Photoselected electron transfer pathways in DNA photolyase

Prytkova, Tatiana; Beratan, David N.; Skourtis, Spiros S.

doi:10.1073/pnas.0605319104

Cited by 67 publications

(108 citation statements)

References 51 publications

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“…Intramolecular electron hopping from the isoalloxazine ring to the adenine moiety is unfavorable due to their redox potentials (ΔG ∼ þ0.1 eV) (14) and moreover we did not observe any fast quenching of FADH − Ã fluorescence without substrate (5). It is thought that the repair reaction by photolyase involves electron tunneling (15)(16)(17). However, the cyclic electrontunneling pathways, forward (k FET ) and backward (k BET ) or return (k ER ), are a matter of some debate.…”

Section: Electron-tunneling Pathways and Functional Role Of Adenine Mmentioning

confidence: 94%

“…One view is that the electron tunneling is mediated by the intervening adenine with a total distance of about 8 Å (15). An alternative model suggests that tunneling occurs directly from the o-xylene ring of FADH − to the 3′ side of CPD with a shortest distance of 4.3 Å (16,17). To test the electron-tunneling directionality, we used a series of substrates, UhiU, UhiT, ThiU and ThiT (chemical structures in Fig.…”

Section: Electron-tunneling Pathways and Functional Role Of Adenine Mmentioning

confidence: 99%

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Dynamics and mechanism of cyclobutane pyrimidine dimer repair by DNA photolyase

Liu

Tan

Guo

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

153

250

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Photolyase uses blue light to restore the major ultraviolet (UV)-induced DNA damage, the cyclobutane pyrimidine dimer (CPD), to two normal bases by splitting the cyclobutane ring. Our earlier studies showed that the overall repair is completed in 700 ps through a cyclic electron-transfer radical mechanism. However, the two fundamental processes, electron-tunneling pathways and cyclobutane ring splitting, were not resolved. Here, we use ultrafast UV absorption spectroscopy to show that the CPD splits in two sequential steps within 90 ps and the electron tunnels between the cofactor and substrate through a remarkable route with an intervening adenine. Site-directed mutagenesis reveals that the active-site residues are critical to achieving high repair efficiency, a unique electrostatic environment to optimize the redox potentials and local flexibility, and thus balance all catalytic reactions to maximize enzyme activity. These key findings reveal the complete spatio-temporal molecular picture of CPD repair by photolyase and elucidate the underlying molecular mechanism of the enzyme's high repair efficiency.DNA repair photocycle | ultrafast enzyme dynamics | thymine dimer splitting | electron tunneling pathway | active-site mutation U ltraviolet (UV) component of sunlight irradiation causes DNA damage by inducing the formation of cyclobutane pyrimidine dimer (CPD), which is mutagenic and a leading cause of skin cancer (1-3). CPD can be completely restored by a photoenzyme, photolyase, through absorption of visible blue light (4). In our early work (5-7), we have observed a cyclic electron-transfer (ET) reaction in thymine dimer (ThiT) repair by photolyase and determined the time scale of 700 ps for the complete repair photocycle (7). However, the central questions of whether the splitting of the cyclobutane ring is synchronously or asynchronously concerted or stepwise and whether the cyclic ET involves specific tunneling pathways were not resolved. Furthermore, the molecular mechanism underlying the high repair efficiency has not been elucidated. Here, using femtosecond spectroscopy and site-directed mutagenesis, we are able to measure the dynamics of all initial reactants, reaction intermediates, and final products with different substrates and with wild-type and active-site mutant enzymes, and thus reveal the complete spatio-temporal molecular picture of thymine dimer repair by photolyase.Photolyase contains a fully reduced flavin adenine dinucleotide (FADH − ) as the catalytic cofactor and electron donor (4). Based on previous studies (4-10), a sequential repair mechanism of thymine dimer splitting is shown in Fig. 1. Previously, we found that the forward ET from FADH − Ã to ThiT occurs in 250 ps (1∕k FET ) and the total decay of intermediate FADH• in 700 ps (1∕k total ) (5, 7). These dynamics usually follow a stretched-exponential decay behavior, reflecting heterogeneous ET dynamics controlled by the active-site solvation (5, 6, 11). However, in that study, no thymine-related species could be detected in the vi...

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Section: Electron-tunneling Pathways and Functional Role Of Adenine Mmentioning

confidence: 94%

Section: Electron-tunneling Pathways and Functional Role Of Adenine Mmentioning

confidence: 99%

Dynamics and mechanism of cyclobutane pyrimidine dimer repair by DNA photolyase

Liu

Tan

Guo

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

153

250

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“…3C). Two water molecules that are halfway between the 3Ј-thymine and the isoalloxazine ring in the A. n. CPD photolyase, and thus cross the path of direct electron transfer between the CPD lesion and the FADH Ϫ cofactor (26), are replaced by the carboxamide group of N431 (Fig. 3B).…”

Section: Resultsmentioning

confidence: 99%

Recognition and repair of UV lesions in loop structures of duplex DNA by DASH-type cryptochrome

