Lecture 5: DNA Repair Flashcards
Define endogenous agents and list them
• Endogenous Agents: formed inside the cell by normal metabolic pathways
- Cellular metabolism
- Replication errors
- Oxygen free radicals
- Certain hydrocarbons
- Base mismatch- insertions/deletion
Define exogenous agents and list them
• Exogenous Agents: come from the surrounding environment
- UV light exposure -> UV-C, UV-B
- Ionising radiation -> gamma rays, X-rays
- Chemical exposure
Explain the types of changes that can occur due to DNA damage
Single Base Changes
- Single bases changes affect the sequence of DNA -> not its overall structure
- > Base alkylation
- > Base deamination
- > Base oxidation
- Doesn’t affect cellular processes- e.g: transcription or replication
- > Can be harmful to future generations -> inheritance of single base change
- Conversion of base= DNA mismatch
- Mismatch only in parent DNA
- In next replication-parent DNA will be used as template + daughter strands will have complementary paired based to original single base change
= altered DNA sequence forever
Structural Distortions
- Structural distortions cause physical change (change structure)
- Disrupt transcription or replication
- Adduct formation ->Benzo[a]pyrene
- Photodimerism -> UV light
- DNA crosslinks ->chemotherapy
- DNA-protein crosslinks ->ethanol metabolism products
- Single strand breaks ->oxygen radicals
- Double strand breaks -> ionizing radiation
State the different in rate of DNA damage and repair in a healthy cell and an unhealthy cell
- Healthy cell-> Rate of DNA damage = rate of repair
- Unhealthy cell -> Rate of DNA damage > rate of repair
Explain how cells respond to DNA damage
- damage detected by sensors
- triggers apical signalling
- mediators aid downstream signalling
- signal reaches effectors
- effectors can either cause apoptosis or checkpoint arrest
- in checkpoint arrest the cells can undergo DNA repair + then proliferate or can not undergo DNA repair + head into senescence
List the different types of DNA repair mechanism
1) Direct enzymatic repair
2) Base excision repair
3) Nucleotide excision repair
4) Mismatch repair
5) Double-strand break repair
- Non-homologous end joining
- Homologous recombination
Describe what direct enzymatic repair is for
- Repair of alkylated bases
- Involves the direct reversal or simple removal of the damage
- Relatively rare
State a common chemical that can cause alkylation of bases and list where it can be found
- Chemicals, such as nitrates can alkylate bases within DNA
- Nitrates found in :
- preservatives in food
- tobacco smoke
- formed in the gastrointestinal tract
Explain how alkylation of bases causes mispairing
- Alkylation of bases (methylation) disrupt pairing of bases
->Deamination of methylated cytosine ->changes to thymidine
= mispairing of O6-methylguanine with thymidine - If methyl groups are not removed-> DNA replication of the mispair
= lead to transition mutations - Transition mutation: point mutation where one purine or pyrimidine changes to another, A to G, C to T
Explain the overall mechanism for direct enzymatic repair
- Alkylation of O6 -methylguanine -> removed by O6 -alkylguanine DNA alkyltransferase
- Enzyme contains two domains-> each domain has an active site containing a cysteine residue
- N-terminal domain - transfers an alkyl group from phosphotriesters to its cysteine residue
- C-terminal domain- transfers an alkyl group from either O6 -alkylguanine or O4 -alkylthymine to another cysteine residue
Describe what base excision repair is for
- Repair of single bases that are modified by deamination, oxidation or methylation
- Can also repair single strand DNA breaks
Explain how the different single base changes can be caused
Oxidation by Reactive Oxygen Species
- Reactive Oxygen Species ROS: superoxide and hydroxyl radicals generated by cellular respiration
- 8-oxoguanine can pair with Cytosine or Adenine
= causing transversion mutation
Single Base Changes – Deamination
- Deamination involves removal of an amino group
- Can be spontaneous (water-mediated) or through nitrites
- Uracyl recognised as an inappropriate base in DNA ->lead to transversion mutation
Explain the mechanism for base excision repair
- DNA glycosylase recognizes a damaged base -> cleaves between the base and deoxyribose in backbone
= remove the base = abasic – apurinic and apyrimidinic (AP) site - An AP endonuclease cleaves the phosphodiester backbone near the AP site
- = a single strand nick
- Short Patch Repair: DNA polymerase β adds one nucleotide to 3’-OH at the nick
- Long patch repair: DNA pol DNA pol δ or ε initiate repair synthesis from the free 3’ OH at the nick
- removing a portion of the damaged strand (with its 5’3’ exonuclease activity)
- replacing it with undamaged DNA
= generating a flap -> removed by Flap endonuclease - The remaining nick is sealed by DNA ligase
Describe what nucleotide excision repair is for
- Repair of bulky lesions or adducts ->lead to DNA distortion
Explain how different structural distortions can be formed
- Polycyclic Aromatic Hydrocarbons can form DNA adducts
- Benzo[a]pyrene is carcinogenic ->constituent of cigarette smoke
Photodimersim - Caused by UV light –>dimer formation between adjacent pyrimidine rings on the same strand
