Byrdin, M.

Kinetics of cyclobutane thymine dimer splitting by DNA photolyase directly monitored in the UV (Article)

2011-06-07T00:00:00Z

CPD photolyase uses light to repair cyclobutane pyrimidine dimers (CPDs) formed between adjacent pyrimidines in UV-irradiated DNA. The enzyme harbors an FAD cofactor in fully reduced state (FADH-). The CPD repair mechanism involves electron transfer from photoexcited FADH-to the CPD, splitting of its intradimer bonds, and electron return to restore catalytically active FADH-. The two electron transfer processes occur on time scales of 10-10and 10-9s, respectively. Until now, CPD splitting itself has only been poorly characterized by experiments. Using a previously unreported transient absorption setup, we succeeded in monitoring cyclobutane thymine dimer repair in the main UV absorption band of intact thymine at 266 nm. Flavin transitions that overlay DNA-based absorption changes at 266 nm were monitored independently in the visible and subtracted to obtain the true repair kinetics. Restoration of intact thymine showed a short lag and a biexponential rise with time constants of 0.2 and 1.5 ns. We assign these two time constants to splitting of the intradimer bonds (creating one intact thymine and one thymine anion radical T○-) and electron return from T○-to the FAD cofactor with recovery of the second thymine, respectively. Previous model studies and computer simulations yielded various CPD splitting times between <1 ps and <100 ns. Our experimental results should serve as a benchmark for future efforts to model enzymatic photorepair. The technique and methods developed here may be applied to monitor other photoreactions involving DNA.

The cryptochromes: Blue light photoreceptors in plants and animals (Article)

2011-06-02T00:00:00Z

Cryptochromes are flavoprotein photoreceptors first identified in Arabidopsis thaliana, where they play key roles in growth and development. Subsequently identified in prokaryotes, archaea, and many eukaryotes, cryptochromes function in the animal circadian clock and are proposed as magnetoreceptors in migratory birds. Cryptochromes are closely structurally related to photolyases, evolutionarily ancient flavoproteins that catalyze light-dependent DNA repair. Here, we review the structural, photochemical, and molecular properties of cry-DASH, plant, and animal cryptochromes in relation to biological signaling mechanisms and uncover common features that may contribute to better understanding the function of cryptochromes in diverse systems including in man. Copyright

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.

What makes the difference between a cryptochrome and DNA photolyase? A spectroelectrochemical comparison of the flavin redox transitions (Article)

2009-01-21T00:00:00Z

Cryptochromes and DNA photolyases are highly homologous flavoproteins that accomplish completely different tasks. While plant cryptochrome1 functions as blue light photoreceptor that triggers various morphogenic reactions, photolyases repair UV-induced DNA damages. Both enzymes share the photoactive cofactor, noncovalently bound FAD. For photolyase, the reaction mechanism involves electron transfer to the substrate from the excited-state of fully reduced flavin. For cryptochrome, photoexcitation of the oxidized flavin leads to formation of the semireduced radical FADḢ. Key parameters for the redox state of the flavin in the cell are the midpoint potentials E1 and E2 for the oxidized/semireduced and semireduced/fully reduced transitions, respectively. A link between cryptochrome function and its cofactor's redox states has been suggested early on, but no reliable determinations of midpoint potentials have been available. Here we report spectroelectrochemical titrations of cryptochrome1 from Arabidopsis thaliana and photolyases from both E. coli and Anacystis nidulans at pH 7.4. For the cryptochrome, we obtained E1 ≈ E2 ≈ -160 mV vs NHE, strongly deviating from the photolyases where FADḢ could not be oxidized up to 400 mV, and E2 ≈ -40 mV. Functional and evolutionary implications are discussed, highlighting the role of an asparagine-to-aspartate replacement close to N5 of the flavin.

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.