R7 DNA damage and repair Flashcards
What is a DNA lesion ?
breaks or other chemical changes that affect the structure of the helix, ultimately preventing transcription
What are some examples of DNA lesions ?
ss breaks, ds breaks, bulky adduct (covalently linked), interstrand crosslink, base mismatch, base alkylation
What effect can UV have on DNA ?
cause thymines to form dimers (bond to one another)
What effect can ionising radiation have on DNA ?
ss and ds breaks
What effect can alcohol have on DNA ?
interstrand crosslinks
What effect can oxidative damage have on DNA ?
oxidised bases, ROS - reactive oxygen species
What effect can mechanical stress have on DNA ?
ds breaks during mitsosis
What process is the biggest source of DNA lesions
DNA replication
How does the unpacking of chromatin affect the chances of lesions ?
Increases, as the proteins in chromatin protect from UV and IR
How can DNA polymerase increase the chance of lesions occurring ?
May miss or add nucleotides or mismatch them, damaged nucleotides can be used, ribonucleotides are incorporated or not removed from RNA primers
How do unpaired bases affect the chances of lesions occurring ?
they are exposed to bases and ROS
How is ssDNA affected by lesions ?
more prone to breakage due to mechanical stress
What happens if lesions are not repaired or repaired incorrectly ?
Mutation(s) will occur
What type of lesions does the cell recognise ?
DNA free ends (not telomeres), ssDNA accumulation and persistence, base pairing incorrectly, the bases not being A,T,G,C, not having deoxyribose
How is an extra nucleotide base repaired by the cell
Nuclease removes the structural damage then polymerase fills the gap then ligase seals the nick
How is a thymine dimer repaired by the cell ?
Photolyase (light-activated enzymes) cleaves the bond between thymines during times of high UV
How does an extra nucleotide/ mismatch affect the replication process ?
they will be paired up on one strand leading to a mutation, this would be the same for mismatch
How may a RNA nucleotide affect the replication process?
cause mismatch in the daughter strand
How may an oxidised guanine affect the replication process?
oxo-G can pair with A, when repaired by cut and patch the G will be removed and replaced with a T, leading to a mutation in both strands
What is the cut and patch mechanism ?
Nuclease removes a nucleotide, polymerase adds one, ligase seals the nick
How can a dsBreak be caused?
broken replication fork, unseperated sister chromatids in mitosis, ionising radiation, some chemicals (chemotherapeutic)
How is an ICL caused ?
endogenous metabolites (acetaldehyde), chemicals (chemotherapeutic drugs)
How is a dsbreak repaired ?
Ligation of broken ends, homologous recombination (the breakage is compared to a non-broken homologous chromosome and copied)
What can ligation of a dsbreak cause ?
addition or deletion at the site of breakages which is error prone
What can homologous recombination cause ?
Erroneous repair
What is erroneous repair in terms of DSB ?
- telomeres added to a break (causing the loss of the acentromeric arm and a shorter chromosome),
- reciprocal translation - the broken end joins to another strand
- dicentric chromosomes - another chromosome binds to the broken end, making an unstable double chromosome, the chromosome then has 2 centromeres so the spindle fibres pull at them both, leading to more breakages
What is GCR ?
Gross chromosomal rearrangement:
- large deletions, large insertions, large inversions, translocation
arises from erroneous DSB repair
what is aneuploidy ?
more than one abnormal chromosome
What is common in cancer cells that may be caused by erroneous dsb repair ?
aneuploidy, GCR
How is ICLs repaired ?
they cannot be transcribed as the DNA strands cannot be separated
in proliferating cells it may block sister chromatid segregation in mitosis
may be converted to DSB then repaired as a DSB
sometimes induced in anti-cancerous therapy
What is the bacterial SOS response ?
During serious DNA damage, ssDNA accumulates and activates SOS regulons
This causes:
- replication to continue, cell division inhibitors are activated, filamentous cells with many chromosome copies are made
- homologous recombination genes activated
- trans lesion synthesis (TLS) DNA polymerase genes activated allowing for mutagenesis (gene mutation in base order). the polymerase uses the damage template strand adding the wrong bases
- delayed cell division allows time to repair DNA before dividing
What is mutagenesis ?
The change in base order leading to a mutant forming
Where are the DNA damage checkpoints in the cell cycle?
G1 - so DSB cannot go into S
Intra-S checkpoints - replication fork problems means the late origin is not fired
G2 - there is ssDNA, mitosis is then blocked
What is the DNA damage response based on ?
PTMs, transcription activation plays a minor role
Why is blocking the next stage of the cell cycle important ?
Allows time for repair
What two processes must happen before a mutation is acquired ?
lesion formation, escaping repair
How can we work on the probability of mutations?
multiple the probability of lesions, and probability of escaping correct repair
external stimuli may change the probability, UV increases the chance of lesions
How can mutation rates be increased ?
Exposure to high UV, then placed in the dark so photolyase isn’t activated
what can increase the chance of acquiring lesions ?
- DNA polymerase loses its proofreading activity
- Wrong nucleotide incorporated
- Helicase loses function, forks break more often
- Mitochondria are leaking, more ROS in the nucleus
What increases the probability of escaping repair ?
- Mutations lead to higher mutation rates due to higher probability of unrepaired or incorrectly repaired damage.
- Mutations in DNA damage checkpoint genes
- Mutations in chromosome segregation genes (mitotic spindles)
What is somatic mosaicism ?
the acquiring of mutations in somatic cells leading to only some areas being affected - cannot be inherited
BRCA is an example of this
Why might a replication fork be stalled ?
DNA damage (ss breaks, ds breaks, abbducts), G-quadruplexes (lots of G), fork collision, not enoguh free nucleotides, helicase and polymerase loose function, R-loops (RNA, DNA hybrids), failure for topioisomerase, lack of checkpoints, DNA-binding proteins, crosslinks
