Damage, repair and recombination of DNA sequences Flashcards
What 3 things can damage DNA?
1) Oxidation (H removed or O added)
2) Hydrolysis (Water splitting bonds)
3) Uncontrolled random methylation to a base
How does oxidation damage DNA?
Opens up bonds (double bonds)
Affects the ability of the bases to bond with its pair
How does hydrolysis damage DNA?
Can release amino groups on the bases (DEAMINATION)
OR
Can separate a purine base from the deoxyribose sugar (DEPURINATION)
How does uncontrolled methylation damage DNA?
Methylation of the base at N atom - affects the ability of the bases to interact with their counterparts
What is oxidation?
H removed
OR
O added
What is the result of DEPURINATION?
Liberation of a purine (guanine or adenine) from its sugar attachment:
- Un-purinated sugar has no coding potential
- Gap in the DNA sequence (read by DNA polymerase as no DNA information
- Deletion of a bas pair in that position
What is the result of DEAMINATION?
Removal of a amino group from the base:
- Changes the identity of the base (cytosine to uracil)
- Causes wrong base pairing (adenine instead of guanine)
In deamination, what is the base change?
Cytosine to Uracil
What is BASE Excision Repair and what is the process of it?
Repair of point mutations (deamination and depurination):
1) Uracil DNA glycosylase recognises uracil in the DNA as foreign - cleaves uracil from the sugar
2) AP endonuclease cuts the inside of the DNA and removes the SUGAR
3) Followed by cleavage of the phosphate in the DNA backbone by phosphodiesterase (removes the PHOSPHATE)
4) Single stranded break and loss of nucleotide is formed - this is a signal for DNA polymerase (as this is a primer-template junction)
5) DNA polymerase repairs the gap
6) DNA ligase seals the nick
What causes the formation of pyrimidine dimers?
UV radiation
What are pyrimidine dimers?
What does this cause?
Formation of covalent bonds between the carbon atoms of two adjacent pyrimidines (cytosine or thymine)
- Distorts the structures of the pyrimidines - they can no longer pair properly with purine counterparts
- No longer can form correct H bonds
- Arrests DNA replication or cause mis-reading of the DNA sequence by DNA polymerase
What is the NUCLEOTIDE Excision Repair?
What is the process of repairing a pyrimidine dimer?
Repairs many types of DNA damage (including pyrimidine dimers)
1) Covalently linked dimers distort the helical structure of DNA - are readily detected
2) Recruits excision nuclease - Cleaves single-stranded DNA segment containing pyrimidine dimer defect
3) DNA helicase liberates the DNA fragment by disrupting the H bonding
4) Causes a gap in the DNA - primer template junction recognised by DNA polymerase
5) DNA ligase seals the nick
What disorder does DEFECTIVE Nucleotide Excision Repair machinery cause?
How?
Xeroderma pigmentosum (XP) - Extreme sensitivity to sunlight-induced skin cancer
Mutations in 7 different genes can cause this (including XPA, XPC, XPD, XPF, XPG)
These genes all encode enzymes which participate in NER pathway for the correction of UV-induces DNA lesion
SO, cause a defect in the repair of UV induced dimers
What is up-regulated in response to UV light?
What happens if exposed to TOO MUCH UV light?
Enzymes involved in the Nucleotide Excision Repair Pathway
(Genes: XPA, XPC, XPD, XPF, XPG)
If too much light - repair system becomes overloaded and can lead to mutations that cannot be repaired
What are the homologues of XP genes in E.coli?
Uvr protiens (UvrA, UvrB, UvrC, UvrD)
How is the Nucleotide Excision Repair system targeted to transcribed DNA sequences?
It is PHYSICALLY COUPLED to RNA polymerase,
As RNA actively transcribes genes - NERS moved along with it
So, os constantly surveying the DNA to find any spontaneous mutations occuring
Where is NERS targeted to?
Protein coding genes by RNA pol
Why are double stranded DNA breaks dangerous?
Can break a chromosome in half and can cause the loss of the chromosome to one of the daughter cells (not segregated)
What 2 methods can repair dsDNA breaks?
1) Non-homologous End Joining
2) Homologous Recombination (HR)
What is the process of Non-homologous End Joining?
