Lecture 8 DNA Repair 1 DR/MMR/BER Flashcards
DNA damage and DNA repair
Diff restoration method depending on damage
Direct repair (DR) for methylated bases and pyrimidine dimers
Base excision repair (BER) for modified bases and abasic sites
Nucleotide excision repair (NER) for a range of bulky lesions including pyrimidine dimers
Mismatch repair (MMR) for misplaced bases short insertions and deletions resulting from DNA synthesis
Recombinational repair for homologous recombination, interstrand crosslink, dsDNA breaks and problems with DNA replication
Direct repair (DR)
Elimination of DNA damage by chemical reversion without nucleotide template or breakage of phosphodiester backbone or DNA synthesis. Error free process preserves genetic info. Simple - uses little energy and safe - minimises chance of mistakes by repair.
Limited to:
A) repair of ss breaks
B) repair of pyrimidine dimers
C) repair of methyl groups
Direct repair: SS DNA breaks
Breaks occur during DNA recombi and replication, DNA repair or exposure to X-rays or gamma radiation.
Not a problem for non dividing cells but can interfere with transcription. Breaks close to each other on opposing strands or replication of an SS break can generate potentially more hazardous dsDNA break
DNA ligase repairs SS DNA breaks. Bacterial DNA ligases use NAD. Bacteriophage and eukaryotic DNA ligases use ATP as an energy cofactor. DNA ligases cannot join free SS DNA molecules.DNA ligase requires a 3’OH group and 5’ -P group, larger gaps cannot be repaired.
Phage T4 DNA ligase can join blunt ended dsDNA useful in cloning
Direct repair: photoreactivation - restoration of pyrimidine dimers
1949 Albert Kelner was studying survival of Streptomyces griseus spores after UV irradiation but kept getting anomalous results. UV irradiated spores exposed to light showed signs of increase in survival and recovery.
Renato Dulbecco found similar results in phage also publishing in 1949 and called the process photoreactivation.
10 years later it was discovered that photoreactivation caused by DNA photolyase or photo reactivating enzyme. The first DNA repair process to be discovered
Enzyme repairs cyclobutane pyrimidine dimers (CPD) e.g. T<>T, T<>C,C<>C. DNA photolyases are found in viruses bacteria yeast plants invertebrates and many vertebrates.
DNA photolyase requires 2 light harvesting cofactors to absorb light energy (350-450nm blue light):
5,10 methenyl tetrahydrofolate &
1,5 fihydroflavin adenine dinucleotide (FAD)
Thus light can be used to repair UV damage (280-315nm)
Catalytic FAD cofactor is in a reduced state so that when the absorbed blue light energy is transferred the FADH- becomes excited and donates an electron to the dimer addition of this electron to the pyrimidine dimer breaks the covalent bonds and the pyrimidine molecules are returned to their undamaged states. Once the damage is repaired the electron is transferred back to the FADH- cofactor returning it to it’s reduced state regaining it’s catalytic activity
Direct repair: repair of methyl groups - e.g. O6- methylguanine
Methylating agents such as methyl methane sulphonate (MMS) react with DNA to produce both O alkylated and N alkylated products. On of these products is O6-methylguanine which can mispair with thymine. A methyltransferase recognises the distortion in the DNA backbone. Works best on dsDNA. Methyltransferase accepts the methyl group onto a cysteine in the protein, which results in its inactivation. Hence it is known as a suicide methyltransferase, since it’s activity is lost in the process of repair.
