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[ONCG2003] Colorectal Cancer & Ovarian Cancer > 2: Excision Repair Pathways > Flashcards

2: Excision Repair Pathways Flashcards

1
Q

Examples of exogenous damage to DNA?

A
  • UV
  • X-rays
  • Chemicals
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2
Q

What is meant by “endogenous damage to DNA”

A

Damage that occurs within the organism/genome, for example, metabolism, DNA replication errors, and fork stalling

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3
Q

What are the classes/types of DNA damage?

A
  • 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
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4
Q

What are the three excision repair pathways?

A
  • Nucleotide excision repair (NER)
  • Base excision repair (BER)
  • Mismatch repair (MMR)
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5
Q

What are the 3 key steps/mechanisms of all excision repair pathways?

A
  • IDENTIFICATION (of damaged DNA)
  • REMOVAL (of damaged segment)
  • REPAIR (of damaged segment)
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6
Q

What is a small adduct? What is the consequence if it goes unrepaired?

A

A small chemical that gets added to a base.
If unrepaired, it may cause a mismatch during DNA replication.

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7
Q

Give an example of a small adduct and the cause o it?

A

Oxidative stress may cause an addition of oxygen to guanine, to 8-oxoguanine

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8
Q

What kinds of DNA damage might Base Excision Repair correct?

A
  • oxidation
  • deamination
  • simple alkylation
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9
Q

T or F: Base excision repair only corrects mutagenic base lesions?

A

False, it can correct both mutagenic and cytotoxic lesions

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10
Q

What does 8-oxoguanine incorrectly pair to instead of cytosine?

A

Adenine

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11
Q

What does cytosine become after it undergoes deamination, and which repair pathway corrects it?

A

Cytosine -> Uracil (H2O in, NH3 out)
Corrected by base excision repair

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12
Q

What kinds of damage can alkylation generate?

A
  • promutagenic bases
  • cytotoxic lesions
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13
Q

What is an example of a promutagenic base lesions caused by alkylation that can be repaired by BER?

A

O6-methylguanine (6-meG)
Allows pairing with T instead of C

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14
Q

What are 2 examples of cytotoxic lesions caused by alkylation and which repair pathway fixes them?

A
  • N7-methylguanine (7-meG)
  • N3-methyladenine (3-meA)
    Blocks DNA polymerases

Corrected by base excision repair

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15
Q

Briefly outline the steps of the base excision repair pathway

A
  • Recognition of DNAdamage by DNA glycolysase
  • Assembling complexes, and incision of damage by AP Endonuclease (APE)
  • Resynthesisto replace damage site by polymerase and ligase
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16
Q

What is the difference between mono- and bi-functional glycosylases?

A

Monofunctional just excises the base, whereas I functional excises the base and cuts the backbone

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17
Q

How do monofunctional glycosylases work?

A

Uses H2O for nucleophilic attack on N-glycosidic bond

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18
Q

How do bifunctional glycosylases work?

A

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

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19
Q

What does APE1 do

A

Ensures backbone is cleaved and termini are correct so following enzymes can bind.

20
Q

What does APE1 do following a monofunctional DNA glycosylase?

A

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

21
Q

What does APE1 do following a bifunctional glycosylase?

A

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

22
Q

What is the functional of DNA polymerase beta in the BER pathway?

A
  • “tidies up” the 5’-deoxyribosephosphate end created by APE1 after the action of monofunctional glycosylase, to a 5’P end
  • adds the missing nucleotide
23
Q

What is the role of DNA ligase III

A

Seals the nick between 3’OH 5’P
Common in all pathways

24
Q

What is the role of XRCC1

A

Acts as ‘scaffolding’ for proteins to bind during the action of DNA ligase III

25
Q

What happens if the DNA ends are resistant to DNA poly Beta?

A

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.

26
Q

What is the difference between short- and long-patch base excision repair?

A

Short - single nucleotide gap generated and filled
Long - a gap of 2-10 nucleotides generated and filled

27
Q

When might short- and long- patch BER be used?

A

Short -in proliferating and non-proliferating cells
Long - following replication in proliferating cells

28
Q

What proteins are used in short-patch BER?

A

DNA glycosylase
APE1
DNA polyB
DNA ligase 1 or 3
(PARP1, XRCC1)

29
Q

What proteins are used in long-patch BER?

A

DNA glycosylase
APE1
DNA poly e
PCNA
FEN1
Ligase 1

30
Q

What are the consequences if a bulky adduct goes unrepaired?

A

Distorts DNA helix
Blocks DNA replication
Stalls transcription

31
Q

What factors may induce bulky DNA lesions?

A

UV irradiation
Environmental mutagens
Chemotherapeutic agents

32
Q

What does nucleotide excision repair correct?

A

Bulky DNA lesions
Eg pyrimidine dimers formed from covalent bonds

33
Q

T or F: in NER, enzymes that recognise adducts are specific to each type of damage

A

False: one set of enzymes can recognise many substrates, they are all bulky and destabilize the DNA duplex

34
Q

Outline the process of nucleotide excision repair

A
  • 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
35
Q

What are the two types of nucleotide excision repair

A

Global genome repair and transcription coupled repair

36
Q

What is the difference between GG-NER and TC-NER

A

Global genome - contains XPC, detects DNA damage anywhere in genome
Transcription coupled - detected by polyII, repair performed on transcribed strand. Contains CSA and CSB.

37
Q

Which proteins are specific to transcription coupled NER?

A

CSA and CSB

38
Q

Describe the formation and action of the pre-incision complex in NER

A
  • 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
39
Q

List proteins that make up the pre-incision complex in NER

A

XPD
TFIIH
XPA
ERCC1
XPF
XPG
PCNA
RNA poly o/e/k

40
Q

How may dna damage occur during replication

A
  • DNA polymerase adding incorrect base
  • Slippage can occur in areas of repeated nucleotides
41
Q

What does mismatch repair correct?

A

Errors in DNA replication:
- single base mismatches
- small IDLs
- larger IDLs

42
Q

Describe how strand slippage may result in DNA damage during dna replication

A
  • 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.
43
Q

Describe the steps of mismatch repair

A
  • 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
44
Q

Which heterodimer recognises these DNA damages, and what proteins make up the dimer:
1. Single base mismatch
2. Small IDLs
3. Larger IDLs

A
  1. MutSa: MSH2/MSH6
  2. MutSa: MSH2/MSH6
  3. MutSb: MSH2/MSH3
45
Q

Describe the 3 different types of MutL heterodimers and what they do

A
  • MutLa: intrinsic endonuclease activity
  • MutLb: accessory factor for MutLa
  • MutLy: is used instead of MutLa in resolution of meitotic recombination intermediates
46
Q

Which hereditary syndrome is associated with MSI in CRC and ovarian cancer?

Which mutations are common in this syndrome?

A

Lynch syndrome, aka hereditary non-polyposis colorectal cancer (HNPCC)

Mutations in MSH2 or MLH1

47
Q

What is the consequence of having mutations in MLH1 or MSH2

A
  • inability to correct frame shift mutations
  • causes microsatellite instability
  • replication errors are not repaired, leading to accumulation of further mutations

[ONCG2003] Colorectal Cancer & Ovarian Cancer (7 decks)

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