Dynamics and mechanism of repair of ultraviolet-induced (6–4) photoproduct by photolyase

Abstract

One of the detrimental effects of UV radiation on DNA is the formation of the (6-4) photoproduct (6-4PP) between two adjacent pyrimidines1. This lesion interferes with replication and transcription and may result in mutation and cell death2. In many organisms a flavoenzyme called photolyase uses blue light energy to repair the 6-4PP3. The molecular mechanism of the repair reaction is poorly understood. Here, we use ultrafast spectroscopy to show that the key step in the repair photocycle is a cyclic proton transfer between the enzyme and the substrate. By femtosecond synchronization of the enzymatic dynamics with the repair function, we followed the function evolution and observed direct electron transfer from the excited flavin cofactor to the 6-4PP in 225 ps but surprisingly fast back electron transfer in 50 ps without repair. Strikingly, we found that the catalytic proton transfer between a histidine residue in the active site and the 6-4PP, induced by the initial photoinduced electron transfer from the excited flavin cofactor to 6-4PP, occurs in 425 ps and leads to 6-4PP repair in tens of nanoseconds. These key dynamics define the repair photocycle and explain the underlying molecular mechanism of the enzyme’s modest efficiency.

Crystal structure of a prokaryotic (6-4) photolyase with an Fe-S cluster and a 6,7-dimethyl-8-ribityllumazine antenna chromophore

“…His366 is conserved in FeS-BCPs, in eukaryotic (6-4) photolyases, and in animal cryptochromes; CPD photolyases have an Asn at the homologous position. The His366 homolog of eukaryotic (6-4) photolyases is essential for DNA repair; it serves as a donor/acceptor in catalytic proton transfer, which accompanies the light-driven electron transfer (22,24). His366 in PhrB is stabilized by van der Waals contacts with Leu370 and Met410 (Fig.…”

The molecular origin of high DNA-repair efficiency by photolyase

“…2a and Supplementary Fig. 1a), Ae À ðt=tÞ b (A, amplitude; t, decay time constant; and b, stretched parameter), due to the modulation of ET by active-site solvation on similar timescales [11][12][13]19,20 . Using t h i ¼ ðt=bÞGð1=bÞ and knowing the deactivation lifetimes (t LT ) in nanoseconds in the absence of substrate (Supplementary Fig.…”

“…1a) [11][12][13][14] . We observed two consecutive competitions of these elementary steps that make key contributions to the final repair efficiency: the first one is the forward electron transfer (ET, t FET ) from the excited cofactor flavin (FADH À *) to substrate thymine dimer (To 4T), the major UV-induced DNA photoproduct 15 , against the deactivation process (t LT ), leading to the first-branching quantum yield F FET of 0.85.…”

Determining complete electron flow in the cofactor photoreduction of oxidized photolyase

“…protein electron transfer | flavin photoreduction | femtosecond dynamics | electron flow directionality | reduction potential funnel P hotolyase and cryptochrome are evolutionally related and contain a flavin adenine dinucleotide (FAD) as the catalytic cofactor with a unique bent structure in the active sites, but the two perform different functions: photolyase repairs UV-damaged DNA and cryptochrome functions as a photoreceptor for regulation of plant growth or synchronization of circadian rhythm (1)(2)(3)(4)(5). The active state of the cofactor in vivo is in the anionic hydroquinone form (FADH -) in photolyase (6), but currently the redox status of flavin in cryptochrome is under debate with some studies suggesting flavin to be in oxidized (FAD), whereas others claiming anionic (FAD -/FADH -) states for the functional form in vivo (7)(8)(9)(10).…”