DNA Damage And Repair Flashcards
Name some sources of dna damage
◦ Ionising radiation ◦ Alkylating agents ◦ UV light ◦ V(D)J recombination ◦ Free radicals ◦ DNA replication errors
Which components of DNA can be damaged?
- All 3 components of DNA can be damaged
* DNA damage from environment/normal metabolism=1*10^6 molecular lesions per cell per day
Briefly outline the DNA damage response
• DNA damage response (DDR)
◦ Proteins detect damage
◦ Signalling cascade to activate a pathway to respond to damage
‣ Signals>Sensors>Transducers>Effectors
◦ Pathways can result in
‣ Senescence
‣ Cell cycle transitions
‣ Apoptosis
‣ Transcription
‣ DNA repair
◦ The pathway activated depends on the type of damage
Name some sensors, transducers and effectors
• Sensors ◦ Rad17/9/1/1 complex ◦ MRN complex ◦ DNA-PK • Transducers (these listed are central DDR kinases) ◦ ATM ◦ ATR/ATRIP ◦ DNA-PKcs • Effectors ◦ 53BP1 ◦ TopBP1 ◦ CHK1 ◦ CHK2 ◦ P53
What is checked at each cell cycle checkpoint?
• Checkpoints - temporary cycle arrest, provides time for DNA damage to be repaired ◦ G1checkpoint ‣ Is environment favourable? ‣ ENTER S ◦ G2 checkpoint ‣ Is all DNA replicated? ‣ Is all DNA damage repaired? ‣ ENTER MITOSIS ◦ Checkpoint in mitosis ‣ Are all chromosomes properly attached to the spindle? ‣ PULL DUPLICATED CHROMOSOMES APART
What are senescence and apoptosis
• Senescence and apoptosis
◦ Occur if DNA damage levels are too high or persist
◦ Senescence = permanent cell cycle arrest (G0)
◦ Apoptosis = programmed cell death
How is DNA repaired
• DNA damage repair
◦ Ideal scenario = repair DNA damage AND maintain cell function
◦ There are different repair pathways
◦ The pathway used depends on the type of damage and cell cycle phase
Name 2 DSB repair pathways
Non-homologous end joining
Homologous-directed repair
Explain non-homologous end joining
◦ DNA double strand breaks are most genotoxic (damaging to DNA) lesion
‣ KU70/80 added to DSB ends to protect ends
‣ DNA-PKcs recruited to form DNA-PK complex - ends can be processed if required to remove damaged DNA components
‣ XRCC4, DNA ligase IV and XLF recruited
‣ Ligation of 2 broken ends back together
What are the problems with non-homologous end joining?
◦ This is the easiest pathway for repair
◦ However is error prone so can lead to mutation - rather “primitive” - the wrong ends can be joined together - there is no way to check that these broken ends are the ones that need to be joined
Give an overview of homology-directed repair and give an example
◦ Multiple steps
◦ Uses homologous template (eg sister chromatid)
◦ S and G2/M phase
◦ All HDR pathways initiated by DSB end resection
◦ Several downstream sub-pathways
Example: Holliday junction resolution
What are the steps in Holliday junction resolution?
‣ Double strand break
‣ DSBs undergo nuclease degradation
‣ Form two 3’ ended single stranded tails
‣ Tails may be 100s of BP long
‣ In a process mediated by recombination proteins, one of the 3’ ended tails from broken duplex (shown in grey) “envades” homologous double stranded DNA e.g. Sister chromatid (shown in pink)
‣ Displaced strand which has the same bases as invading tail forms a loop (shown below)
‣ Loop structure + Envading strand = displacement loop (or D loop)
‣ 3’ end of tail strand acts as primer for DNA synthesis where complementary pink strand is template
‣ Synthesis occurs to extend tail, using complementary strand as template
‣ The D loop moves along as tail extends
‣ The replication bubble (D loop) dissociates
‣ The tail extension is caught by the 3’ end of the other single stranded tail (from the other side of the break)
‣ This join serves as a primer to extend the second tail and fill in the bases (complementary base pairing) across to the other side of the break by replication and ligation
‣ DNA has been repaired
How can DNA damage be the basis of cancer cell killing?
◦ Cross-links: displaying ◦ DNA alkylation: nitrogen mustards ◦ Protein-DNA addicts: camptothecins (CPT) ◦ Strand breaks: radiation, bleomycin ◦ PARP inhibition ◦ dNTP pools: hydroxyurea (HU
What are some current cancer therapies?
◦ DNA repair defects in cancers make them sensitive to more DNA damage = cause of cancer can be its weakness
◦ Normal cells often affected too (often affects other rapidly dividing cells such as hair)
‣ Radiation: skin problems, fatigue, heart problems,
‣ Chemotherapy: hair loss, fertility problems, nausea
◦ Can promote tumour evolution and secondary cancers
◦ Need to develop better strategies to increase cancer cell death and lower side effects and limit cancer evolution
How can cancer cells die if one repair pathway is blocked?
- For some cancer cells, if one pathway is blocked, this causes cell death as alternative pathways are mutated
- Normals cells have alternate pathways so cells survive • For some cancer cells, if one pathway is blocked, this causes cell death as alternative pathways are mutated
- Normals cells have alternate pathways so cells survive
