Maintenance Of Genomic Integrity

Question 1

How does damage to the DNA occur?

Answer 1

Copying errors during DNA replication - greatest number
Spontaneous depurination
Exposure to different agents - UV light, tobacco products

Question 2

5 major types of DNA repair

Answer 2

Direct reversal of damage
Base excision - corrects DNA damage caused by reactive oxygen species deamination, hydroxylation, spontaneous depurination
Nucleotide excision repair - removes adducts that produce large distortions of DNA
Homologous recombination repair and non homologous recombination repair- repairs DNA double strand breaks
DNA mismatch repair - repairs copy errors made during replication

Question 3

Guanine methylation

Answer 3

Example 7-methyl-guanine nitrogen at point 7 is methylated
This type of damaged is caused to damage the cancer cells in order to try to treat the cancer
7 methyl guanine causes a large distortion in the DNA causing a problem in DNA replication -> leads to cell death which is the treatment goal

Question 4

Guanine methylation another example - ethyl Methane sulphonate

Answer 4

Guanine normally pairs with cytosine
O-6 alkyl guanine pairs with thymine
Thymine pairs with adenine
There is an overall transition from G-c to A-T

This mutation does not cause cell death -> it does cause a transition mutation this is a dangerous mutation can cause cancer if not removed

Question 5

UV induced DNA lesions

Answer 5

Thymine dimer - 2 thymines in the DNA are liked by C=C double bonds
CPD cyclobutane pyramidine diner contains a 4 membered ring arising from the coupling of the C=C double bond of pyramidines
These diners interfere with base pairing during DNA replication, leading to mutations
(6-4) photo products - pyrimidine dimer
Occurs less frequently more mutagenic

Question 6

DNA repair

Answer 6

Cells have several repair systems and are usually constitutive
Approx 100 genes involved
Many different substrates
Broadly speaking repair involves either:
Enzymatic reversal
Removal and replacement of damage

Question 7

Types of enzyme reversal

Answer 7

UV induced dimers-> monomerisation (break cyclobutane bond leaving 2 normal thymine)
By action of visible light and photolyase
O^6 alkyl guanine removal of alkyl group via the action of alkyl transferase -> returns the structure back to normal
Strand break in sugar phosphate back bone -> ligation by lighting enzyme

Question 8

Base excision repair (BER) substrates

Answer 8

Spontaneous hydrolytic depurination of DNA
Deamination of cytosine
Formation of DNA adducts after exposure to reactive small metabolites

Question 9

BER process

Answer 9

Altered DNA base is excised in free form by a DNA glycoslyase and the resulting abasic site is corrected by the concerted action of an apurinic endonuclease, a DNA polymerase and a DNA ligand.

1) removal of base
2) removal of a purification site -sugar and phosphate
3) addition of new nucleotides
4) ligation

Question 10

DNA glycosylases

Answer 10

There are many glycosylases each one recognises a different type of base damage

Question 11

Nucleotide excision repair

Answer 11

Operates on double stranded DNA
Cannot act on sign,e stranded DNA - in replication
Non specific, it recognises distortions rather than specific adducts
Will remove and repair large adducts e.g. Thymine dimer

Question 12

Principle of NER

Answer 12

Endonuclease
Exonuclease - removes several or tens of nucleotides
Polymerase
Ligase

Question 13

Daughter strand gap repairs

Answer 13

During synthesis of new DNA/RNA replication gaps are left opposite dimers as they cannot be fixed until in a double strand
Tolerance mechanism
Dimers are then removed later from the double stranded DNA by excision repair

Question 14

Xeroderma pigmentosa

What causes it

Answer 14

Autosomal recessive - rare 
Extreme sun sensitivity
Skin tumours 
Dimer not removed new thymine not put into the DNA - no DNA repair 
Defect in DNA NER

Question 15

How many types of XP are there ?

