DNA Damage & Repair Flashcards
Name the 5 types of DNA damage
- Single-strand break
- Double-strand break
- Bulky adducts (a piece of DNA covalently bonded to a (cancer-causing) chemical)
- Base mismatches (inc. insertions and deletions)
- Base alkylation
Name the two causes of DNA damage, giving examples for each
Environmental Damage
- Chemical (i.e. carcinogens) – chemical damage originates from a wide range of sources (dietary, lifestyle, occupational, medical [i.e. treatments – chemotherapy etc.])
- Non-ionising radiation – UV
- Ionising radiation – cosmic particles (sub-atomic particles), x-rays
Endogenous Damage (DNA damage is a natural phenomenon)
- ROS
- Spontaneous deamination
- Replication errors
List 5 potential consequences of DNA repair failures
- Cancer
- Neurodegeneration
- Immune Dysfunction
- Sterility
- Ageing (Progeria)
What are the 3 major sources of chemical damage (to DNA)?
- Hydrolytic Attack
- Oxidative Damage
- Alkylation
- **Methylation **
Outline the following types of hydrolytic attack on DNA? Explain any potential conseqences.
- Depurination
- Depyrimination
- Deamination
- Strand Breaks
- Depurination – removal of a purine base (this does occur naturally/i.e. it is a normal physiological process).
- Depyrimination – removal of pyrimidine base
- **Deamination - **Cytosine Deamination: C –> U (remember U is normally only found in RNA). Can be reveseved (i.e. if the U is replaced with a C)
- Strand Breaks
Concerning Oxiative Damage:
(a) What is a conseuqence of free radicals?
(b) Name three types of oxidative damage and their effects.
(a) Free Radicals from normal metabolism attach purine and pyrimidine rings caused strand breaks
(b) There are over 100 oxidative modifications of DNA, examples include:
- G –> 8-hydroxyguanin (therefore pairs with A instead of C)
- Loss of planar ring (of bases) resulting in non-coding bases
- Helical distortion – addition of covalent bonds between base and sugar-phosphate backbone (distorts the helix)
Methylation is an example of Alkalation - outline this process and discuss potential complications of G –> meG conversion.
Methylation
- Distorts the DNA structure via alkylation (there are many environmental alkylating agents
Methylation of G: G –> meG
- Consequences – methyl group distorts the DNA structure (can be corrected by dealkylation)
What is endogenous DNA damage?
Damage that is not due to chemical changes
List and explain the two main types of endogenous DNA damage, give examples for each,
Replication Errors – consequence = mismatches or small loops (of bases) resulting in helix distortion
- Nucleotide insertions/deletions
- Incorporation of wrong nucleotide
- Incorporation of damaged nucleotides
Radiation Damage – consequence = CPDs (cyclobutane pyrimidine dimers) resulting in helix distortion
- Ionising radiation (cosmic rays and X-rays)
- UV
Name and briefly outline the 6 major DNA repair mechanisms
- Direct Repair - Reversal of DNA damage
- Base Excision Repair (BER) – removes single damaged base (main protector against metabolic damage)
- Nucleotide Excision Repair (NER) - removes damaged nucleotides (particularly CPDs, thymine dimers and large chemical adducts)
- Mistmactch Repair (MMR) – removes mismatched base pairs and insertion/deletion loops
- Homologous Recombination Repair (HR) – double-strand break and cross-link repair
- Non-Homologous End Joining (NHEJ) – DS break repair
Concerning direct repair:
(a) which organisms can perform this type of repair?
(b) what are the two main methods of direct repair?
(c) outline the major mammalian direct repair pathway.
(a) Bacteria and Yeast mainly (can be performed by mammals but this is not common given the simplicity of the repair mechanism)
(b) There are two main methods –
- Photoreactivation of CPDs – either non-enzymic (200-300nm irradiation) or enzymic (photolyases)
- Reversal of alkylation damage
(c) There is one mammalian direct pathway – O6-methylguanine-DNA methyltransferase (however it is not commonly used)
What is the purpose of base excitions repair (BER)?
Briefly outline the steps involved
Removes base damage caused by hydrolysis, ROS etc.
- DNA glycosylases identify and remove the damaged base
- AP endonuclease and T-phosphodiesterase cut the sugar-phosphate backbone
- Gap is filled by DNA polymerase b
- Nick is sealed by DNA ligase
NB: There are various BER pathways. The steps for BER are generic (i.e. regardless of the damage) however the glycosylases are specific
What is the purpose of nucleotide excision repair (NER)?
Briefly outline the steps involved.
Removes damaged nucleotides, particularly CPDs.
- Damaged bases are detected
- Helicases and nucleases act to open up and cut either side of the mutated base; 24-32bp oligonucleotide is removed
- Gap is repaired by DNA polymerase e or d
- Nick is sealed by DNA ligase
What is the purpose of transcription coupled NER? Outline the differences between “normal” NER and transcription coupled NER.
- Transcribed regions are repaired better than untranscribed regions.
- Preferential repair of transcribed strand (5-10 fold)
- XPC not required
- Damaged recognised by stalled RNA pol
- Additional protein required (not used in global NER) esp CSA and CSB
Outline the bacterial MMR (mismatch repair) process
- MutS recognises mismatch
- MutL binds MutS and activates MutH
- MutH nicks unmethylated (newly replicated) strand
- Intervening DNA excised and resynthesised – this repairs correct strand