What does this cause>
Trims back the overhang of the broken DNA to make the ends of the DNA flush
Then, quick stick back together of 2 molecules which looks like they should be together
Causes a loss of DNA nucleotides
Is Non-homologous End Joining effective?
Not really:
- No sequence specificity (if 2 or more chromosomes break - randomly sticks back together)
- Can scramble chromosomes (mutagenic)
- Also causes a loss of DNA nucleotides as trims back the overhangs before sticking them back together
What is the last line of defence when DNA is broken?
Why?
Homologous recombination (after BER and NER)
As breaks in the DNA formed by BER or NER weaken the DNA structure - is then prone to becoming a dsDNA break
In which cells does HR repair DNA breaks?
DOUBLE STRANDED breaks in EVERY cell (dividing or not)
What is often recruited to the site of DNA damage, along with NER, BER machinery?
HR machinery
How is HR different to Non-Homologous end joining?
HR is an accurate repair mechanism
As it uses the information from the DNA sequence in the UNDAMAGED sister chromatid to repair the broken one
NHEJ doesn’t do this - random stick back together
What is the process of HR to repair double stranded DNA breaks?
1) Broken DNA molecule is resected at its 5’ end of both the DNA stands - creating a single stranded overhang of the 3’ tails
2) DNA-binding protein RecA promotes STRAND INVASION of one of the 3’ overhangs into the sister chromatid (undamaged chromosome)
3) This disrupts the base pairing in the sister chromatid and forms a recombination intermediate (base paring between the sister chromatid and the 3’ overhang - by RecA)
4) DNA pol begins to extend the 3’ end of the broken DNA using the sequence information from the sister chromatid
5) Migration of the branch point until the newly synthesised DNA of the damaged DNA fully overlaps the region of resection
6) Newly synthesised DNA dissociates from the sister template and re-anneals to its original partner strand - forms a primer-template junction, allowing the synthesis of the other strand by DNA pol
7) Forms a pair of single stranded nicks, which are repaired by DNA ligase
During HR, what is the point of invasion by the 3’ overhang into the unbroken sister chromatid called?
The branch point
What is a ‘heteroduplex’, formed in HR?
A double stranded molecule of nucleic acid originated through the genetic recombination of 2 single complimentary strands, derived form a different source (Eg. homologous chromosome)
What mutations in the genes encoding components of the HR DNA repair mechanisms cause cancer?
What cancers do they cause?
1) BRCA2
- Breast
- Ovarian
- Prostate
2) ATM
- Leukaemia
- Lymphoma
3) Fsnconi Anaemia
- Leukaemia
What happens once the HR recombination is defective in BRAC2-mutated cancers?
These cancerous cells become dependant on the OTHER pathways for correct DNA repair
What is the treatment for BRAC2-mutated cancers and why?
Inhibitors of the BER and NER mechanisms
These tumours depend on these to repair their DNA
Cells suffer so much DNA damage - they are unable to make adequate repairs to their DNA and they die
What is ‘synthetic lethality’ and where is it present?
When the simultaneous perturbation of 2 genes result in cellular death
Present in BRAC2-mutated cancers when they are treated with inhibitors of BER and NER mechanisms
What are PARP inhibitors?
Treat ovarian and prostate cancer by blocking BER and NER in the BRAC2 tumour
Why is homologous recombination used in meiotic cells?
The get the ‘crossing-over’ of paired chromosomes - to get genetic diversity
What is the process of HR in gametes?
1) Spo11 endonuclease forms a double stranded break in one of the 2 molecules to be recombined
2) Mre11 endonuclease resects back the 5’ ends - to create 3’ overhangs
3) RecA promotes strand invasion and the formation of a branch point
4) DNA polymerase extends the branch point
5) There is a meosis specific ligation event where the 3’ end of the newly synthesised molecule is joined to the 5’ end of the broken DNA strand, before it has dissociated from the template - forming a SECOND branch point
6) Holiday junction is formed
7) Ligation also occurs in the OTHER strand of broken DNA
In HR in gametes, what determines if recombination occurs?
How can this happen?
How the holiday junction is resolved
Can happen in 1 of 2 ways:
1) ONLY internal stands are broken and rejoined - NO genetic exchange
2) BOTH internal and external strands are broken and rejoined - genetic exchange!!
