Villette, S.

DNA repair by photolyase: A novel substrate with low background absorption around 265 nm for transient absorption studies in the UV (Article)

2010-01-19T00:00:00Z

CPD photolyase enzymatically repairs the major UV-induced lesion in DNA, the cyclobutane pyrimidine dimer (CPD), by photoreversion of the damage reaction. An enzyme-bound reduced flavin (FADH-) cofactor functions as photosensitizer. Upon excitation, it transiently transfers an electron to the CPD, triggering scission of the interpyrimidine bonds. After repair completion, the electron returns to the flavin to restore its functional reduced form. A major difficulty for time-resolved spectroscopic monitoring of the enzymatic repair reaction is that absorption changes around 265 nm accompanying pyrimidine restoration are obscured by the strong background absorption of the nondimerized bases in DNA. Here we present a novel substrate for CPD photolyase that absorbs only weakly around 265 nm: a modified thymidine 10-mer with a central CPD and all bases, except the one at the 3′ end, replaced by 5,6-dihydrothymine which virtually does not absorb around 265 nm. Repair of this substrate by photolyases from Anacystis nidulans and from Escherichia coli was compared with repair of two conventional substrates: a 10-mer of unmodified thymidines containing a central CPD and an acetone-sensitized thymidine 18-mer that contained in average six randomly distributed CPDs per strand. In all cases, the novel substrate was repaired with an efficiency very similar to that of the conventional substrates (quantum yields in the order of 0.5 upon excitation of FADH-). Flash-induced transient absorption changes at 267 nm could be recorded on a millisecond time scale with a single subsaturating flash and yielded very similar signals for all three substrates. Because of its low background absorption around 265 nm and the defined structure, the novel substrate is a promising tool for fast and ultrafast transient absorption studies on pyrimidine dimer splitting by CPD photolyase.

Observation of an intermediate tryptophanyl radical in W306F mutant DNA photolyase from Escherichia coli supports electron hopping along the triple tryptophan chain (Article)

2007-09-04T00:00:00Z

DNA photolyases repair UV-induced cyclobutane pyrimidine dimers in DNA by photoinduced electron transfer. The redox-active cofactor is FAD in its doubly reduced state FADH-. Typically, during enzyme purification, the flavin is oxidized to its singly reduced semiquinone state FADH°. The catalytically potent state FADH-can be reestablished by so-called photoactivation. Upon photoexcitation, the FADH° is reduced by an intrinsic amino acid, the tryptophan W306 in Escherichia coli photolyase, which is 15 Å distant. Initially, it has been believed that the electron passes directly from W306 to excited FADH°, in line with a report that replacement of W306 with redox-inactive phenylalanine (W306F mutant) suppressed the electron transfer to the flavin [Li, Y. F., et al. (1991) Biochemistry 30, 6322-6329]. Later it was realized that two more tryptophans (W382 and W359) are located between the flavin and W306; they may mediate the electron transfer from W306 to the flavin either by the superexchange mechanism (where they would enhance the electronic coupling between the flavin and W306 without being oxidized at any time) or as real redox intermediates in a three-step electron hopping process (FADH°* ← W382 ← W359 ← W306). Here we reinvestigate the W306F mutant photolyase by transient absorption spectroscopy. We demonstrate that electron transfer does occur upon excitation of FADH° and leads to the formation of FADH-and a deprotonated tryptophanyl radical, most likely W359°. These photoproducts are formed in less than 10 ns and recombine to the dark state in ∼1 μs. These results support the electron hopping mechanism.

Electron hopping through the 15 Å triple tryptophan molecular wire in DNA photolyase occurs within 30 ps (Article)

2006-08-17T00:00:00Z

Photoreduction of the semi-reduced flavin adenine dinucleotide cofactor FADḢ in DNA photolyase from Escherichia coli into FADH - involves three tryptophan (W) residues that form a closely spaced electron-transfer chain FADḢ-W382-W359-W306. To investigate this process, we have constructed a mutant photolyase in which W359 is replaced by phenylalanine (F). Monitoring its photoproducts by femtosecond spectroscopy, the excited-state FADḢ* was found to decay in ∼30 ps, similar as in wild type (WT) photolyase. In contrast to WT, however, in W359F mutant photolyase the ground-state FADḢ fully recovered virtually concomitantly with the decay of its excited state and, despite the presence of the primary electron donor W382, no measurable flavin reduction was observed at any time. Thus, W359F photolyase appears to behave like many other flavoproteins, where flavin excited states are quenched by very short-lived oxidation of aromatic residues. Our analysis indicates that both charge recombination of the primary charge separation state FADH-W382 ̇+ and (in WT) electron transfer from W359 to W382 ̇+ occur with time constants <4 ps, considerably faster than the initial W382→FADḢ* electron-transfer step. Our results provide a first experimental indication that electron transfer between aromatic residues can take place on the time scale of ∼10-12 s.