1 MCB 502A-2015. Lecture #13 DNA repair: NER and BER. Chromosomal Lesions One-strand DNA damage and its repair Photoreactivation The 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 inefficient non-enzymatic reactions to develop an 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 decided 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”) to break (lyse) chemical bonds. As with any enzyme, before we can purify and characterize it, we need an assay for its activity. Pyrimidine dimers in DNA can be detected in several ways: 1) by inactivation of “transforming principle” (DNA). 2) by thin-layer chromatography of the products of complete DNA hydrolysis (100°C + strong acid). Pyrimidine dimers stay dimeric, all other bases will be monomers. 3) by complete enzymatic hydrolysis of irradiated DNA (enzymes cannot hydrolyze sugar-phosphate bond between the two pyrimidines of a dimer) with subsequent affinity chromatography for sugar-phosphates (activated charcoal). Once we measure the amount of pyrimidine dimers in DNA, we can incubate this dimer-containing DNA with a photolyase ± light and measure the amount of dimers left after the reaction: (Fig. 3-3 of Friedberg, p. 95). You can see that adding photolyase to DNA in the dark does not help: you have to shine light on the reaction for photoreactivation to happen. It can be shown that in the dark, the enzyme binds to pyrimidine dimers tightly but does nothing. The enzyme binds pyrimidine dimers equally well in both duplex and ssDNA, suggesting that photolyase recognizes dimers directly, rather than indirectly via changes in the overall DNA conformation. Since the action spectrum of the enzyme in vivo lies on the border of visible and near-UV light, — that is, at wavelengths that do not reverse pyrimidine dimers directly, researchers always suspected some light-catching and energy-transforming cofactors involved. Indeed, two chromophores are non-covalently associated with the enzyme: one is FADH- (dihydroflavin adenine dinucleotide), the other is a reduced pterin with appended polyglutamate residues: (Draw schematically as in Fig. 3-5 Friedberg, p. 98). The division of labor between the two2 chromophores is as follows: the pterin antennae catches the light, becomes excited and transfers the energy to FADH- cofactor. The excited FADH- extracts an electron from a Trp residue of the enzyme (this is the element adopted from the non-enzymatic sensitized photoreversal) and uses it to split the pyrimidine dimer, eventually returning the electron back where it was taken: (Draw schematically as in Fig. 3-8 Friedberg, p. 102). There are only 10-20 photolyases per E. coli cell. The in vitro turnover rate is 50 monomerized dimers per minute per enzyme molecule, which is impressive. Unfortunately, the in vivo turnover rate is only 50 monomerized dimers per minute per cell, which is disappointingly slow. The reason for the discrepancy is unknown. Not surprisingly, the contribution of photoreactivation to survival of E. coli after UV can be only described as "modest". Photolyase is one of the few DNA repair enzymes that reverses DNA modification directly. Another example is the Ada protein which picks methyl groups from DNA-phosphates and from O-6-methylguanine. The direct reversal of DNA modification is the least “invasive” way to repair DNA, — however, chemical considerations limit its utility to a few specialized cases. The uncertainty principle of DNA repair and how to get around it Recall that one cannot study the cell cycle in a population of cells without synchronization. Also recall that the better we synchronize the cell cycle in bacteria (by blocking protein synthesis), the more it becomes perturbed, therefore different from the normal cell cycle! That was a classic uncertainty principle, applied to the cell cycle research. A similar situation is encountered with the DNA repair studies. To understand the repair mechanism, ideally we would like to know how the cell repairs a few “pure” DNA lesions. However, in order to reliably detect DNA lesions and their subsequent repair in vivo with physical techniques, we need to introduce massive amounts of DNA damage. This perturbs the normal cell metabolism, because the more we damage DNA, the more damage to other cellular components, like RNA, proteins and membranes, will contribute to the overall phenomenon. Even more importantly, cellular responses to massive DNA damage will differ from repair of a few DNA lesions. For example, if cells feel that they cannot repair all DNA lesions, they activate programs of self-elimination. In eukaryotic cells, these programs induce apoptosis, — programmed cell death. In prokaryotic cells, these programs induce cell lysis via activation of dormant genetic elements (prophages). Is it possible, without treating cell with DNA-damaging agents, to ask the cell to perform a considerable (= readily detectable by physical assays) DNA repair effort? Sounds like another paradox, but in fact it is not that difficult: we just need to damage DNA outside the cells and then to introduce this DNA efficiently into the cells. Perhaps the most efficient way to introduce DNA into a cell is
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