Lecture 6- DNA repair Flashcards
Give some example of how DNA molecules can be damaged
- Thermal degradation
- Metabolic byproducts/oxidation
- Environmental substances
- Radiation
Which bases are purines and which are pyrimidines?
G and A = purines
T and C = pyrimidines
Describe the base pairings
- A pairs with T (2 hydrogen bonds)
* C pairs with G (3 hydrogen bonds so are more thermally stable)
Which group on thymine doesn’t hydrogen bond?
Methyl group
What happens to cytosine in the presence of water?
- deamination
- loses amino group and converted to carbonyl group
- looses NH3
- causes cytosine to be converted to uracil which then hydrogens bonds with adenine
Name 3 ways DNA bases can be damaged
- Deamination of cytosine
- Oxidative damage
- Hydrolytic attacks
What is a transition mutation?
refers to a point mutation that changes a purine nucleotide to another purine, or a pyrimidine nucleotide to another pyrimidine.
Why are transition mutations more likely to occur than transversions?
Substituting a double ring structure for another double ring structure is more like than substituting a single ring for a double ring (and vice versa)
What is a transversion mutation?
Transversion substitution refers to a purine being replaced by a pyrimidine, or vice versa
What is a frameshift mutation?
Frameshift mutation is a type of mutation involving the insertion or deletion of a nucleotide in which the number of deleted base pairs is not divisible by three. If a mutation disrupts this reading frame, then the entire DNA sequence following the mutation will be read incorrectly
What is photolytic conversion?
Light energy modifying the chemistry of the carbon-carbon double bond
How is depurination resolved?
By the BER pathway
Describe the BER pathway
- Uracil DNA glycosylase remove particular base (deaminated cytosine) leaving DNA helix with a missing base
- AP endonuclease and phosphodiesterase remove sugar phosphate creating a single-nucleotide gap
- DNA polymerase adds/replaces nucleotide into the gap so the U is restored to a C
- DNA ligase seals the nick
How do BER glycosylases identify errors?
Using base-flipping strategy
Describe the nucleotide excision repair pathway
- Excision nuclease cuts out the region around the pyrimidine dimer, removing the damaged section of DNA
- DNA helicase helps with this and unwinds the DNA into single strands
- Left with DNA helix with 12-nucleotide gap
- DNA polymerase and DNA ligase then add nucleotides and seal the gap by using the template strand
How are pyrimidine dimers removed?
By the nucleotide/short patch excision repair pathway
Describe the trans-lesional DNA synthesis pathway
- Covalent modifications occur to sliding clamp when polymerase encounters DNA damage
- This causes the DNA polymerase to be released
- The sliding clamp then allows the loading of translesion polymerase by assembly factors
- Translesion DNA polymerase then does some DNA synthesis to ‘mend’ the damaged DNA
What are the 2 mechanisms to repair double stranded breaks?
- Nonhomologous end joining
2. Homologous recombination
Why are double strand breaks particularly hazardous and what causes them?
No template strand to facilitate repair
Caused by: ionising radiation, replication errors and oxygen radicles
How does nonhomologous end joining repair double stranded DNA breaks?
- The ends of the DNA break are recognised by Ku heterodimers and can be processed and made shorter
- The ends are then joined together by ligation and repaired
- Can cause a deletion of part of the DNA sequence which can lead to a missense mutation
- Also required DNA-PK and ATM protein kinases function
How does homologous recombination repair double stranded DNA breaks?
- Processing of 5’ ends by nuclease at the point of the DNA break to create sticky ends
- Strand exchange occurs by complementary base pairing
- Using the information from the sister chromatids homologous recombination occurs
- Invalid strand is released/broken and the double helix reforms
- DNA synthesis continues using strand from damaged DNA as template to fill gaps from the break
- DNA ligation
- Damaged repaired accurately using sister chromatid as a template
What are the steps to perform a biochemical analysis of complementation groups?
- Make cell lysates
- Use artificially damage DNA templates
- Use repair assay to determine enzymatic function for each group
- Repair the transcribed strand is faster than the non-transcribed strand
What does trans-lesional DNA polymerase lack and cause when performing trans-lesional DNA synthesis?
LACK: precision in template recognition and substrate base choice and exonucleolytic proof reading activity.
CAUSE: most base substitutions and single nucleotide deletion mutations