RecQ helicases and XPD in NER Flashcards
(29 cards)
1
Q
Which outcome of HR is promoted by BLM?
A
Non-crossover
2
Q
What is the role of WRN?
A
Removes structures at telomeres so they can be replicated
3
Q
What is the function of helicases? (3)
A
- Disruption of hydrogen bonds between DNA and/or RNA strands
- Translocation of the enzyme along ssDNA fuelled by ATP hydrolysis
- 5’-3’ or 3’-5’ (describing the direction of translocation NOT which strand is being displaced from the duplex)
4
Q
What are 2 examples of helicase families within superfamily 2?
A
- RecQ family
- Fe-S family
5
Q
What is the founding member of the RecQ helicase family? (3)
A
- E. coli RecQ helicase
- Involved in many pathways including processing of DSBs to make 3’ overhangs and working with TopIII to catenate/decatenate DNA
- Best known yeast homologue is Sgs1
6
Q
What are genetic disorders associated with RecQ helicase mutations? (3)
A
- Werner syndrome (WRN)
- Bloom syndrome (BLM)
- Rothmund-Thomson syndrome (RECQ4)
7
Q
What are the characteristics of Werner syndrome? (3)
A
- Accelerated ageing
- Cardiovascular disease
- Cancer predisposition
8
Q
What are the characteristics of Bloom syndrome? (3)
A
- Stunted growth
- Prone to diabetes
- Cancer predisposition
9
Q
What are the characteristics of Rothmund-Thomson syndrome? (4)
A
- Stunted growth
- Light sensitivity
- Cataracts
- Cancer predisposition
10
Q
What is shown by the differences in symptoms between RecQ genetic disorders? (3)
A
- Some overlap but members of the family have distinct roles and can’t substitute for each other
- Lack helicase function = chromosomal instability = cancer (all)
- Rare autosomal recessive disorders
11
Q
What direction does WRN work? (2)
A
- Translocation 3’-5’ along DNA and displacement of the other strand 5’-3’
- Entire RecQ family is same direction
12
Q
What happens to recombination in Bloom syndrome? (3)
A
- Increased recombination (hyperrecombination) in cells deficient in BLM
- Sign of genome fragility and somatic mutations
- High levels of recombination between homologous chromosomes can lead to LoH and cancer
13
Q
What is the role of BLM in recombination? (4)
A
- Promotes DSB processing by Exo1
- Regulation of Rad51-dependent D-loop formation
- Promotion of synthesis-dependent strand annealing (avoids crossovers)
- Promotion of non-crossover outcome
14
Q
How does BLM unwind D loops? (2)
A
- Kicks of Rad51 from the invading DNA strand
- Suppresses mutagenic recombination events as inappropriate recombination leads to chromosomal instability (only doing recombination if completely necessary)
15
Q
How does BLM work with TOPOIIIα? (3)
A
- BLM+TOPOIIIα (+RMI1-2) = the BLM dissolvasome which promotes dissolution of dHJ (non-crossover)
- BLM branch migrates the 2 HJs towards eachother and then TOPOIIIα (type I topoisomerase) creates a nick in one strand of the DNA duplex to decatenate
- Bloom syndrome cells lacking BLM therefore do more resolution than dissolution, leading to LoH
16
Q
How is WRN involved in HR? (4)
A
- WRN-deficient cells show defects in HR (vs hyperrecombination for BLM-deficient cells)
- WRN helps stimulate resection in the early stages of recombination
- Redundant with BLM at some stages
- Also involved in NHEJ depending on the cell cycle phase
17
Q
How do RecQ helicases respond to replication fork stalling? (2)
A
- Either reversal of the fork (Chicken foot structure) using BLM helicase activity which allows lesion bypass/translesion synthesis/lesion repair
- Or cause conversion to a recombination intermediate and break-induced replication (BLM regulates resection and strand invasion)
18
Q
What is the shelterin complex? (2)
A
- 3’ overhang of the telomere end invades the double-stranded region in a D loop-like structure
- Shelerin protein complex (including TRF2) associates with the telomere for protection and to differentiate from a DSB
19
Q
How does WRN interact with telomeres? (3)
A
- WRN associates with the shelterin complex during S phase
- WRN is important for unwinding the D loop at the telomere so that replication can go all the way to the end of the chromosome
- Otherwise the replication fork would terminate which would cause (even greater) gradual telomere loss
20
Q
What is G quadraplex DNA? (4)
A
- Secondary structure in DNA caused by bonds between G bases
- Prevents transcription or replication machinery from proceeding
- Found throughout the genome but especially in the G-rich strand of telomeres
- Both WRN and BLM involved in unwinding these structures to minimise loss of DNA during replication
21
Q
How is BLM linked to the symptoms of Bloom syndrome? (3)
A
- BLM loss causes upregulation of HR
- Causes cancer via LoH
- Involved in telomere replication (BLM expression elevated in many cancers) but not linked to premature ageing (?)
22
Q
How is WRN linked to the symptoms of Werner syndrome? (3)
A
- WRN loss causes downregulation of HR
- Causes cancer via deficiencies in recombination resulting in translocations and deletions
- Causes telomeres to get shorter faster resulting in genomic instability because WRN is important in dismantling D-loop structures during telomere replication, therefore linked to premature ageing unlike BLM
23
Q
What direction do the Fe-S helicases work? (3)
A
- 5’-3’ direction
- Have a reactive iron which may be involved in DNA binding
- E.g. XPD
24
Q
What is caused by mutations in XPD? (2)
A
- Xeroderma pigmentosum (and others)
- Disorders have in common an extreme sensitivity to sunlight because XPD is involved in nucleotide excision repair
25
What is nucleotide excision repair (NER)? (2)
- NER is responsible for repairing bulky adducts caused by UV damage of DNA which distort the helical structure of the duplex
- Cyclobutane pyrimidine dimers disrupt base pairing and block transcription/replication fork etc
26
How does XPD helicase normally exist in the cell? (2)
- Part of the core subcomplex of TFIIH which is a basal transcription factor important for initiation of transcription
- TFIIH also has the CAK (CDK-activating complex) subcomplex which means it interacts with the cell cycle
27
What are the main components of NER? (4)
- 2 important translocases for NER are XPB (3'-5') and XPD (5'-3') which are both ATP dependent
- Damage recognition done by XPC scanning (general pathway) or stalled transcription machinery (transcription-coupled pathway)
- Verification of the lesion by XPD by unwinding the DNA helix to test the distortion caused by the damage
- Excision of the piece containing the damage (24-32 nt), resynthesis and ligation
28
What cellular processes is XPD involved in? (3)
- Arch domain of XPD is involved in dsDNA separation as well as interacting with the transcription machinery
- XPD is also important in transcription but just acts as a scaffold there
- XPD helicase activity is only required in NER not in transcription which is why some mutations cause defects in NER but not transcription
29
How do XPD mutations cause skin cancer?
Mutations in XPD = defective NER = cells can't repair UV damage = hypersensitivity to sunlight and skin cancer
