MCB 502A-2014. Lecture #13DNA repair: NER and BER. Chromosomal LesionsOne-strand DNA damage and its repairMisincorporation is only one source for potential mutagenic DNA changes. Another, sometimes the dominant source, is DNA modification and damage by chemical and physical agents. We havealready discussed the point that DNA has been chosen over RNA for its greater chemical stability. Indeed, the RNA backbone is prone to hydrolysis due to the proximity of the 2’-OH. This hydrolysis occurs rapidly in alkaline pH, but is a problem 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 DNA is the decreased stability of the N-glycosydic bond compared to RNA: DNA in solution is more prone to lose bases from sugars to form abasic sites,although it is usually not a problem at pH around neutral. A significant degree of chemical protection comes with the double-strandedness of DNA,as its major reactive groups are protected by stacking interactions and hydrogen bonding betweenbases. However, the cells are working factories, and active metabolism generates a variety of reactive chemicals that can modify even well-protected DNA bases. In addition, there are physical and chemical assaults, like UV-light of the sun rays, or DNA damaging substances (hydrogen peroxide, bleomycin) produced by other cells, that even the chemically-stable DNA cannot avoid. If one considers the variety of these DNA modifying/damaging possibilities versus the length of the chromosomal DNA, the genome does not look so invincible. To compensate for the threat, the cells employ powerful mechanisms to repair modified or damaged DNA. On the other hand, no mechanisms of general RNA repair are known at this time. Before we discuss DNA repair mechanisms, I would like to make clear the distinction between mutations (changes in DNA sequence)and DNA lesions (changes in DNA chemistrythat require correction), as the students oftenthink these are synonyms. In fact, they are twovery different phenomena, as should be clearfrom the table on the right, so do not mix them!UV irradiation damages DNAIt has been known for a long time that UV irradiation kills bacterial cells. The 1954 study had shown that, out of the three biopolymers with precise composition — RNA, DNA and proteins, — DNA synthesis is preferentially inhibited after cells were irradiated with UV. Thus, researchers suspected chemical changes in UV-treated DNA, — and such changes were indeed found. Recall the absorption spectrum of DNA with a maximum around 260 nm (this is "hard" 1Level Mutation DNA lesionDNA chemistry normal abnormalChromosome cycle normal blockedCell phenotype changed unchangedReparable? No YesRelation to the other No PotentiallyUV), corresponding to the absorption of bases. When DNA bases absorb UV photons, they become activated and can react with each other. The small distances between the neighboring bases in the DNA stacks facilitate these reactions. In particular, UV irradiation induces intra-strand crosslinks between adjacent pyrimidine bases. The major product is the so-called cyclobutane pyrimidine dimer (but there are two more chemically-distinct dimers). These intra-strand crosslinks distort DNA (for example, the normal 34° rotation angle between the adjacent bases is replaced by 0° angle in the cyclobutane dimer). It is calculated that 30 pyrimidine dimersform per E. coli genome-equivalent at the UV-light dose of 1 Joule/m2. When wild type E. coli cells are irradiated with various doses of UV, the dose-dependence ("the survival curve") features resistance shoulder (complete survival) up to 30 J/m2, demonstrating that the cell can repair without consequences up to 900 PDs in its DNA. Beyond this UV dose, exponential loss of viability ensues, so that survival at 80 J/m2 is only 0.001. At this dose, the chromosome must be riddled with UV-induced lesions (2,400 per genome-equivalent, or one lesion per every 2,000 bp). Mutants were sought with increased or decreased UV-resistance, but only UV-sensitive mutants were found, indicating that E. coli has multiple pathways to resist UV damage, but no pathways that would further sensitize it to UV damage. The multiple UV-sensitive mutants of E. coli were mapped to a dozen individual loci, but they grouped to only three major categories by epistatic analysis. The first category comprised moderately-sensitive mutants and was represented by a single locus phr. The second category comprised highly-sensitive mutants and was represented by four separate loci with a mnemonic name uvr for UV-resistance. The third category comprised mutants with UV-sensitivity ranging from moderate to high and was represented by a motley crew of the rec genes, whose products are implicated in homologous recombination. Thus, genetic analysis revealed an unexpected complexity of DNA repair of UV damage. PhotoreactivationThe phr gene encodes a curious enzyme called photolyase that uses visible light to break pyrimidine dimers back into monomers in the process called “photoreactivation”. Photoreactivation was discovered when scientists were trying to reduce the mysterious irreproducibility of the UV-light killing: to their surprise they found that UV-irradiated cells recover significant viability if exposed to visible light right after UV! Subsequent characterization of the phenomenon yielded a remarkable story of how Nature exploits inefficientnon-enzymatic reactions to develop a very efficient catalyst to do the same job. First of all, there is a direct photoreversal of UV-photoproducts by the same UV-light. The wavelength dependence is different, though: the maximum for pyrimidine dimer formation is~260 nm, whereas the maximum for direct photoreversal is ~235 nm. Second, there is sensitized photoreversal, mediated by aminoacid tryptophan, by tryptophan-containing oligopeptides (like Lys-Trp-Lys), as well as by tryptophan-containing small DNA-binding proteins, like gp32 of T4. The mechanism of this reaction is thought to involve electron transfer from the excited indole ring of tryptophan to the dimer. It is the latter mechanism of photoreversal that the Nature 2decided to upgrade to enzymatic photoreactivation of UV-inactivated DNA. The enzyme involved, the product of the phr gene, is a 54 kDa monomeric protein in E. coli, called photolyase, because it uses light (“photo”)
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