2: Excision Repair Pathways Flashcards
Examples of exogenous damage to DNA?
- UV
- X-rays
- Chemicals
What is meant by “endogenous damage to DNA”
Damage that occurs within the organism/genome, for example, metabolism, DNA replication errors, and fork stalling
What are the classes/types of DNA damage?
- loss of base (apurinic/apyrimidinic site)
- small adducts, eg addition of O2 or methyl group
- Bulky adducts, eg addition of large chemical group
- Single strand breaks
- Double strand breaks
- Mismatched bases
- Crosslinks
What are the three excision repair pathways?
- Nucleotide excision repair (NER)
- Base excision repair (BER)
- Mismatch repair (MMR)
What are the 3 key steps/mechanisms of all excision repair pathways?
- IDENTIFICATION (of damaged DNA)
- REMOVAL (of damaged segment)
- REPAIR (of damaged segment)
What is a small adduct? What is the consequence if it goes unrepaired?
A small chemical that gets added to a base.
If unrepaired, it may cause a mismatch during DNA replication.
Give an example of a small adduct and the cause o it?
Oxidative stress may cause an addition of oxygen to guanine, to 8-oxoguanine
What kinds of DNA damage might Base Excision Repair correct?
- oxidation
- deamination
- simple alkylation
T or F: Base excision repair only corrects mutagenic base lesions?
False, it can correct both mutagenic and cytotoxic lesions
What does 8-oxoguanine incorrectly pair to instead of cytosine?
Adenine
What does cytosine become after it undergoes deamination, and which repair pathway corrects it?
Cytosine -> Uracil (H2O in, NH3 out)
Corrected by base excision repair
What kinds of damage can alkylation generate?
- promutagenic bases
- cytotoxic lesions
What is an example of a promutagenic base lesions caused by alkylation that can be repaired by BER?
O6-methylguanine (6-meG)
Allows pairing with T instead of C
What are 2 examples of cytotoxic lesions caused by alkylation and which repair pathway fixes them?
- N7-methylguanine (7-meG)
- N3-methyladenine (3-meA)
Blocks DNA polymerases
Corrected by base excision repair
Briefly outline the steps of the base excision repair pathway
- Recognition of DNAdamage by DNA glycolysase
- Assembling complexes, and incision of damage by AP Endonuclease (APE)
- Resynthesisto replace damage site by polymerase and ligase
What is the difference between mono- and bi-functional glycosylases?
Monofunctional just excises the base, whereas I functional excises the base and cuts the backbone
How do monofunctional glycosylases work?
Uses H2O for nucleophilic attack on N-glycosidic bond
How do bifunctional glycosylases work?
Uses an amino group of lysine chain, forming an intermediate with cleaves the DNA backbone 3’ to the lesion.
Leaves a 5’ phosphate and a 3’-a,b-unsaturated aldehyde
Excises the base and cuts the backbone
What does APE1 do
Ensures backbone is cleaved and termini are correct so following enzymes can bind.
What does APE1 do following a monofunctional DNA glycosylase?
APE1 recognises the AP site generated by the monofunctional glycosylase
APE1 cleaves the backbone, resulting in the formation of a single nucleotide hap flaked by 3’-hydroxyl and 5’-deoxyribosephosphate (5’-dRP) ends
What does APE1 do following a bifunctional glycosylase?
The backbone is cleaved by the glycosylase
APE1 cleaves the 3’-a,b-unsaturated aldehyde to generate a 3’hydroxyl end alongside the 5’P
What is the functional of DNA polymerase beta in the BER pathway?
- “tidies up” the 5’-deoxyribosephosphate end created by APE1 after the action of monofunctional glycosylase, to a 5’P end
- adds the missing nucleotide
What is the role of DNA ligase III
Seals the nick between 3’OH 5’P
Common in all pathways
What is the role of XRCC1
Acts as ‘scaffolding’ for proteins to bind during the action of DNA ligase III
What happens if the DNA ends are resistant to DNA poly Beta?
DNA polymerase delta/epsilon adds bases to the ‘OH end, into the single nucleotide gap.
