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BMD211-Human Molecular Biology > Lecture 5: DNA Repair > Flashcards

Lecture 5: DNA Repair Flashcards

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1
Q

Define endogenous agents and list them

A

• Endogenous Agents: formed inside the cell by normal metabolic pathways

  • Cellular metabolism
  • Replication errors
  • Oxygen free radicals
  • Certain hydrocarbons
  • Base mismatch- insertions/deletion
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2
Q

Define exogenous agents and list them

A

• Exogenous Agents: come from the surrounding environment

  • UV light exposure -> UV-C, UV-B
  • Ionising radiation -> gamma rays, X-rays
  • Chemical exposure
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3
Q

Explain the types of changes that can occur due to DNA damage

A

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
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4
Q

State the different in rate of DNA damage and repair in a healthy cell and an unhealthy cell

A
  • Healthy cell-> Rate of DNA damage = rate of repair

- Unhealthy cell -> Rate of DNA damage > rate of repair

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5
Q

Explain how cells respond to DNA damage

A
  • 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
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6
Q

List the different types of DNA repair mechanism

A

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

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7
Q

Describe what direct enzymatic repair is for

A
  • Repair of alkylated bases
  • Involves the direct reversal or simple removal of the damage
  • Relatively rare
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8
Q

State a common chemical that can cause alkylation of bases and list where it can be found

A
  • Chemicals, such as nitrates can alkylate bases within DNA
  • Nitrates found in :
  • preservatives in food
  • tobacco smoke
  • formed in the gastrointestinal tract
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9
Q

Explain how alkylation of bases causes mispairing

A
  • 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
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10
Q

Explain the overall mechanism for direct enzymatic repair

A
  • 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
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11
Q

Describe what base excision repair is for

A
  • Repair of single bases that are modified by deamination, oxidation or methylation
  • Can also repair single strand DNA breaks
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12
Q

Explain how the different single base changes can be caused

A

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

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13
Q

Explain the mechanism for base excision repair

A
  1. DNA glycosylase recognizes a damaged base -> cleaves between the base and deoxyribose in backbone
    = remove the base = abasic – apurinic and apyrimidinic (AP) site
  2. An AP endonuclease cleaves the phosphodiester backbone near the AP site
  3. = a single strand nick
  4. Short Patch Repair: DNA polymerase β adds one nucleotide to 3’-OH at the nick
  5. 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
  6. The remaining nick is sealed by DNA ligase
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14
Q

Describe what nucleotide excision repair is for

A
  • Repair of bulky lesions or adducts ->lead to DNA distortion
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15
Q

Explain how different structural distortions can be formed

A
  • 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
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16
Q

List the human diseases that are associated with defects in NER genes

A

 xeroderma pigmentosum (XP)
 Cockayne syndrome (CS)
 PIBIDS A

17
Q

State what these diseases that are associated with defects in the NER genes have in common

A

increased sensitivity to sunlight

18
Q

Name the 2 NER pathways

A

Global and Transcription coupled NER

19
Q

State the number of NER genes that can be defected to cause XP

A

XP caused by defects in 7 different NER genes: XPA to XPG

20
Q

Explain the mechanism for GNER

A
  1. XPC- detects helix distortion +stabilises the bend
  2. 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
  3. XPF endonuclease- adds to the complex
  4. Dual incision is made: on the 3’ side by XPG + on the 5’ side by XPF
    The excised oligonucleotide is typically 25-30bp long
  5. DNA polymerase δ/ε- fills in the gap
  6. DNA ligase III- seals the nick
21
Q

Explain the mechanism for TNER

A
  • Recognition of distortion different from global NER

 RNA polymerase is stalled= leads to recruitment of TFIIH

22
Q

Explain why the difference in repair mechanism for TNER

A
  • 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
23
Q

Describe what mismatch repair is for

A
  • Repair of mispaired bases or short deletions or insertions

- Specific to newly synthesised (daughter) DNA strand

24
Q

Explain the mismatch repair mechanism in prokaryotes

A
  1. 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
25
Q

Explain the mismatch repair mechanism in eukaryotes

A
  1. MSH2-MSH6 complex- binds to the mismatch
    + identifies newly synthesized strand
  2. MLH1 endonuclease +other factors, such as PMS2- bind to the newly synthesised strand
    + recruit a helicase +exonuclease
    = remove several nucleotides including the lesion
  3. DNA Pol  -fills gap
  4. DNA ligase- seals new segment of DNA to rest of daughter strand
26
Q

Describe what double-strand break repair is for

A
  • Repair of DNA double strand breaks or interstrand crosslinks – lesions which have no template for repair
27
Q

Explain the Non-homologous end joining mechanism

A
  • 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
28
Q

Explain the Homologous recombination pathways mechanism

A
  1. DSB ends degraded by nuclease at 5’ ends to form 3’ ended ss tails
  2. 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
  3. Invading strand + complementary region share polarity- go in 5’-3’= forms loop = D-loop
  4. 3’ end of invading strand= acts as primer for DNA synthesis, complementary strand= template
  5. Replication happens in transient bubble- short sequences new DNA is formed + the damaged sequence is replaced + template is used as a track
  6. Bubble moves along until section of DNA that is complementary to the section in the other strand that is involved in DSB is formed
  7. Replication bubble dissociates
  8. Newly synthesised segment of DNA caught by other 3’ ended ss tail
  9. New segment of DNA acts as template for the part of DNA on the other strand involved in DSB
  10. 3’ -OH end of new segment of DNA acts as primer
  11. DNAP fills gap of the other strand involved in the DSB
  12. DNA ligase joins the new segments of DNA with the rest of the DNA strands
29
Q

Give examples of diseases caused by each type of DNA damage and their sensitivities

A
  • 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

BMD211-Human Molecular Biology (5 decks)

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