Maintenance Of Genomic Integrity Flashcards
How does damage to the DNA occur?
Copying errors during DNA replication - greatest number
Spontaneous depurination
Exposure to different agents - UV light, tobacco products
5 major types of DNA repair
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
Guanine methylation
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
Guanine methylation another example - ethyl Methane sulphonate
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
UV induced DNA lesions
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
DNA repair
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
Types of enzyme reversal
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
Base excision repair (BER) substrates
Spontaneous hydrolytic depurination of DNA
Deamination of cytosine
Formation of DNA adducts after exposure to reactive small metabolites
BER process
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
DNA glycosylases
There are many glycosylases each one recognises a different type of base damage
Nucleotide excision repair
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
Principle of NER
Endonuclease
Exonuclease - removes several or tens of nucleotides
Polymerase
Ligase
Daughter strand gap repairs
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
Xeroderma pigmentosa
What causes it
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
How many types of XP are there ?
Is each mutation controlling a different thing does this change the outcome of the disease
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
Normal NER step 1
2 proteins recognise the DNA damage and bind to it
XPC recognises the 6-4 product not CPD
XPC and XPE recognise CPD
NER step 2
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
NER step 3
Recruitment of XPF and XPG nucleases which cleave DNA surrounding the lesion
Lesion removed
NER step 4
DNA polymerase and accessory factors are required to resynthesise the DNA across the excised region
Mutation and cancer in XP
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
XP variants
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
Repair of double strand breaks includes
BRCA1 and BRCA2
How similar are BRCA1 and BRCA2
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
What are BRCA1 and BRCA2 involved with repair of
DSB
The 2 processes DSB are repaired by
Non- homologous end joining
Homologous recombination repair
What happens in homologous recombination repair
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
What regulates the availability of RAD51
BRCA2
BRCA2 interaction with RAD51
Direct interaction
Binding of RAD51 occurs through the eight BRC repeats in BRCA2
BRCA2 controls intracellular movement and function of RAD51
What is release of RAD51 triggered by
DNA damage by phosphorylation of RAD51 or BRCA2
What is also required for HRR
BRCA1
What is the BRCA1 mechanism
Through the interaction and removal of 53BP1 at sites of DSB, prior to resection and recombination
What happens in homologous recombination repair
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
Non homologous end joining
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
Treating HRR deficient tumours with PARP1 inhibitors
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
What is a DNA mismatch repair
This repairs copy errors made during DNA replication
Hereditary non polyposis colorectal cancer
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
Mutator phenotype hypothesis
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
