DNA damage and repair Flashcards
What is the definition of DNA damage?
change to regular chemical structure of the DNA double helix
Give four examples types of DNA damage?
- break in phosphodiester backbone
- loss of base from deoxyribose sugar
- alteration to the structure of the base
- mis-matched base pairs
How can DNA damage lead to a mutation?
if damaged DNA undergoes further rounds of replication, this can result in a fixed mutation
What is the definition of a mutation?
permanent heritable change in the sequence of an organism’s genome
Give the two types of mutations and give examples of each
Point mutation = alteration, insertion, deletion of one or more bases at a time
Chromosome mutation = translocation, deletion, insertions
What is a transition point mutation?
purine replaced by a purine
or
pyrimidine replaced by a pyrimidine
What is a transversion point mutation?
Purine replaced by a pyrimidine, or vice versa
What is a missense point mutation?
codon now codes for a different amino acid
What is a nonsense point mutation?
codon now codes for a stop codon
What are the stop codons?
TAA, TAG, TGA
What are the three types of effects of base-pair substitution mutations?
Neutral = substituted amino acid has similar chemical properties to the original amino acid
Silent = base substitution in third position so codon encodes for the same amino acid (degeneracy of DNA)
Frameshift = caused by insertion or deletion results in change in all downstream codons
What is the difference between a forward mutation, a reverse/reversion mutation, and a suppressor mutation?
forward mutation = wild type gene changes to a mutant gene
Reversion mutation = mutant gene changes back to wild type gene
Suppressor mutation = changes sequence at a different location from the original mutation in a way that compensates for the original mutation and restores function
What are the two types of reversion mutations that can occur?
- True reversions = restores sequence to code for the wildtype amino acid
- partial reversions = changes the amino acid sequence but restores the function of the protein
What are the two types of suppressor mutations that can occur?
Intragenic = occurs within the same gene
Intergenic = occurs within a different gene
What does spontaneous mean in terms of mutation?
Occurs without exposure to exogenous agents
What does Lamarckism tell us about mutations?
- tells us that they are adaptive
i. e. they develop as a response to a particular environment to allow adaption
Are mutations random or adaptive? why?
Random
- mutations occur spontaneously throughout growth but may confer a selective advantage in a particular environment, allowing the effect of the mutation to be observed
What experiment was carried out by Luria and Delbruk? what did this show about mutation?
1) Grow E.coli in a batch culture
2) plate out some of the batch culture on nutrient medium and isolate single colonies and re-suspend them in liquid media
3) plate the individual colonies as a lawn and add T4 bacteriophage
4) count the number of colonies = colony growth = resistance
Showed:
- large variation in number of resistant colonies between individual plates = number of resistant colonies depended on when, during growth in batch culture, the mutation occurred.
How do random spontaneous mutations arise?
Occurs due to replication of pre-mutagenic DNA
What are the two main causes of endogenous premutagenic damage to DNA? give examples of each
DNA replication errors:
1) nucleotide or template tautomerism causing mismatches
2) replication slippage (causes incorporation or deletion of extra bases)
Endogenous DNA damage (within the cell):
1) base deamination
2) base loss (depurination, depyrimidination)
3) base modification due to exposure to metabolic products (alkylation, oxidative damage)
What four mechanisms are used to ensure the high fidelity of DNA synthesis during replication?
1) base pairing
2) DNA polymerase - base selection, 3’-5’ exonuclease activity
3) Accessory proteins (single stranded binding protein)
4) post-replication mismatch repair
True or false: mismatches are pre-mutagenic lesions?
True (a further round of replication would created a fixed mutation)
Tautomerised adenine resembles which other nitrogenous base? what is the result of this?