Pokorný

Klar

Hennecke

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

156

181

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DNA photolyases and cryptochromes (cry) form a family of flavoproteins that use light energy in the blue/UV-A region for the repair of UV-induced DNA lesions or for signaling, respectively. Very recently, it was shown that members of the DASH cryptochrome subclade repair specifically cyclobutane pyrimidine dimers (CPDs) in UV-damaged single-stranded DNA. Here, we report the crystal structure of Arabidopsis cryptochrome 3 with an in-siturepaired CPD substrate in single-stranded DNA. The structure shows a binding mode similar to that of conventional DNA photolyases. Furthermore, CPD lesions in double-stranded DNA are bound and repaired with similar efficiency as in single-stranded DNA if the CPD lesion is present in a loop structure. Together, these data reveal that DASH cryptochromes catalyze light-driven DNA repair like conventional photolyases but lack an efficient flipping mechanism for interaction with CPD lesions within duplex DNA.Arabidopsis ͉ DNA repair ͉ photolyase C ryptochromes (cry) and DNA photolyases form a unique family of flavoproteins, with members present in all kingdoms of life (1). This family is divided into several subclades according to sequence similarity and function. DNA photolyases are enzymes that repair cytotoxic and mutagenic DNA lesions induced by UV-B, specifically cis-syn cyclobutane pyrimidine dimers (CPDs) or 6-4 pyrimidine-pyrimidone lesions (6-4 photoproduct) by using light energy in the UV-A/blue region (2). The catalytic cofactor of DNA photolyase is flavin adenine dinucleotide (FAD) in its fully reduced form (FADH Ϫ ), and this is present in a U-shaped conformation, as shown in several DNA photolyase structures (3-7). Catalysis involves electron transfer from the excited catalytic cofactor to the UV-B photoproduct, splitting the cyclobutane or oxetane rings, and electron backtransfer to the semireduced FADH°(1). Excitation of FAD is accomplished either by direct photon absorption or by Förster-type energy transfer from an antenna cofactor (1). Despite considerable sequence and structural similarity with DNA photolyases and common cofactor compositions, cryptochromes generally lost repair activity but gained photoreceptor function operating in the same waveband region as DNA photolyases (1). In plants, cryptochromes trigger several developmental processes, such as deetiolation and photoperiodic flower induction, and they entrain the circadian clock (8). In animals such as Drosophila, cryptochromes also function as photoreceptors for light input to the clock (9) and in magnetoreception (10), whereas mammalian cryptochromes are central components of the circadian clock without proven photoreceptor function (11). The more recently discovered subclade of the cryptochrome/ photolyase family, named cry DASH, includes members in plants, cyanobacteria, eubacteria, and vertebrates (12-15). It has been suggested that they represent photoreceptors because they lacked repair activity for CPDs in double-stranded DNA (dsDNA), despite their DNA-binding activity (12, 13). Positive support fo...

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“…The computed average electronic coupling and the proposed electron transfer pathways between FADH − and the dimer are tested by predicting the rate of the photoinduced ET reaction which is known from experiment. The calculations show that the directionality of the π → π * charge-transfer transitions of the flavin isoalloxazine ring play a central role in determining the ET rate and the ET pathways [3] COMPUTATIONAL METHODS…”

Section: Introductionmentioning

confidence: 99%

Flavin Charge Transfer Transitions Assist DNA Photolyase Electron Transfer

Skourtis¹,

Prytkova²,

Beratan³

et al. 2007

AIP Conference Proceedings

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This contribution describes molecular dynamics, semi-empirical and ab-initio studies of the primary photo-induced electron transfer reaction in DNA photolyase. DNA photolyases are FADH − -containing proteins that repair UV-damaged DNA by photo-induced electron transfer. A DNA photolyase recognizes and binds to cyclobutatne pyrimidine dimer lesions of DNA. The protein repairs a bound lesion by transferring an electron to the lesion from FADH − , upon photoexcitation of FADH − with 350-450 nm light. We compute the lowest singlet excited states of FADH − in DNA photolyase using INDO/S configuration interaction, time-dependent densityfunctional, and time-dependent Hartree-Fock methods. The calculations identify the lowest singlet excited state of FADH − that is populated after photo-excitation and that acts as the electron donor. For this donor state we compute conformationally-averaged tunneling matrix elements to empty electron-acceptor states of a thymine dimer bound to photolyase. The conformational averaging involves different FADH − -thymine dimer confromations obtained from molecular dynamics simulations of the solvated protein with a thymine dimer docked in its active site. The tunneling matrix element computations use INDO/S-level Green's function, energy splitting, and Generalized Mulliken-Hush methods. These calculations indicate that photo-excitation of FADH − causes a π → π * charge-transfer transition that shifts electron density to the side of the flavin isoalloxazine ring that is adjacent to the docked thymine dimer. This shift in electron density enhances the FADH − -to -dimer electronic coupling, thus inducing rapid electron transfer.

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Photoselected electron transfer pathways in DNA photolyase

Cited by 67 publications

References 51 publications

Dynamics and mechanism of cyclobutane pyrimidine dimer repair by DNA photolyase

Dynamics and mechanism of cyclobutane pyrimidine dimer repair by DNA photolyase

Recognition and repair of UV lesions in loop structures of duplex DNA by DASH-type cryptochrome

Flavin Charge Transfer Transitions Assist DNA Photolyase Electron Transfer

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