Creates a FLAP structure which can be excised by FEN1/PCNA , ends can be sealed by ligase I.
What is the difference between short- and long-patch base excision repair?
Short - single nucleotide gap generated and filled
Long - a gap of 2-10 nucleotides generated and filled
When might short- and long- patch BER be used?
Short -in proliferating and non-proliferating cells
Long - following replication in proliferating cells
What proteins are used in short-patch BER?
DNA glycosylase
APE1
DNA polyB
DNA ligase 1 or 3
(PARP1, XRCC1)
What proteins are used in long-patch BER?
DNA glycosylase
APE1
DNA poly e
PCNA
FEN1
Ligase 1
What are the consequences if a bulky adduct goes unrepaired?
Distorts DNA helix
Blocks DNA replication
Stalls transcription
What factors may induce bulky DNA lesions?
UV irradiation
Environmental mutagens
Chemotherapeutic agents
What does nucleotide excision repair correct?
Bulky DNA lesions
Eg pyrimidine dimers formed from covalent bonds
T or F: in NER, enzymes that recognise adducts are specific to each type of damage
False: one set of enzymes can recognise many substrates, they are all bulky and destabilize the DNA duplex
Outline the process of nucleotide excision repair
- RECOGNITION: repair recognition complexes recognise DNA damage
- ASSEMBLING complexes and INCISION of the damage, via DNA helicases and nucleuses
- RESYNTHESIS to replace damage site via polymerase and ligase
What are the two types of nucleotide excision repair
Global genome repair and transcription coupled repair
What is the difference between GG-NER and TC-NER
Global genome - contains XPC, detects DNA damage anywhere in genome
Transcription coupled - detected by polyII, repair performed on transcribed strand. Contains CSA and CSB.
Which proteins are specific to transcription coupled NER?
CSA and CSB
Describe the formation and action of the pre-incision complex in NER
- TFIIH has helicase activity to unwind DNA around adduct
- XPD subunit stalls at damage site
- recruits further proteins to form pre-incision complex
- XPA positions proteins and recruits ERCC1 and XPF to perform 5’ incision
- DNA synthesis initiated. XPG performs 3’ incision
List proteins that make up the pre-incision complex in NER
XPD
TFIIH
XPA
ERCC1
XPF
XPG
PCNA
RNA poly o/e/k
How may dna damage occur during replication
- DNA polymerase adding incorrect base
- Slippage can occur in areas of repeated nucleotides
What does mismatch repair correct?
Errors in DNA replication:
- single base mismatches
- small IDLs
- larger IDLs
Describe how strand slippage may result in DNA damage during dna replication
- newly synthesised loop slips out in an area of single, di-, or tri-nucleotide repeats
- an extra nucleotide is added to the strand
Or - template strand loops out
- a nucleotide is omitted on the new strand.
Describe the steps of mismatch repair
- MutS (a or b) heterodimer binds mismatch and clamps shut (uses ATP), following along after DNA polymerase during replication
- PMS2 makes single strand break
- Exo1 excises new DNA
- Polymerase copies template
- Ligase seals ends
Which heterodimer recognises these DNA damages, and what proteins make up the dimer:
1. Single base mismatch
2. Small IDLs
3. Larger IDLs
- MutSa: MSH2/MSH6
- MutSa: MSH2/MSH6
- MutSb: MSH2/MSH3
Describe the 3 different types of MutL heterodimers and what they do
- MutLa: intrinsic endonuclease activity
- MutLb: accessory factor for MutLa
- MutLy: is used instead of MutLa in resolution of meitotic recombination intermediates
Which hereditary syndrome is associated with MSI in CRC and ovarian cancer?
Which mutations are common in this syndrome?
Lynch syndrome, aka hereditary non-polyposis colorectal cancer (HNPCC)
Mutations in MSH2 or MLH1
What is the consequence of having mutations in MLH1 or MSH2
- inability to correct frame shift mutations
- causes microsatellite instability
- replication errors are not repaired, leading to accumulation of further mutations
