what factors can cause DNA damage
- chemical modifications
- UV exposure
- reactive oxygen species
- cosmic radiation
- errors in DNA replication
what is the estimated rate of base changes per generation
due to errors in DNA replication, 70-150 base changes per generation
example of a chemical modification for DNA damage
tobacco smoke
- contains benzyl-pyrene
- benzyl-pyrene can from an adduct of Guanine, meaning it adds onto guanine when tobacco is smoked
- this distorts the guanine base and adds a bulge in the DNA double helix
example of UV exposure for DNA damage
-if you have 2 thymines in a DNA chain and UV radiation is applied, 2 carbons from each of the thymines can form a bond between each other
- this creates a thymine dimer, which adds a bulge in the double helix and decreases its flexibility
describe DNA mutations
changes to the DNA code, e.g. T instead of C
what are the types of DNA mutations
- synonymous: doesn’t change amino acid
- missense: changes the amino acid
- nonsense: changes the amino acid to STOP
- frameshift (in/del): changes all amino acids after that point
- deletion (del): skips a stretch of amino acids
what’s an example of a cytosine mutation
- in a normal mutation where cytosine becomes deaminated and forms uracil, the cell can detect the error and fix it
- with a methylated cytosine, it deaminates to form thymine, which the cell does not expect and cannot fix - this causes a problem
what does incorrect base pairing cause during DNA replication
distortion of the DNA structure
how does the cell prevent distortion of the DNA structure via incorrect base pairing
- DNA polymerases are proof-reading enzymes that correct as they go
- it adds nucleotides towards the 3’ end
- but it also has exonuclease activity, meaning it can “unpick” in the opposite direction
what does the cell do if it can’t correct DNA damage during DNA replication
it has other mechanisms:
- BER: base excision repair
- MMR: mismatch repair
- NER: nucleotide excision repair
what fixes the exchange of a cytosine base to a thymine base
base excision repair
describe how base excision repair works
1) it knows it needs to repair as the T, which should be a C, is incorrectly paired with a G
2) DNA glycosylase will remove the incorrect T base, leaving an abasic (no base) site
3) the APEI endonuclease cleaves the abasic site, removing the DNA backbone too
4) DNA polymerase B also removes the backbone, then replaces it with the correct nucleotide
5) DNA ligase seals the new nucleotide in
what do mismatch repairs fix
- small mismatches and ‘slippages’ of repeated DNA
- since base excision repair is only for the cytosine thymine fix, any other error is fixed by mismatch repairs
describe how mismatch repair works
1) the MutS protein complex with MSH enzymes recognise the error by looking at the mismatched bases
2) to know which of the bases are incorrect, it looks for ss nicks on the newly synthesised DNA strand
3) MutL complex: MLH endonuclease initiates repair by creating a nick (cut) in the mismatched strand and many other bases surrounding it
4) DNA exonuclease then removes a segment of nucleotides starting from that nick
5) DNA polymerase then repairs the gap, adding the (correct) complementary nucleotides, and DNA ligase seals them in
what do nucleotide excision repairs fix
- repairs larger DNA errors/lesions
- cuts out a larger region than base excision repair
- e.g. thymine dimers
how does nucleotide excision repairs recognise the error
two pathways to recognise:
1) global genomic repair NER: XPC and Rad23B enzymes recognise the bulge in the helix - this can take place at anytime
2) transcription-coupled NER: happens during transcription when DNA is being used as a template - RNA polymerase recognises the error
describe how nucleotide excision repair works
1) error is found via global genomic repair NER or transcription-coupled NER
2) helicase unwinds the DNA
3) RPA, a ssDNA binding protein, keeps the DNA unwound
4) XP endonuclease cuts at the damage strand, excising 24-32 base pairs
5) DNA polymerase replaces the bases and DNA ligase seals them in
what’s an example of a large DNA error
- double stranded DNA breaks
- this is when both strands of the DNA helix collapse at the same or nearby site
what causes double stranded DNA breaks
- the replication fork collapses during DNA replication
- ionising radiation
- chemical damage
are double stranded DNA breaks common
yes: 5-10% of cells in culture have a DSB
how are double stranded DNA breaks repaired
- non-homologous end joining
- homologous recombination repair
how does non-homologous end joining repaired DBS
sticks the broken ends together, worries about the rest later
describe how non-homologous end joining works
1) DNA-PK + KU protein complex recognises the error and binds to the DSB ends
2) artemis, an exonuclease, trims the ends so that they are symmetrical
3) DNA ligase then joins the ends together
what’s an issue with non-homologous end joining
the exonuclease artemis removes bases freely to make the 2 DNA strands symmetrical, leading to potential frameshifts and other disruptions to the gene
