DNA Repair Flashcards
What can DNA damage be?
spontaneous or mutagen-induced
Give examples of spontaneous DNA damage
- depurination
- deamination
Give examples of mutagen-induced DNA damage
- pyrimidine dimers
- alkylation
- substitution
- deletions/insertions
- frameshift mutations
- DSBs
What are the 2 DNA related point mutations?
- transitions (purine or pyrimidine is replaced by another)
- trasversions (purine is replaced by a pyrimidine or vice versa)
What happens if depurination or deamination are left uncorrected?
deletion or substitution of base pairs during DNA replication
What does nitrous acid do?
oxidatively deaminate primary amines, producing transition mutations
What recognises unnatural nucleotides?
specific DNA glycosylases
When does DNA mispairing occur?
when methylated C is converted to T by deamination
When do pyridine and thymine dimers form?
when cells are exposed to UV irradiation
What are alkylating agents?
chemicals that add an alkyl group to another molecule
What do intercalating agents do?
generate insertion/deletion mutations and increase the distance between 2 consecutive base pairs
What does replication of DNA with insertion/deletion mutations do?
generate deletion or insertion of one or more nucleotides in the newly synthesised DNA i.e. frameshift mutation
What is Ames test used for?
to assess mutagenicity of compounds using bacterial strain Salmonella typhimurium which is unable to grow and form colonies in a medium lacking histidine
What are the 5 main DNA repair pathways?
- direct repair
- excision repair
- mismatch repair
- recombination repair
- non homologous end joining
What are the 4 types of DNA repair systems in bacteria?
- repair of synthesis errors
- repair of DNA modifications
- repair of replication fork barriers by translesion synthesis
- repair of breaks in DNA by homologous recombination and non-homologous end joining
What does proofreading by DNA pol do?
decrease errors introduced during DNA synthesis by 1000-fold
What does mismatch repair in E. coli do?
correct errors that remain after proofreading and reduce errors by a further 100-fold
What does mismatch repair involve in E. coli?
the MutSLH system, which depends on the methylation of selected A residues in GATC to distinguish newly synthesised vs template DNA
What are the 5 steps of mismatch repair in E. coli?
- MutH binds to the dam site and MutS recognises that there is a mismatch
- MutL bind to the MutS (ATPS)
- after the MutS has recognised the damage, it communicates to MutH which becomes activated as a nuclease
- it cleaves the newly synthesised unmethylated strand and acts in the same way as an NER
- DNAPIII then synthesises a new strand, ensuring the correct nucleotides are used
What are the targets for Dam methylase after replication?
GATC sequences
What does photoreactivation repair do?
remove pyrimidine dimers via a light-dependent reaction
Where does photoreactivation repair occur?
bacteria but not placental mammals
What are photolyases?
photo-reactivating enzymes made up of 2 tightly bound chromophores that repair pyrimidine dimers by using light energy and a very rapid photochemical reaction
What are the 5 steps of photoreactivation repair?
- enzyme binds to DNA containing pyrimidine dimer
- absorbs visible light (300-500nm) using second chromophore
- energy transferred to FADH2
- FADH2 transfers electron to pyrimidine dimer
- pyrimidine dimer splits into monomeric form
What does BER do?
remove only the damaged base via DNA glycosylase
What does DNA glycosylase do?
cleave the glycosidic bond leaving an apurinic or apyrimidinic site
What are the 4 enzymes involved in BER?
- AP endonuclease
- DNA pol I
- DNA ligase
- DNA glycosylase
What does NER involve in E. coli?
- ATP
- UvrABCD proteins
What is UvrA?
ATPase that recognises damage with 2 ATP binding sites that binds damaged DNA
What is UvrB?
monomer with helicase motifs but no ATPase or helicase activity that is activated by UvrA
What is UvrC?
nuclease monomer that binds ssDNA non-specifically and does not interact with UvrA or B in solution
What are the 4 steps of NER in E. coli?
- UvrA recognises damage and binds with UvrB
- UvrA is released and UvrC binds
- UvrC nicks DNA on both sides of damage
- UvrD unwinds region, releasing damaged strand
Give examples of conditions in which UV-induced DNA lesions cannot be repaired
- xeroderma pigmentosum
- Cockayne syndrome
What happens when a lesion is encountered during replication?
DNA Pol III is replaced by error-prone translesion DNA polymerase, Pol IV or Pol V
What does translesion DNA polymerase do?
extend DNA synthesis beyond thymine dimer independent of base pairing and has no proofreading exonuclease activity
Where is DSB repair by non-homologous end joining (NHEJ) common?
mammalian somatic cells
What does Ku do?
splice the 2 fragments together at the break site
What are the 4 stages of NHEJ?
- end recognition by Ku heterodimers
- processing of DNA ends
- limited repair synthesis
- ligation
How can the replication fork collapse?
through homologous recombination repair
When are DSBs produced?
- replication fork encountering an ss nick in the template DNA
- ionising radiation
- replication errors
- oxidising agents
