in eukaryotes, BUT methylation not used to distinguish parental vs newly synthesized strand.
    • How parental and newly synthesized strands distinguished still unclear.
    • Mutations in human homologs (e.g., MutSa and MutLa) increases rate of colorectal cancer.
    • In mice, mutations induce lymphoma, GI, skin, and other tumors

DNA Repair Mechanisms: Error-Prone

  • SOS system – bacteria
    • Used at replication forks that stalled because of unrepaired DNA damage