Lecture 5: DNA Repair Pathways

1. Mutations and DNA damage

• Spontaneous mutations

  • due to natural process in cells

    • Ex- DNA rep errors

• Inuced mutations

  • due to interactions of DNA with outside agent that leads to DNA damage

    • UC radiation

• mutations lead to genetic variation that led to evolutionary changes

Transitions and transversions

• Transition mutations → replace one pyrimidine base with another, or one purine base with another

• Transversion mutations→ rep pyrimidine wt purine base and vs

• Point mutations→ mutation that alter a single nucleotide

  • silent mutation

  • missense mutations

  • nonsense

Point mutation (sub) types

• Silent mutations→ change nu without changeing AA sequence

• Missense mutations→ change in protein-coding region that lead to change in AA

  • EX; sickle cell anemia At→ TA

• Nonsense→ nu sub that leads to stop codon (premature termination of protein synthesis

-know the picture adn what final pattern looks like

Insertions or deletions can cause frameshift mutations

• changes the mRNA, which leads to creation of nonfunctional protein

• If it is not in pairs fo 3, it wil cause frameshift mutation

Repeat expansion found in 2 diff mechanism

• Unequal crossing over

• Slippage during DNA replication

3 classes of DNA damage

• single base change

  • Deamination

• structural distortions

• DNA backbone damage

Single Base Change

• Deamination

  • most frequence kind of hydrolytic damage

    • deamination of 5-methylctosine generates thymine

  • alkylation agents(ex: nitrosamines) lead to creation of O6-methylgyanosine

    • mispairs with thymine

    • GC→GT→AT point mutation after DNA Rep

Structural distrtion

UV radiation cleates cyclobutane ring btw thymines →T-T dimer impacting double helix and block transcription and replication

DNA backbone damage

• Formation of abasic sites

  • loss of nitrogenious bae

  • generates spontaneously by creation of unstable base adducts

• Double-standard DNA breaks

  • due to ionizing radiation and a lot of chemical compounds

  • leads to DNA damage

2.lesion bypass

Translesion synthesis (TLS)

error prone DNA polymerase replace replicative polymerases and copy paste with damaged DNA

• DNA polymerase (n) performs translesion syn past TT dimers by inserting AA.

3. Direct reversal of DNA damage

Reversal of thymine-thymine dimers by DNA

• DNA photolyase use energy of blue light to break covalent bonds holding 2 pyrimidines together

• humans do not have photoreactive pathway

-BUT

• DNA methyltransferase does damage reversal

4. Repair of single base changes and structural distortions by removal of DNA damage.

Pathways for repair of single base changes and structural distortion

• single base change

  • base excision repair

  • mismatch repair

• structural distortion

  • nucleotide excision repair

• single base change

  • base excision repair

  • Mismatch repair

• Base excision repair

  • DNA glycosylase recognizes and excises the damaged base

  • endonuclease Cleves the phosphodiester bond at either 3’ or 5’ of abasic site

    • removes 1-10 nucleotides

  • DNA polymerase replaces missing nucleotides

  • DNA ligase seals gap

• Mismatch repair pathway in mammalian cells

  • Mismatch error due to DNA duplication

    • rec by MutS”alpha”/ MitL”alpha”

  • Single-strand break of 5’ or 3’ generated by EXO1 with help from PCNA and RFC

  • 5’→3’ or 3’→5’ exonuclease activity of EXO1 removes mismatch and other nucleotides

  • 5’-3’ repair synthesis mediate by DNA polymerase

  • remaining gap ligased by DNA ligase 1

Recurring theme in DNA Repair

• Hand-off of damaged DNA from a complex with nuclease activity to a complex with polymerase activity to a complex with ligase activity.

• structural distortion

  • nucleotide excision repair

• nucleotide excision repair

  • Repair of structural distortion

    • from T-T dimers from UV irradiation

  • GGR pathway (Global genome repair)

    • repair of lesions in whole genome

  • TCR pathway (Transcription couples repair): repair of lesions in the transcribed strand of active genes.

Xeroderma pigmentosum and related disorders: defects in nucleotide excision repair

• autosomal recessive disorder

• inc risk of sunlight-induces skin cancer

• Defects in nucleotide excision repair or in T-T dimer translesion repair.

5. Double-strand break repair

• due to oxygen species, ionizing radiation, and the free radical chemicals made

• repaired by homologous recombination or nonhomologous end-joining

Repair by recombination

• Homologous recombination

  • repair double strand break by getting genetic material from undamaged homologous chromosomes

• Nonhomologous end-joining (NHEJ)

  • rejoins double-strand break via direct ligation of DNA end withNO any prior sequence homology

  • HOM recomb has a big role in doub-str break repair in prokaryotes and single-cell eukaryotes

  • double strand break primarily repaired through NHEJ in mammalian cells

    • homologous recombination primarily servs to repair double strand breaks at the replication fork.

Homologous recombination

• many roles in eukaryotic organisms

  • crossing over in meiosis

  • transposition

  • mating-type switching in yeast

  • antigen-switching in trypanosomes

  • dna repair

Model for mammalian DNA double-strand break repair by homologous recombination

• Steps

  • Double-strand break (DSB) is induced by ionizing radiation.

  • End-processing and recognition: Recruitment of MRN (Mre11-
    Rad50-Nbs1) to the DSB. The 3′, 5′ exonuclease activity of Mre11
    generates 3′ ssDNA tails that are recognized by Rad52.

  • Strand invasion and DNA synthesis. The 3′ tails invade
    homologous intact sequences to generate a hybrid molecule.
    Missing sequence information at the DSB is restored by DNA
    synthesis.

  • Branch migration

  • Holliday junction resolution and ligatio

Holliday junctions

• 1960s→ RObin made a model of general recombination based on genetic data from fungi

  • 2 duplexes by exzyme complex called Resolvasome

    • in E.Coli RuvABC resolves Holliday junctions

    • Rad51C required for Holliday junction processing in mammalian cells

Hereditary breast cancer syndromes: mutations in BRCA1 and BRCA2

• 5-10% all cases

• mutation on BRCA1 and BRCA2" “tumor suppressor genes”

• risk of breast (ovarian) cancer

  • BRCA1: 50-87%

  • BRCA2: 15-44%

Nonhomologous end-joining

• 2 broken ends is ligated together even if they came from same chromosome

• double-strand break repair leads to mutations and Indels (inserts/deletions)

Model for mammalian DNA double-strand break repair by nonhomologous end-joining

1. Double-strand break.

   • induced by ionizing radiation.

2. End recognition.

   • Broken ends are recognized by heterodimers of
     Ku70/Ku80.

3. End processing.

   • endonuclease Artemis activated by the DNA-dependent protein kinase catalytic subunit (DNA-PKCS). DNA polymerase (pol) “u” or pol “y” fill-in gaps and extend 3′ or 5′ overhangs.
     

4. End bridging

   • Ligase complex XRCC4-DNA ligase IV is recruited to the damaged site and forms a bridge.

5. Ligation:

   • broken ends ligated by the XRCC4-DNA ligase IV complex.