Resembles guanine
result: cytosine added opposite the adenine tautomer and if replicated again results in a T-A to C-G mutation
How do DNA looping-out errors spontaneously generate addition or deletion mutants?
when the new strand loops out , results in an additional base in the new strand
When the parental strand loops out, this results in the loss of a base from the new strand
Where do DNA looping-out errors typically occur?
repetitive dna sequences
What is depurination? what are results?
endogenous DNA damage: hydrolysis of bond between sugar and base results in removal of the purine base, leaves an apurinic (SP) site premutagenic lesion
- results in random base substitution or base-skipping during replication leading to substitution or deletions - further round of replication causes mutation
Which base loss endogenous damage is more common: depurination or depyrimidination?
Depurination
What is deamination? what are results using cytosine as an example?
Endogenous DNA damage: removal of the amino group from a base.
Cytosine deamination results in uracil, which can be repaired but if not, when replicated, the uracil will base pair with an adenine, changing the C-G to a T-A fixed mutation.
What is the results of deamination of methylated cytosine (5-MeC)?
Produces thymine
results in G-C to A-T mutation
What is alkylation? what are results using guanine as an example?
endogenous DNA damage: addition of methyl or ethyl group to sugar or base by endogenous alkyl donors
results: guanine able to be methylated at the O6 position to produce O6-methylguanine which can base pair with thymine. If this premutagenic lesion is replicated, this produces a G-C to A-T mutation.
What is oxidative DNA damage? what are results using guanine as an example?
ROS attack bases
ROS attack of guanine can produce 8oxoG premutagenic lesion, which can base pair with either C or A depending on the isomeric form. If base pairs with A, this can result in a GC to AT mutation
Results of ROS damaged bases can either have no effect, be mutagenic, or leads to strand breakage/block replication
Why is there a high amount of oxidative DNA damage in mitochondrial DNA?
ROS leak from the ETC
What are the two main causes of exogenous mutagens? give examples of each
Chemical mutagens:
- base analogues
- base modifying agents (alkylating agents)
- Intercalating agents
Physical mutagens:
- ionising radiation (X-rays)
- UV radiation
What is an example of a base analogue? what are the results of this mutagen?
5-bromouracil (5BU)
results: behaves like thymine normally, but when tautomerised, it behaves like cytosine and base pairs with guanine.
changes AT to CG transition mutation
What are the effects of the base-modifying agent nitrous acid?
exaggerate endogenous deamination processes by creating acidic conditions that promote deamination
What are the results of G, C, and A deamination DNA damage?
Deaminated G produces Xanthine which base pairs with cytosine so has no effect
Deaminated C produces U, which base pairs with adenine so produces a CG-AT transition mutation
Deamination A produces Hypoxanthine, which base pairs with C so produces a AT-CG transition mutation
Give three examples of base-modifying agents
- nitrous acid
- hydroxylamine
- methylmethane sufonate (MMS)
What are the effects of the base-modifying agent hydroxylamine? give an example
hydroxylates and changes the bases
- E.g. hydroxylation of cytosine results in a CG-AT transition mutation
What are the effects of the base-modifying agent MMS? Give an example
methylation of bases, which blocks replication or introduces mutations upon further replication
- E.g. methylation of guanine results in O6-methylguanine that base pairs with thymine to produce a GC-AT transition mutation
Which base is particularly susceptible to DNA damage?
guanine
What is an intercalating agent? give an example of an intercalating agent? what are the results of this mutagen?
Have a flat, planar structures that insert into the minor groove of the helix, resulting in partial unwinding and insertion or deletion of additional bases upon replication.
E.g. ethidium bromide
What is benzopyrene and how does it become a carcinogen?
Benzopyrene is a procarcinogen in the environment but metabolised in the organism into a carcinogen benzopyrene dihydrodiol epoxide. It is this BPDE that binds to the DNA and induces pre-mutagenic lesions.
Which UV radiation is most able to cause affect DNA, why?
UVC because DNA absorbs UV radiation with a peak absorbance at 254 nm (within the UVC absorbance range)
What is the wavelength range for UVC, UVA and UVB?
UVC = 200-280 nm UVB = 280-320 nm UVA = 320-400 nm
