L9: maintaining genome integrity Flashcards
What are the sources of DNA damage?
Endogenous biochemical processes (major source)
- Deamination
- Depurination, depyrimidation
- Tautomeric shift – misincorporation
- Reactive oxygen species, free radicals – base modification and strand breaks
Environmental
- Ionising radiation – single and double strand breaks, base and sugar damage and generation of free radicals
- Alkylating agents – base modification (e.g. in cigarette smoke, cooked meat)
- Crosslinking agents – intra and inter strand
- UV commonest environmental mutagen
What are the proofreading DNA polymerases?
- If you’ve got an abberant base, you’re going to copy the DNA and incorporate this abberant nucleotide
- What cells need to do is they need to be able to recognise that there is this misincorporated nucleotide and then they can backtrack, change this base and carry on going
- So it is important to have proofreading polymerases that check the nucleotides that are incorporated
What are the consequences of unrepaired DNA damage?
- Modified bases can block transcription and replication – cell death, premature senescence (aging)
- Modified bases with altered base pairing properties – misincorporation during translation – affect cell function or viability – cell death, premature senescence (aging)
- DNA replication is required to convert a modified or damaged base (DNA lesion) into a permanent change (i.e. into a mutation)
- Modified bases with altered base pairing properties – misincorporation at DNA replication – mutation – affect cell function or viability – cell death, premature senescence (aging), cancer
- Strand breaks (particularly DSBs) – genome instability at cell division – affect cell function or viability – cell death, premature senescence, cancer
How is genome of the skin stem cells protected?
- organisation minimises progressive accumulation of mutations (necessary for cancer)
- In skin: stem cells are deep within the tissue and they move further and further to the top of the skin
- In addition there’s another protective mechanism in our skin: melanin.
- Melanin is very good at protecting our cells from the damage of UV, people with eumelanin are more protected than people with pheomelanin, because eumelanin is able to absorb the free radicals generated by the UV light
What are the biochemical mechanisms of protection by eliminating/neutralising genotoxic agents?
- Stem cells have high levels of Mdr1 (multi-drug resistance) – a membrane protein that pumps out genotoxics (so do cancer cells – resistance to chemotherapeutics!)
- Detoxification of ROS e.g. by superoxide dismutase, catalase
- Free radical scavengers e.g. Vitamin C/E (soak up)
- Glutathione-S-transferases – detoxify many carcinogens by linking them to glutathione (convert into safer forms)
- Frequent inactivation of GSTP gene in many cancers (increased rate of mutation driving carcinogenesis)
What are the possible DNA repair pathways?
- Nucleotide excision repair (NER)
o Inherited NER gene mutations in xeroderma pigmentosum (paradigm for inherited DNA repair deficiency/cancer susceptibility disorders)
o UV, alkyl, oxidative, intra strand cross links - Base excision repair (BER) [SSBR]
o alkyl, oxidative, depurination, depyrimidination, [SSBs]
o Inherited mutations in MUTYH gene (BER of oxidative damage) cause Multiple Adenoma Phenotype (MAP)
o Inherited SSBR defect in neurodegenerative disorder- spinocerebellar ataxia with axonal neuropathy - Mismatch repair (MMR)
o Inherited MMR gene mutations in hereditary non-polyposis colon cancer (HNPCC). - DNA double strand break repair by non-homologous end joining (NHEJ)
o DSBs
o Non-homologous (error prone)
o Inherited LIG4 mutation- immunedeficiency, lymphoid malignancy - DNA double strand break repair by homologous recombination
o DSBs, [ICLs]
o Inherited BRCA1 and BRCA2 mutations in hereditary breast cancer
What is the mechanism of base excision repair?
Base excision repair
- DNA glycosylase marks a bit of DNA that is distorted
- AP endonuclease is then recruited that removes the bad base
- Then another enzyme repairs that DNA sequence
- And finish off with a DNA ligase that glues the DNA together
What is the nucleotide excision repair pathway?
- Deals with the broadest range of lesions – UV, bulky adducts, intra-strand cross links.
- The importance of DNA repair in protecting against cancer was first revealed in 1968 when NER was found to be defective in the human skin cancer prone inherited disease xeroderma pigmentosum (XP).
- NER has been extensively characterised biochemically (reconstituted in vitro) and genetically – around 30 proteins involved.
- By study of human XP mutant cell lines and rodent UVS ERCC mutant lines (excision repair cross complementing – defect can be complemented by transfer of functional ERCC gene).
What are the two types of nucleotide excision repair?
- Global genome repair: operates at low level over entire genome
- Transcription coupled repair: operates at a higher level on the transcribed strands of active genes (UV-induced lesions block transcription!)
what is the phenotype of xeroderma pigmentosum?
Xeroderma pigmentosum phenotype
- Rare autosomal recessive
- Parchment skin (xeroderma) and freckles (pigmentosum) on sun-exposed areas
- Progressive degenerative eye and skin alterations – mean onset 2 years of age
- >1000-fold increased risk of skin cancers
- 20-fold increased risk of internal tumours
- Progressive neurologic abnormalities
How lesions are incorporated in the nucleotide excision repair pathway?
- Polymerase slippage during replication of repeat sequences
- If polymerase slips you tend to incorporate one too many or one too few of these individual nucleotides
what is the mismatch repair pathway?
- Machinery that recognises polymerase slippages
- Two main proteins: MutS and MutL
- These proteins look to see if there’s any slippage
How is MMR deficiency and hereditary Non-Polyposis Colon Cancer (HNPCC) linked?
- HNPCC – hereditary ~3% of colon cancers
- Accelerated cancer progression, adenoma – carcinoma in 2-3 years (usually 8-10 years)
- Germ line mutations in a MMR gene – mostly MSH2 or MLH1 – predisposed to colon cancer
- Patients inherit 1 defective allele
- In tumours there is loss of heterozygosity (LOH) – the remaining functional allele is lost
- Leads to high rates of mutation which drive carcinogenesis
- If you are unable to fix these little bubbles that form in the DNA, you start changing the number of these individual repeats
Describe repair of double strand breaks by homologous recombination (HRR)
- One of the most important
- Can repair DNA with the highest fidelity
- Also one of the most complicated
- Uses the good copy of your DNA sequence to repair the bad copy of your sequence
- Uses another DNA sequence as a template
describe repair of double strand breaks by non-homologous end joining (NHEJ)
- Used out of desperation, if there’s no other way
- Problem: doesn’t have a correct template and you get high frequency of mutations following from NHEJ
