L9, DNA repair defects (XP and NER) Flashcards
1
Q
Clinical features of XP:
A
- Rare, AD
- ~2.3 per million live births in Europe
- Dermatological: Dry skin, abnormal pigmentation, skin cancer predisposition, eye abnormalities, susceptible to sunburn (~50%)
- Neurological: ~30% of patients; progressive neurological degeneration with neuronal degeneration
- First characterised by skin cancer predisposition
2
Q
How does UV radiation damage DNA?
A
- Pyrimidine dimers (preferentially absorbed) -> covalent bonds between adjacent T or C nts
- -> Helix distortion, blocking DNA synthesis leaving a gap opposite the site of damage which is filled by translesion polymerases (Low fidelity!)
3
Q
2 potential products of UV irradiation:
A
Two types of pyrimidine dimer:
- Crosslinking of 6, 5 positions -> cyclobutane pyrimidines aka CPDs (small distortion)
- Linking of 6, 4 position -> 6, 4 photoproducts (large distortion)
4
Q
5 broad steps in NER pathway:
A
- DNA damage recognition
- DNA unwinding open pre-incision complex
- Double DNA incision
- Oligonucleotide excision and repair synthesis
- DNA ligation
5
Q
Cellular features of classical XP patients:
A
- Increased sensitivity to DNA damaging agents
- Increased mutability
- Reduced DNA repair synthesis (aka unscheduled DNA synthesis, UDS)
6
Q
How were the different complementation groups for XP identified?
A
- in vivo assay
- ?
- Complementation groups identified by analysis of UDS in heterodikaryons produced by fusing cells from different patients
- UDS is able to be restored when different groups match
7
Q
What are complementation groups between XP patients?
A
- Different complementation groups have a defect in a different gene
- 8 possible groups
- Groups A through G are NER deficient
- XP-V is NER proficient
8
Q
Clinical features of XP-A, B, D, F, G:
A
- Severe sunburn from early age
- Early diagnosis allows preventative UV protection, reducing skin cancers
- Neurological abnormalities
9
Q
XP-C, E clinical symptoms:
A
- Normal sunburn for skin type - later diagnosis -> delayed preventative UV protection - increased skin cancers
- No neurological abnormalities
10
Q
Review: 5 steps in NER pathway and where the various XP defects fall:
A
- DNA damage recognition - XP-C, E
- DNA unwinding open pre-incision complex - XP-B, D, A
- Double DNA incision - XP-F and XP-G
- Oligonucleotide excision and repair synthesis
- DNA ligation
11
Q
XP-C defective cells/role of XPC:
A
- Phenotypically heterogenous for UV^S
- XPC copurifies with human homolog of RAD23 as well as centrin 2
- Binds diverse, helix distorting, damage containing substrates -> preferentially binds 6-4PPs over CPDs
- Binds to undamaged strand, detects free bases opposite damage
12
Q
XP-E defective cells/ role of XPE:
A
- Only mildly UV^s
- Near normal NER
- XPE found complexed with DDB1 in UV-DDB
- DDB binds specifically to damaged DNA, ‘flips out’ damaged bases
- Creates ssDNA to facilitate XPC binding
- XPE expression is induced in p53 dependent manner in response to UV irradiation
13
Q
How do XPB and XPD function in NER ?
A
- Both are ATP dependent DNA helicases
- Subunits of TFIIH
- Recruited to damage by interaction with XPC
- RPA binds undamaged strand
14
Q
What is the role of XPA in NER:
A
- Verifies damage
- Positions repair machinery correctly around damaged site
- Interacts with RPA, TFIIH and XPF-ERCC1
15
Q
What are the roles of XPF and XPG in NER?
A
- XPF-ERCC1 induce nick at 5’ side of damage
- XPG induces nick adjacent to 3’ side
- Both must interact with TFIIH and XPA
