Cancer and DNA repair- 4 lectures Flashcards
2 types of DNA damage
endogenous- spontaneous damage from defective repair etc
exogenous- from radiation, sunlight etc
biggest mutation burden
SSBs- 55k per cell per day
example of 2 types of DNA damage which aren’t DSBs or SSBs
cytosine deamination
depurination/depyrimidination
2 ways of identifying DNA repair proteins
looking for abnormal karotypes in mutants
mutational signatures- tracking specific mutations in sequences (you can do a lot of mathsy stuff out of this)
photoylases
direct DNA repair enzymes. convert pyrimidine dimers into normal base pairs using photons
alkyltransferases
transfer methyl groups to cysteine- also direct DNA damage reversal
base excision repair (BER)- early steps
DNA glycosylase does the excision of a lesion
nick is then created
2 types of BER
short patch- repairs single BP gaps by cutting out the BP
long patch- several nucleotides need to be replaced- damaged DNA is a ‘flap’ which is removed
NER meaning
nucleotide excision repair
rough steps of NER
detection of unpairing and abnormal DNA structure
incisions made at both sides of a lesion
excision w helicases
DNA synthesis and ligation
what is transcription coupled NER
a version of NER which happens during transcription- triggered by RNA Pol II detecting damage
similar events to regular NER
what is ICL repair
interstrand cross-link repair
Falconi anaemia
aplastic anaemia with short stature, hypogonadism, skin pigmentation
Due to defective ICL DNA damage repair
examples of pathways used in ICL
DNA polymerase synthesis, hom. recomb. and NER- she’s diverse
examples of when detection occurs
DNA replication or transcription, or independently of other pathways
2 pathways for DSB repair
NHEJ, HR
difference between DSB repair paths
NHEJ can occur at any point in the cell cycle, more error-prone?
HR requires a sister chromatid, so is restricted to S/G2- the sister chromatid makes it more accurate
3 pathways of HR
single-strand annealing
double holliday junction (DHJ)
synthesis-dependent strand-annealing
single-strand annealing HR
3’OH generation, annealing of the homologous sequence, removal of ‘flaps’ and ligation
-often leads to deletions
double holliday junction pathway
resection, one end then ‘invades; the sister chromatin
second end captured in a D-loop, 2 HJs are formed
can lead to gene conversion of recombination, depending on how cleavage occurs
dissolution doesn’t lead to crossing over, resolution can
synthesis-dependent strand-annealing pathway
resection, invasion of sister chromatid, polymerase then fills in and completes the repair with no impact on the sister chromatid
strand invasion protein
Rad51
how is HR chosen over NHEJ
MRN displaces recruited Ku, preventing the pathway from occuring
NHEJ
non-homologous end joining
initial recruited protein for NHEJ
Ku70/80 dimer (i think)
C-NHEJ
canonical NHEJ, evolutionarily conserved and pretty much error-free
general steps of NHEJ
recognition of ends, terminal processing, synthesis and ligation
C-NHEJ steps
Ku end binding
processing and damage removal- artemis (endo and exonuclease), pol u polymerases
ligation- complex w Lig4, promoted by the DNA-PKcs that binds early on
A-NHEJ
alternative NHEJ, used mostly during mitosis when there aren’t a lot of other options
A-NHEJ proteins
very biochemically distinct
MRN complex does resection, PARP and Lig3 involved in later steps
why does A-NHEJ lead to deletions?
uses microhomology to bring together strands- but these overlap regions can be really small, hence errors
NHEJ other role
important in antibody gene rearrangement (V(D)J) recombination
important in early stages of immunoglobin/TCR production- helps variability
class switch recombination
changes the type of antibody being produced- IgGs to IgMs etc
mechanism of V(D)J recombination
generation of DSBs by RAG proteins, processing by NHEJ leading to a join
replicative polymerases- lowest to highest fidelity
pol alpha < pol beta < pol epsilon
which polymerases are involved in proofreading?
polymerases epsilon and gamma
how does proofreading work?
the terminus with the incorrect nucleotide flips into the exonuclease site of the polymerase- nucleotide is removed
3 responses to misincorporation
dissociation- when a regular exonuclease will come and remove the nucleotide
extension- polymerase carries on regardless, potential mutation
proofreading by polymerases
example of a common misincorporation
rNTP instead of dNTP, causing backbone issues as RNA is less stable and this can cause nicks to form
can also lead to stalling and fork breakage
what is RER?
ribonucleotide excision repair
rough steps of RER
RNAse makes an incision, extension by pol gamma, flap is cleaved and nick is ligated
what is MMR
mismatch repair
MMR steps
incorrectly inserted nucleotides are removed by a complex binding to the lagging strand,
proteins are recruited and identify the daughter strand,
exonuclease removes ssDNA around the mismatch,
pol gamma resynthesis,
ligation
3 ways bypass without repair can occur
template switching- fork just goes past the damage
translesion synthesis polymerases are recruited- this replaces the normal polymerase, the catalytic subunit can accommodate damage better
repriming by PrimPOl- allows gap filling and repair by pol gamma
what is break induced repair?
mechanism of DNA repair following a break in the replication fork which acts similarly to HR
BIR steps
resection of the cut end
strand invasion
extension of D loop
lagging strand synthesis by Pol gamma
why are stable dNTP levels important?
high dNTP concentration can reduce polymerase fidelity and delay entry to S phase
low dNTP concentration can lead to fork arrest, telomere and mtDNA defects, cause epigenetic breakdown, encourage rNMP incorporation
what maintains dNTP levels?
regulation of proteins ribonucleotide reductase (generates dNTPs) and SAMHD1 (keeps levels low)
example of a damage checkpoint
p53, a tumour suppressor, is important in the G1/S and G2/M checkpoints
proteins needed to detect damage and encourage it to be fixed
sensors
transducers
effectors
how can checkpoints be identified?
genetics (e.g. looking at repair mutants after causing damage, looking at conservation of proteins)
how can a DSB lead to delay in cell cycle continuation?
MRN complex acts as a sensor
downstream phosphorylation of effectors- e.g. Chk2
this phosphorylation of Chk2 blocks phosphatase activity, so CDK remains phosphorylated and inactive, so no cell cycle progression
other ways of getting G2-M blocks other than DSBs
replication fork stalling
low dNTPs and template damage can lead to this fork stalling
another molecule which can trigger a block to mitotic entry
ATR, which eventually phosphorylates Chk1, can also help stabilise forks or stimulate dNTP synthesis, reduce origin firing (less forks becoming activated)
what does p53 do?
induces apoptosis of stressed cells
cellular stress leads to p53 stabilisation, rather than its degradation, and p53 can then encourage transcription of apoptotic proteins such as PUMA and NOXA
what kind of damages can lead to apoptosis by the p53 pathway?
DSBs, HR defects, telomere dysfunction