UVA: 320-400 nm – majority of UV light reaching earth, little damage
UVB: 295-320 nm - ~10% of UV light reaching earth, responsible for most of DNA damage
UVC: 100-295 nm – majority stopped by ozone layer, harmful for DNA - DNA helix is distorted
- Transcription may be blocked
- Cyclobutane pyrimidine dimers = produce a kink in DNA
List the human diseases that are associated with defects in NER genes
xeroderma pigmentosum (XP)
Cockayne syndrome (CS)
PIBIDS A
State what these diseases that are associated with defects in the NER genes have in common
increased sensitivity to sunlight
Name the 2 NER pathways
Global and Transcription coupled NER
State the number of NER genes that can be defected to cause XP
XP caused by defects in 7 different NER genes: XPA to XPG
Explain the mechanism for GNER
- XPC- detects helix distortion +stabilises the bend
- XPC and 23B- recruit TFIIH helicase
= opens DNA double helix
Simultaneously: RPA- binds to the undamaged strand of DNA
: XPG endonuclease- binds to the “preincision” complex - XPF endonuclease- adds to the complex
- Dual incision is made: on the 3’ side by XPG + on the 5’ side by XPF
The excised oligonucleotide is typically 25-30bp long - DNA polymerase δ/ε- fills in the gap
- DNA ligase III- seals the nick
Explain the mechanism for TNER
- Recognition of distortion different from global NER
RNA polymerase is stalled= leads to recruitment of TFIIH
Explain why the difference in repair mechanism for TNER
- Actively transcribed genes ->more efficiently repaired than non- transcribed DNA
- Active genes are in euchromatin structure ->transcription may be more vulnerable to DNA damage
- Active gene - more likely to be important for survival
- DNA damage blocks transcription
- Transcription coupled nuclear excision repair: repairs damage in the transcribed strand of active genes
Describe what mismatch repair is for
- Repair of mispaired bases or short deletions or insertions
- Specific to newly synthesised (daughter) DNA strand
Explain the mismatch repair mechanism in prokaryotes
- Identification of parent strand:
- MutH - recognises the parental strand by binding GmeATC
- MutS- binds mismatch
- MutL- links H to S
- Before replication: both (parental) strands have adenine methylated
- Immediately after replication: the parental strand is methylated + daughter strand is not
2. MutH cleaves the unmethylated strand on the 5’ side of the G in the GATC sequence
3. The combined action of DNA helicase II+ SSB+ an exonuclease- removes a segment of the new strand between the MutH cleavage site and a point just beyond the mismatch
4. DNA polymerase III – fills resulting gap
5. DNA ligase- seals the nick
Explain the mismatch repair mechanism in eukaryotes
- MSH2-MSH6 complex- binds to the mismatch
+ identifies newly synthesized strand - MLH1 endonuclease +other factors, such as PMS2- bind to the newly synthesised strand
+ recruit a helicase +exonuclease
= remove several nucleotides including the lesion - DNA Pol -fills gap
- DNA ligase- seals new segment of DNA to rest of daughter strand
Describe what double-strand break repair is for
- Repair of DNA double strand breaks or interstrand crosslinks – lesions which have no template for repair
Explain the Non-homologous end joining mechanism
- happens in g1
1. Resection may occur: degradation at the ends of the break of ~10bp
2. Ku heterodimer (protein)- recognises DSB and binds around broken ends- leaves DNA ends exposed
3. Ku recruits DNA-PKcs
4. DNA-PKcs -recruits artemis (nuclease) to bind + artermis is phosphorylated
5. Artermis- trims any ss tails at the break
6. Ligase IV + XRCC4 + XLF/Cernunnos form a complex (ligase)- ligates DSB
Explain the Homologous recombination pathways mechanism
- DSB ends degraded by nuclease at 5’ ends to form 3’ ended ss tails
- One of the 3’ ended ss tails interact with homologous duplex
Ss tail invades homologous duplex at region of homology = heteroduplex with invading ss + complementary region in homologous duplex - Invading strand + complementary region share polarity- go in 5’-3’= forms loop = D-loop
- 3’ end of invading strand= acts as primer for DNA synthesis, complementary strand= template
- Replication happens in transient bubble- short sequences new DNA is formed + the damaged sequence is replaced + template is used as a track
- Bubble moves along until section of DNA that is complementary to the section in the other strand that is involved in DSB is formed
- Replication bubble dissociates
- Newly synthesised segment of DNA caught by other 3’ ended ss tail
- New segment of DNA acts as template for the part of DNA on the other strand involved in DSB
- 3’ -OH end of new segment of DNA acts as primer
- DNAP fills gap of the other strand involved in the DSB
- DNA ligase joins the new segments of DNA with the rest of the DNA strands
Give examples of diseases caused by each type of DNA damage and their sensitivities
- hereditary nonpolyposis colorectal cancer ->DNA mismatch repair -> UV+ chemical exposure
- XP-> NER-> UV+ point mutations
- Bloom’s syndrom-> DBS by homologous recombination -> mild alkylating agents
- Fanconi anemia-> DBS homologous recombination-> DNA crosslinks + ROS
- Hereditary breast cancer-> DBS by homologous recombination-> none
