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)