Cancer and DNA repair- 4 lectures

Question 1

2 types of DNA damage

Answer 1

endogenous- spontaneous damage from defective repair etc
exogenous- from radiation, sunlight etc

Question 2

biggest mutation burden

Answer 2

SSBs- 55k per cell per day

Question 3

example of 2 types of DNA damage which aren’t DSBs or SSBs

Answer 3

cytosine deamination
depurination/depyrimidination

Question 4

2 ways of identifying DNA repair proteins

Answer 4

looking for abnormal karotypes in mutants
mutational signatures- tracking specific mutations in sequences (you can do a lot of mathsy stuff out of this)

Question 5

photoylases

Answer 5

direct DNA repair enzymes. convert pyrimidine dimers into normal base pairs using photons

Question 6

alkyltransferases

Answer 6

transfer methyl groups to cysteine- also direct DNA damage reversal

Question 7

base excision repair (BER)- early steps

Answer 7

DNA glycosylase does the excision of a lesion
nick is then created

Question 8

2 types of BER

Answer 8

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

Question 9

NER meaning

Answer 9

nucleotide excision repair

Question 10

rough steps of NER

Answer 10

detection of unpairing and abnormal DNA structure
incisions made at both sides of a lesion
excision w helicases
DNA synthesis and ligation

Question 11

what is transcription coupled NER

Answer 11

a version of NER which happens during transcription- triggered by RNA Pol II detecting damage
similar events to regular NER

Question 12

what is ICL repair

Answer 12

interstrand cross-link repair

Question 13

Falconi anaemia

Answer 13

aplastic anaemia with short stature, hypogonadism, skin pigmentation
Due to defective ICL DNA damage repair

Question 14

examples of pathways used in ICL

Answer 14

DNA polymerase synthesis, hom. recomb. and NER- she’s diverse

Question 15

examples of when detection occurs

Answer 15

DNA replication or transcription, or independently of other pathways

Question 16

2 pathways for DSB repair

Answer 16

NHEJ, HR

Question 17

difference between DSB repair paths

Answer 17

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

Question 18

3 pathways of HR

Answer 18

single-strand annealing

double holliday junction (DHJ)

synthesis-dependent strand-annealing

Question 19

single-strand annealing HR

Answer 19

3’OH generation, annealing of the homologous sequence, removal of ‘flaps’ and ligation

-often leads to deletions

Question 20

double holliday junction pathway

Answer 20

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

Question 21

synthesis-dependent strand-annealing pathway

Answer 21

resection, invasion of sister chromatid, polymerase then fills in and completes the repair with no impact on the sister chromatid

Question 22

strand invasion protein

Answer 22

Rad51

Question 23

how is HR chosen over NHEJ

Answer 23

MRN displaces recruited Ku, preventing the pathway from occuring

Question 24

NHEJ

Answer 24

non-homologous end joining

Question 25

initial recruited protein for NHEJ

Answer 25

Ku70/80 dimer (i think)

Question 26

C-NHEJ

Answer 26

canonical NHEJ, evolutionarily conserved and pretty much error-free

Question 27

general steps of NHEJ

Answer 27

recognition of ends, terminal processing, synthesis and ligation

Question 28

C-NHEJ steps

Answer 28

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

Question 29

A-NHEJ

Answer 29

alternative NHEJ, used mostly during mitosis when there aren't a lot of other options

Question 30

A-NHEJ proteins

Answer 30

very biochemically distinct MRN complex does resection, PARP and Lig3 involved in later steps

Question 31

why does A-NHEJ lead to deletions?

Answer 31

uses microhomology to bring together strands- but these overlap regions can be really small, hence errors

Question 32

NHEJ other role

Answer 32

important in antibody gene rearrangement (V(D)J) recombination important in early stages of immunoglobin/TCR production- helps variability

Question 33

class switch recombination

Answer 33

changes the type of antibody being produced- IgGs to IgMs etc

Question 34

mechanism of V(D)J recombination

Answer 34

generation of DSBs by RAG proteins, processing by NHEJ leading to a join

Question 35

replicative polymerases- lowest to highest fidelity

Answer 35

pol alpha < pol beta < pol epsilon

Question 36

which polymerases are involved in proofreading?

Answer 36

polymerases epsilon and gamma

Question 37

how does proofreading work?

Answer 37

the terminus with the incorrect nucleotide flips into the exonuclease site of the polymerase- nucleotide is removed

Question 38

3 responses to misincorporation

Answer 38

dissociation- when a regular exonuclease will come and remove the nucleotide extension- polymerase carries on regardless, potential mutation proofreading by polymerases

Question 39

example of a common misincorporation

Answer 39

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

Question 40

what is RER?

Answer 40

ribonucleotide excision repair

Question 41

rough steps of RER

Answer 41

RNAse makes an incision, extension by pol gamma, flap is cleaved and nick is ligated

Question 42

what is MMR

Answer 42

mismatch repair

Question 43

MMR steps

Answer 43

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

Question 44

3 ways bypass without repair can occur

Answer 44

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

Question 45

what is break induced repair?

Answer 45

mechanism of DNA repair following a break in the replication fork which acts similarly to HR

Question 46

BIR steps

Answer 46

resection of the cut end strand invasion extension of D loop lagging strand synthesis by Pol gamma

Question 47

why are stable dNTP levels important?

Answer 47

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

Question 48

what maintains dNTP levels?

Answer 48

regulation of proteins ribonucleotide reductase (generates dNTPs) and SAMHD1 (keeps levels low)

Question 49

example of a damage checkpoint

Answer 49

p53, a tumour suppressor, is important in the G1/S and G2/M checkpoints

Question 50

proteins needed to detect damage and encourage it to be fixed

Answer 50

sensors transducers effectors

Question 51

how can checkpoints be identified?

Answer 51

genetics (e.g. looking at repair mutants after causing damage, looking at conservation of proteins)

Question 52

how can a DSB lead to delay in cell cycle continuation?

Answer 52

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

Question 53

other ways of getting G2-M blocks other than DSBs

Answer 53

replication fork stalling low dNTPs and template damage can lead to this fork stalling

Question 54

another molecule which can trigger a block to mitotic entry

Answer 54

ATR, which eventually phosphorylates Chk1, can also help stabilise forks or stimulate dNTP synthesis, reduce origin firing (less forks becoming activated)

Question 55

what does p53 do?

Answer 55

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

Question 56

what kind of damages can lead to apoptosis by the p53 pathway?

Answer 56

DSBs, HR defects, telomere dysfunction