MCB 502A-2014. Lecture #13DNA repair: NER and BER.Chromosomal LesionsOne-strand DNA damage and its repair— UV irradiation damages DNA— Photoreactivation— The uncertainty principle of DNA repair and how to get around it— Nucleotide excision repair of UV-lesions— The mechanism of nucleotide excision repair— Base excision repair of damaged, modified or inappropriate bases— Repair avoidanceChromosomal Lesions— DNA modifications do not change behavior of chromosomes— Lesions affecting the behavior of chromosomes— Chromosomal lesions block the chromosome cycleOne-strand DNA damage and its repair-1— Misincorporation is only one source for potentialmutagenic DNA changes.— Another, sometimes the dominant source, is DNAmodification and damage by chemical and physicalagents.— We have already discussed the point that DNA hasbeen chosen over RNA for its greater chemical stability.— Indeed, the RNA backbone is prone to hydrolysis dueto the proximity of the 2’-OH.— This hydrolysis occurs rapidly in alkaline pH, but is aproblem in long RNA molecules even under neutral pH.— Spontaneous hydrolysis of the DNA backbone is 106-times slower.— The small price of a greater backbone stability in DNAis the decreased stability of the N-glycosydic bondcompared to RNA: DNA in solution is more prone to losebases from sugars to form abasic sites, although it isusually not a problem at pH around neutral.*alkaliDNARNAOne-strand DNA damage and its repair-2— A significant degree of chemicalprotection comes with the double-strandedness of DNA, as its majorreactive groups are protected bystacking interactions and hydrogenbonding between bases.— However, the cells are workingfactories, and active metabolismgenerates a variety of reactivechemicals that can modify evenwell-protected DNA bases.— In addition, there are physical andchemical assaults, like UV-light ofthe sun rays, or DNA damagingsubstances (hydrogen peroxide,bleomycin) produced by other cells,that even the chemically-stable DNAcannot avoid.One-strand DNA damage and its repair-3— If one considers all these DNA modifying/damaging possibilities versus thelength of the chromosomal DNA, the genome does not look so invincible.— To compensate for the threat, cells employ several powerful mechanisms to repairmodified or damaged DNA.— On the other hand, no mechanisms of general RNA repair are known at this time.http://mmbr.asm.org/content/78/1/40/F1.expansion.htmlOne-strand DNA damage and its repair-4— Before we discuss DNA repair mechanisms, I would like to make clear thedistinction between mutations (changes in DNA sequence) and DNA lesions(changes in DNA chemistry that require correction), as the students often thinkthese are synonyms.— In fact, they are two very different phenomena, as should be clear from the tablebelow, in which they are different in every single aspect, so do not mix them!Level Mutation DNA lesionDNA chemistry normal abnormalChromosome cycle normal blockedCell phenotype changed unchangedReparable? No YesRelation to the other No Potentially mutagenicmutationDNA lesionDNA lesion DNA lesionUV irradiation damages DNA-1— It has been known for a long time that UVirradiation kills bacterial cells.— The 1953 study had shown that, out of the threebiopolymers with precise composition — RNA, DNAand proteins, — DNA synthesis is preferentiallyinhibited after cells were irradiated with UV.— Thus, researchers suspected chemical changes inUV-treated DNA, — and such changes were indeedfound.— Recall the absorption spectrum of DNA with amaximum around 260 nm (this is "hard" UV),corresponding to the absorption of bases.— When DNA bases absorb UV photons, theybecome activated and can react with each other. Thesmall distances between the neighboring bases in theDNA stacks facilitate these reactions.http://www.ncbi.nlm.nih.gov/pubmed/13034732http://www.scienceisart.com/A_DNA/UVspectrum_2.htmlUV irradiation damages DNA-2— In particular, UV irradiation inducesintra-strand crosslinks between adjacentpyrimidine bases.— The major product is the so-calledcyclobutane pyrimidine dimer (butthere are two more chemically-distinctdimers).— These intra-strand crosslinks distortDNA (for example, the normal 34°rotation angle between the adjacentbases is replaced by 0° angle in thecyclobutane dimer).— It is calculated that 30 pyrimidinedimers form per E. coli genome-equivalent at the UV-light dose of 1Joule/m2.http://iverson.cm.utexas.edu/courses/310N/MOTD%20Fl05/ThymineDimers.htmlhttp://openi.nlm.nih.gov/detailedresult.php?img=3010660_JNA2010-592980.002&req=4CyclobutanedimerUV irradiation damages DNA-3— When wild type E. coli cells areirradiated with various doses of UV,the dose-dependence ("the survivalcurve") features resistance shoulder(complete survival) up to 30 J/m2,demonstrating that the cell can repairwithout consequences up to 900 PDsin its DNA.— Beyond this UV dose, exponentialloss of viability ensues, so thatsurvival at 80 J/m2 is only 0.0001.— At this dose, the chromosome mustbe riddled with UV-induced lesions(2,400 per genome-equivalent, or onelesion per every 2,000 bp).UV dose (J/m2)0 20 40 60 8010.10.010.0010.0001Fraction survivingResistanceshoulderKilling slopeUV irradiation damages DNA-4— Mutants were sought withincreased or decreased UV-resistance, but only UV-sensitivemutants were found, indicating thatE. coli has multiple pathways toresist UV damage, but no pathwaysthat would further sensitize it to UVdamage.— The multiple UV-sensitivemutants of E. coli were mapped to adozen individual loci, but theygrouped to only three majorcategories by epistatic analysis.UV dose (J/m2)0 20 40 60 8010.10.010.0010.0001Fraction survivingNo UV-resistantcandidatesWTMore UV-sensitive candidatesUV irradiation damages DNA-4— The first category comprisedmoderately-sensitive mutants and wasrepresented by a single locus phr.— The second category comprisedhighly-sensitive mutants and wasrepresented by four separate loci with amnemonic name uvr for UV-resistance.— The third category comprised mutantswith UV-sensitivity ranging frommoderate to high and was represented bya motley crew of the rec genes, whoseproducts are implicated in homologousrecombination.— Thus, genetic analysis revealed anunexpected complexity of DNA repair ofUV damage.UV dose (J/m2)0 20 40 60 8010.10.010.0010.0001Fraction
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