Characterization of the Intermediate in and Identification of the Repair Mechanism of (6‐4) Photolesions by Photolyases

Abstract

Quantum mechanics/molecular mechanics calculations are employed to assign previously recorded experimental spectroscopic signatures of the intermediates occurring during the photo-induced repair of (6-4) photolesions by photolyases to specific molecular structures. Based on this close comparison of experiment and theory it is demonstrated that the acting repair mechanism involves proton transfer from the protonated His365 to the N3′ nitrogen of the lesion, which proceeds simultaneously with intramolecular OH transfer along an oxetane-like transition state. Keywords(6-4) photoproducts; (6-4) DNA photolyases; DNA repair; photo-induced; proton transfer UV radiation causes two of the most abundant mutagenic and cytotoxic DNA photolesions: cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP) [1][2][3][4] . Persistence of these lesions can interfere with essential processes such as transcription and DNA replication leading to mutation, nucleotide misincorporation and eventually to cell death. In fact, (6-4) photoproducts are supposed to be the major players in the formation of skin cancer [5][6][7][8][9] . To maintain genetic integrity, many organisms have developed an elegant mechanism to repair these lesions using photo-activated enzymes called DNA photolyases [10][11][12] . These are highly efficient enzymes utilizing themselves UV/ blue light to eliminate these UV-derived photoproducts. After DNA binding through the dinucleotide flipping mechanism [13] , the enzymatic repair occurs in three sequential steps [5][6][7][8][9][10][11][12][13][14][15][16] : (1) photo-induced electron transfer from the catalytic cofactor, reduced flavine adenine dinucleotide (FADH − ) to the lesion [13][14][15][16][17] (2) electron-induced splitting of the lesion (3) back electron transfer to FADH • (Figure 1). Experimental and theoretical [36][37][38][39][40][41][42][43][44][45][46][47] * shirin.faraji@uni-heidelberg.de.Supporting information for this article is given via a link at the end of the document. HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript studies have been performed intensively over the past years, to trace all steps of the repair. While the functional mechanism of CPD photolyases has been identified [7,8,18,19,23,26,36,37] , the details of the repair mechanism operating in (6-4) photolyases is still a matter of ongoing debate. The initial steps of the repair mechanism, i.e. light absorption, energy transfer [13][14][15] and generation of the catalytic electron [16][17] are now well understood, however, the terminal repair of the 6-4PP after the transfer of the electron is not yet finally determined [40,41,44,47] . Unlike in CPD repair, a single-electron transfer to the 6-4PP is not sufficient [28] for their repair and it was thus suggested that additional factors are required to restore the original bases, which left the field open to hypotheses and speculations. A final proof of the mechanism is hence still missing.The current statu...

Two aspartate residues close to the lesion binding site of Agrobacterium (6‐4) photolyase are required for Mg2+ stimulation of DNA repair

“…Consistently, the PhrB-H366N mutant shows no repair activity [21]. Several studies on (6-4) photolyases suggest that proton transfer occurs from this histidine residue to the damaged DNA as part of the repair process and that this residue is protonated in the ground state [23] and deprotonated for 10 ns or longer [24][25][26][27]. Other works support the hypothesis of a deprotonated active site histidine in the ground state and another reaction mechanism in (6-4) photolyases (for a review of the different mechanisms see [28]).…”

Repair of (6‐4) Lesions in DNA by (6‐4) Photolyase: 20 Years of Quest for the Photoreaction Mechanism

“…Several potential reaction pathways were explored and discussed by Faraji et al . . They finally retained a mechanism here termed “proton‐transfer‐steered OH transfer” (Fig.…”

“…The structural data of Maul et al . described above triggered a wealth of theoretical studies aimed at finding a feasible repair mechanism for (6‐4) PPs that does not imply the thermal formation of an oxetane intermediate prior to photoexcitation. A number of the explored pathways turned out to include steps with unreasonably high activation barriers and were hence abandoned.…”

“…The middle row (proton‐transfer‐steered OH transfer) corresponds to the studies by Faraji et al . . OH transfer is here preceded by the protonation of the N3′ atom of the 3′ base by the protonated His 1 .…”

“…In contrast to the “simple OH transfer” mechanism, the histidine His 1 was assumed to be protonated in the dark (His 1 H + )b. Forward electron transfer from photoexcited FADH − would yield the 5′ base anion radical of the lesion (II b , redrawn from their most recent publication ). Remarkably, the original calculations indicated that the electron transferred from photoexcited FADH − was first localized on His 1 (intermediate II b ’), a possibility also considered by Moughal Shahi and Domratcheva .…”