Ada protein: structure and activity
E.coli Ada protein regulates a set of genes involved in repairing alkylating damage - N terminal half of Ada switches on this ‘adaptive response’ once methylated. Humans have similar enzymes but lack N-terminal 20 kDa portion of Ada
Removal of methyl groups achieved by single step methyltransferase reaction. Ada accepts adducts from the modified oxygen molecule onto internal cysteine residues, directly restoring DNA damage and inactivating the Ada protein. Once modified protein is target for degradation. Ada has 2 active cysteines one for repair of O6- methylguanine and other for restoration of methylphosophotriesters (methyl group on phosphate backbone) addition of the latter acts to switch on the ‘adaptive response’
Mismatch repair MMR
Mismatched occur despite proof reading during DNA replication. Hard to remove as cannot be recognised like damaged bases - mismatch pairs appear ‘normal’
Proof reading: bacterial DNA pol lll backtracks and incorrectly inserted nucleotide is excised by 3’5’ exonuclease activity of epsilon the proof reading sub unit aka dnaQ. Mutation of dnaQ gene results in elevated mutation rate.
MMR removes mismatched pairs in the newly synthesised strand allowing E. Coli to replicate it’s genome of 4.6x10^6 nucleotides 9 times out of 10 without any errors.
Multiple pathways for avoiding and repairing mismatches. Major repair system used in bacteria uses MutHLS proteins and depends on DNA methylation to distinguish between template and newly synthesised strand.
In E. Coli Dam methylase adds methyl(CH3) group to adenine in sequence 5’GATC3’ the short delay in methylation of the newly synthesised strand allows enzymes to identify new DNA and target repair to this strand.
Key enzymes of methyl directed MMR:
MutS - binds DNA mismatch
MutH - cuts unmethylated strand at GATC site
MutL - stimulates MutS and MutH
MutU (UvrD) - unwinds nicked DNA strand nicked by MutH
Dam- methylates GATC sites
MutS clamps on DNA and scans for mismatches until it finds a distortion in the DNA backbone caused by the mispaired bases. MutL and MutH are then assembled. DNA is threaded through this MutHLS complex in a loop until hemimethylated DNA is encountered. MutH cleaves the unmethylated strand, corresponding to newly synthesised daughter strand. DNA helicase ll (UvrD) unwinds from this nick and exonucleases degrade the unwound strand. Gap is filled by DNA pol l or lll and nick sealed by DNA ligase.
Types of damage repaired by base excision repair
Deaminated bases e.g. cytosine to uracil, adenine to hypoxanthine
Spontaneous loss of bases e.g. hydrolytic loss of purines
Oxygen free radical damage e.g. thymine glycol, 7,8 dihydro-8-oxoguanine (8-oxoG)
Methylated bases e.g. O6 methylguanine
DNA glycosylases
Relatively small proteins 20-30kDa in size. Target dsDNA with exception of uracil DNA glycosylase which also works on ssDNA. E. Coli has 6 diff types that target specific types of damaged bases. Can be monofunctional (just glycosylases) or bifunctional ( glycosylase and AP lyase functions)
Uracil DNA glycosylase (UDG)
Uracil gets into DNA in 2 main ways:
Deamination of cytosine to uracil, a reaction thats 4000 times faster in ssDNA making it a significant event in actively transcribed genes at DNA replication forks
Misincorporation of dUTP during DNA synthesis. Bacteria have an enzyme called Dut which has dUTPase activity degrading dUTP to dUDP reducing the pool of available dUTP.
UDG flips uracil out of the double helix before cleavage. The bacterial and human enzymes are closely related base flipping is a common feature of DNA glycosylases
AP endonucleases
E. Coli has 4 AP endonucleases
Exonuclease lll (Xth) is monofunctional and cleaves by hydrolysis on the 5’ side of AP sites. Xth has the capacity to repair various blocking groups on the 3’ termini of DNA which includes products of AP lyases (sometimes called 3’ repair) the cleavage product of Xth (5’deoxyribose 5 phosphate) can be eliminated by a 5’ phosphodiesterase e.g. RecJ a 5’3’ exonuclease
Endonuclease IV ( Nth) is bifunctional and has DNA glycosylase activity on urea and thymine glycol and cleaves on the 3’ side of AP sites. Bifunctional AP endonucleases cleave by a beta elimination reaction (i.e. are AP lyases)
In both cases a single nucleotide gap is created which can be filled in by any DNA pol and nick sealed by DNA ligase.
