DNA repair
Direct Repair
it is the elimination of damage using chemical reversion that does not require a nucleotide template, breaking the backbone or DNA synthesis
Repair SS breaks
The break arises from replication, recombination or radiation
can by hazardous if breaks close on opposing strands or if replicated (since can create ds break)
Uses DNA ligase
energy from ATP except bacteria which use NAD
requires 3' OH and5' P
Phage T4 DNA ligase can join blunt ended dsDNA
Photoreactivation
Restores cyclobutane pyrimidine dimers with DNA photolyase
DNA photolyase requires folate and flavin as light harnessing cofactors
reduced FAD becomes excited when it absorbs blue light energy and donates an electron to the pyrimidine dimer, which breaks the covalent bonds. the electron is then transferred back
Repair of O-methylguanine
Methylating agents react with DNA to produce O-alkylated and N-alkylated
methyltransferase recognises the distortion in backbone and accepts the methyl group onto a cysteine, this inactivates the protein
found in E.coli Ada protein which regulates repair
Mismatch repair
Bacterial DNA pol III backtracks and excises incorrect nucleotides
Dam methylase adds methyl group to adenine in GATC 5'-3' sequence, which allows identification of new strands
Proteins
MutS- clamps on DNA and finds backbone distortion
MutL- stimulates MutS and MutH
MutH- cleaves the unmethylated strand to the mismatch site
DAN polymerase- synthesises DNA using template strand
UvrD- unwinds from the nick
Base excision repair
Protects DNA from effects of oxidation, alkylation and deamination
fixes single-base lesions
Process
DNA glycosylases recognise damaged base and cleaves bond between base and backbone
AP endonuclease recognises AP site and cleaves backbone on either site
DNA pol and ligase fill in holes
Uracil DNA glycosylase
Uracil gets into DNA when cytosine is deaminated and when dUTP is misincorporated during DNA synthesis
UDG flips U out of the duplex to check before cleavage
AP endonucleases
exonuclease III cleaves on 5' side of AP site
endonuclease IV cleaves on 3' side of AP site
other enzymes process other thins that need to be removed
Nucleotide excision repair (bacteria)
the removal pf damaged base/s as part of a short oligonucleotide
can repair interstrand crosslinks
Proteins
UvrA- recognises distortion in backbone and loads UvrB onto damaged DNA
UvrB- bends DNA
UvrC- binds to UvrB and makes 3' incision and after DNA is straightened it makes incision on 5' strand
UvrD- releases/unwinds oligonucleotide containing damaged base/s and UvrC
Transcription-Coupled repair (bacteria)
DNA lesion that block polymerase is recognised by Mfd enzyme when RNAP is stalled
the region is exposed and UvrA is recruited., then UvrB onto UvrA
the UvrAB complex forms a beta-hairpin and UvrC completes the process
Mutation Frequency decline (bacteria)
cells defective in this gene do not exhibit mutation frequency decline and
it tracts along the DNA an d ejects the RNAP and the transcript from the DNA
it then attracts UvrA to promote repair
PCR
Taq DNA polymerase meant researchers had a thermostable enzyme for repeat PCR cycling, but had no proof reading process so had a higher mutation rate
Requirements: Known DNA sequence, primers, thermostable DNA polymerase, dNTPs, thermal cylinder
Primer characteristics
18-26 nucleotides
similar melting point of DNA, so similar GC bonds
a "GC clamp" at 3' end
minimal secondary structure
minimal repeat sequences or single bases because polymerases make more mistakes
3 steps
Denaturation- high temperature to separate the strands (90-95)
Annealing- cooler temperature to allow primer binding (55-65)
Extension- optimum temperature for DNA polymerase for dNTP incorporation (68-72)
products start to form after the third cycle since in the first 2 DNA extends past primer (no blunt end)