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MCB 502A-2015. Lecture #13!DNA repair: NER and BER. Chromosomal LesionsOne-strand DNA damage and its repair— 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 cycle— Direct versus chromosome cycle-dependent chromosomal lesionsPhotoreactivationPhotoreactivation-1— … phr … curious enzyme called photolyase … — Photoreactivation was discovered … mysterious irreproducibility … UV-irradiated cells recover … if exposed to visible light right after UV! — … characterization … remarkable story … inefficient non-enzymatic reactions … efficient catalyst… .http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Bacterial_Inhibition/Addnl_UV_Expts.htmhttp://www.photobiology.info/Jagger.htmlNo lightBlue lightPhotoreactivation-2— … direct photoreversal …. — … the maximum for direct photoreversal is ~235 nm. — … sensitized photoreversal… tryptophan, … oligopeptides (like Lys-Trp-Lys)… small DNA-binding proteins… — … electron transfer from the excited indole ring ….Photoreactivation-3— … latter mechanism …. Nature decided to upgrade…— … the product of the phr gene… 54 kDa …photolyase… — … before we can purify … need an assay … . http://www.nature.com/onc/journal/v21/n58/fig_tab/1205958f2.htmlPhotoreactivation-4— PD in DNA can be detected…:1) by inactivation of "transforming principle" (DNA). 2) by thin-layer chromatography of the products of complete DNA hydrolysis… 3) by complete enzymatic hydrolysis of irradiated DNA … … affinity chromatography for sugar-phosphates … Strong acidDNasesPhotoreactivation-5— … measure … pyrimidine dimers in DNA, … incubate … with photolyase ± light … amount of dimers left after the reaction… — Adding photolyase to DNA in the dark does not help: … — … in the dark, the enzyme binds… . — … binds PDs… in both duplex and ssDNA…. recognizes dimers directly, … Enzyme or Time (AU)1005025Original PDs in DNA (%)No light+ lightPhotoreactivation-6— … action spectrum …visible and near-UV light… do not reverse PD…, …light-catching and energy-transforming cofactors …— … two chromophores :… FADH- (dihydroflavin adenine dinucleotide), … a reduced pterin (MTHF) with …polyglutamate (tail). CH2(CHOH)CH2O — ADP(Glu)nPhotoreactivation-7— … division of labor … light catching by the pterin antennae… energy to FADH- … — The excited FADH- … to split PD… returning the electron back … T=TMTHFFADH–TrpT=TMTHFFADH–TrpT=TMTHFFADH–Trpe-T=TMTHFFADH–Trpe-T–TMTHFFADH–Trpe-T–TMTHFFADH–Trpe-Blue lightFor those who prefer real structures http://www.pnas.org/content/108/36/14831/F1.expansion.html— … PD to be split is flipped out … — … a common tactics in DNA repair.Photoreactivation-8— … 10-20 photolyases per E. coli cell. The in vitro turnover rate is 50… — … in vivo turnover rate is only 50 monomerized dimers per minute per cell…. The reason for the discrepancy is unknown. — Not surprisingly, the contribution … to survival … after UV … "modest". In vivoIn vitroUV dose (J/m2)0 20 40 60 8010.10.010.0010.0001Fraction survivingphrPhotoreactivation-9— … few … reverses DNA modification directly. — … Ada protein … DNA-phosphates … O-6-methylguanine. — … direct reversal … the least invasive way to repair DNA, — … More about photoreactivation:http://www.photobiology.info/Jagger.htmlhttp://www.nature.com/nrm/journal/v5/n2/full/nrm1312.htmlhttp://femsle.oxfordjournals.org/content/355/1/1Photolyase as a DNA doctor…owie…DNA with PD (pink)http://www.rcsb.org/pdb/101/motm.do?momID=91There, there…Let me take care of your owie…Photolyase is about to split PD in DNASuntanninghttp://www.dailymail.co.uk/home/you/article-2329154/Burning-issue-Why-time-tackle-sun-damage-youth.htmlThe uncertainty principle of DNA repair -1— …no study the cell cycle … without synchronization. — … better we synchronize … the more it becomes perturbed, … normal… — … the classic uncertainty principle...… similar situation … DNA repair ….The uncertainty principle of DNA repair -2— To understand … … repairs a few "pure" DNA lesions. — … detect DNA lesions … physical techniques… massive … DNA damage. — … perturbs the normal cell metabolism… RNA, proteins and membranes, ….http://www.nature.com/nrmicro/journal/v7/n3/fig_tab/nrmicro2073_F2.htmlProtein damage correlates with radiation lethalityThe uncertainty principle of DNA repair -3— … cellular responses to massive DNA damage… — … cannot repair … activate programs of self-elimination. — … apoptosis (in eukaryotes)… — … activation of dormant genetic elements (in prokaryotes)…. http://www.tinnitusformula.com/library/gingko-and-cancer/#.VHyxrYctAiohttp://phage-biotech.com/?page_id=84The uncertainty principle is everywhere…http://stickmenwithmartinis.com/2012/04/18/uncertainty-principle/The uncertainty principle of DNA repair -4— Is it possible… considerable… DNA repair effort? — … paradox… not that difficult… damage DNA outside … introduce … — … the most efficient way… viruses, … — … bacterial cells … bacteriophages … http://en.citizendium.org/wiki/BacteriophageT7The uncertainty principle of DNA repair -5— … bacteriophage… DNA-damaging agent … injects the DNA … were not exposed … — … phages do not … DNA repair genes … host repair systems … — … studied both physically … and genetically…The uncertainty principle of DNA repair -6— .. dsDNA… ssDNA… particular DNA repair reaction … — … photoreactivation… plating … ssDNA bacteriophages… — … corroborated in vitro … purified … Vibrio cholerae photolyase works similar on ssDNA or dsDNA. ssDNAdsDNAhttp://www.pnas.org/content/103/47/17696.fullThe uncertainty principle of DNA repair -7— … DNA repair mutants of the host cells can be selected … DNA-damaged bacteriophages… WTDNA repair
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