Is each mutation controlling a different thing does this change the outcome of the disease

Answer 15

Each involve different mutation of different genes
Each mutations blocks the normal pathway at a different point
All types of XP leads to the same clinical outcome/disease

Question 16

Normal NER step 1

Answer 16

2 proteins recognise the DNA damage and bind to it
XPC recognises the 6-4 product not CPD
XPC and XPE recognise CPD

Question 17

NER step 2

Answer 17

XPA and TFIIH are recruited to the site of damage -> damage verification
TFIIH contains XPB and XPD these are helicases which unwind the DNA surrounding the lesion

Question 18

NER step 3

Answer 18

Recruitment of XPF and XPG nucleases which cleave DNA surrounding the lesion
Lesion removed

Question 19

NER step 4

Answer 19

DNA polymerase and accessory factors are required to resynthesise the DNA across the excised region

Question 20

Mutation and cancer in XP

Answer 20

XP cells show a high mutation rate
Mutation probably due to unexcised dimers and therefore incorrect bases incorporated opposite damage
This mutation represents a step towards cancer development

Question 21

XP variants

Answer 21

Not deficient in NER
Not very sensitive to killing my UV, but cells are hypermutable by UV
Sensitivity to UV can be engaged by caffeine
Defect in replication of DNA following UV exposure of cells - daughter strand gap repair
Deficient in an enzyme DNA polymerase h, which is able to replicate DNA past UV photo products - translesion synthesis

Question 22

Repair of double strand breaks includes

Answer 22

BRCA1 and BRCA2

Question 23

How similar are BRCA1 and BRCA2

Answer 23

Very different primary sequences
BRCA1 c terminal BRCT domains are found in many repair proteins -> either as pairs or with FHA domain a forkhead associated domain
BRCA1 is half BRCA2
BRCA2 contain prominent BRC repeats these repairs mediate binding to RAD51

Question 24

What are BRCA1 and BRCA2 involved with repair of

Answer 24

DSB

Question 25

The 2 processes DSB are repaired by

Answer 25

Non- homologous end joining

Homologous recombination repair

Question 26

What happens in homologous recombination repair

Answer 26

Catalytic activity of RAD51 is central to this form of DSB repair
RAD51 coats SS DNA to form nucleoprotein filament that invades and pairs with homologous DNA duplex - initiating strand exchange

Question 27

What regulates the availability of RAD51

Answer 27

BRCA2

Question 28

BRCA2 interaction with RAD51

Answer 28

Direct interaction
Binding of RAD51 occurs through the eight BRC repeats in BRCA2
BRCA2 controls intracellular movement and function of RAD51

Question 29

What is release of RAD51 triggered by

Answer 29

DNA damage by phosphorylation of RAD51 or BRCA2

Question 30

What is also required for HRR

Answer 30

BRCA1

Question 31

What is the BRCA1 mechanism

Answer 31

Through the interaction and removal of 53BP1 at sites of DSB, prior to resection and recombination

Question 32

What happens in homologous recombination repair

Answer 32

BRCA1 removes 53BP1 in order for repair to being

1) resection exonucleases and endonucleases remove section of DNA in the 5’ damaged end
2) creating a 3’ overhang which BRCA2 covers in RAD51
3) invasion where homologous chromosomes or sister chromatids if in the latter part of the cell cycle are used as templates to replace the missing nucleotides
4) DNA polymerase and DNA ligase repairs the nucleotides back together again

Question 33

Non homologous end joining

Answer 33

Where the DSB the nucleotides on either side are resected and then are simply put back together again
This is used in the making of antibodies and TCR
No RAD51 or BRCA2 used
More prone to errors
The proteins formed will have missing rna so will not function correctly

Question 34

Treating HRR deficient tumours with PARP1 inhibitors

Answer 34

Ssbr in replicstion are normally fixed by PARP
If parp not present or inhibited then the repair cannot be fixed the replication fork collapses and the cell dies
This can be used on cancer cells as if cancerous the single strand break cannot be repaired with out parp causing cell death

Question 35

What is a DNA mismatch repair

Answer 35

This repairs copy errors made during DNA replication

Question 36

Hereditary non polyposis colorectal cancer

Answer 36

Mutation in the mismatch repair genes
Usually insertion deletion loops are repaired which result as a consequence of polymerase slippage during replication
Slippage causes gains or losses in repetitive DNA
Also called micro satellite instability

Question 37

Mutator phenotype hypothesis

Answer 37

Micro satellite instability
Postulates that mismatch repair defects lead to mutation in other genes including those known to play a role in the adenoma - carcinoma sequence
Therefore increase mutation rates is then the cause of accelerated tumourigenesis
The mutator phenotype plays a role in tumour progression rather than initiation