Mechanism and dynamics of DNA photorepair by (6-4) photolyase – (6-4)PhotoMec

In a pluridisciplinary approach, biochemistry, molecular biology and organic synthesis for the preparation of enzymes and substrates are combined with advanced time-resolved optical spectroscopic techniques for functional studies.
A common enzyme is used for all studies: (6-4)photolyase from the frog Xenopus laevis.
For the photorepair reaction, essential innovations like developing a special synthetic substrate for (6-4) photolyase, covering all relevant timescales (from 200 fs to seconds) and inclusion of the difficult, but crucial 265 nm band of intact nucleobases in the transient absorption studies, should contribute to resolve mechanism and dynamics of photorepair of the 6-4PP.
For the photoactivation reaction, an all timescales approach is combined with site-directed mutagenesis to establish the electron transfer pathway and kinetics. We in particular envisage to understand the coupling between electron and proton transfer that probably plays a crucial role in the reduction of FADox to FADH•.

Repair of the 6-4PP by (6-4) photolyase was known to have a rather low quantum yield (3% to 11%, as compared to 50% to 100% for repair of the CPD by CPD photolyase) and its mechanism was not established experimentally. We have now managed to quantify repair of the 6-4PP in series of single-turnover flashes given to dark adapted samples composed of (6-4) photolyase and synthetic DNA strands containing a central 6-4PP. The results obtained strongly suggest that two successive photoreactions are required to repair the 6-4PP. The DNA based intermediate formed by the first photoreaction has spectral features consistent with an oxetane-bridged dimer; in the absence of a second excitation, it decays back to the original 6 4PP in ~100 s. The quantum yields of the 1st and 2nd photoreaction were estimated to be ~7% and ~80%, respectively. The long lifetime of the intermediate should allow achieving a reasonable overall repair yield in a two photon process under natural sunlight.
These experimental results challenge previous theoretical concepts of the repair mechanism that were based on a supposed one-photon process (as established for CPD repair) and paves the way for more detailed experimental and theoretical studies of each of the two photoreactions in repair of the 6-4PP.

Humans and other placental mammals do not possess photolyases and have to repair CPDs and 6-4PPs by the more complex, ATP consuming and light-independent, nucleotide excision repair (NER). Progress in the understanding of photolyases’ repair mechanisms might thus on the long term contribute to the development of bio-inspired repair compounds that could be included in skin care products to prevent skin cancer.
Beyond elucidating a DNA repair mechanism, the present project also aims at providing new insights into the fundamental process of proton-coupled electron transfer that plays a central role in many biochemical reactions and is of utmost interest for research on bio-inspired solar energy conversion.

Yamamoto, J., Martin, R., Iwai, S., Plaza, P., and Brettel, K. (2013): Repair of the (6-4) photoproduct by DNA photolyase requires two photons. Angew. Chem. Int. Ed. 52, 7432-7436.

Repair of DNA damage induced by UV light from the Sun is crucial for survival on Earth. The two most prominent UV lesions are cyclobutane pyrimidine dimer (CPD) and pyrimidine(6-4)pyrimidone photoproduct (6-4PP). To repair them, Nature has developed specific, light-driven enzymes: CPD photolyase and (6-4) photolyase. While knowledge on CPD repair has advanced substantially in the last decade, the repair mechanism for 6-4PP is still poorly understood.
CPD and (6-4) photolyases are homologous proteins of ~55 kDa found in organisms from all kingdoms of life. They carry a flavin cofactor, flavin adenine dinucleotide (FAD), which plays the role of the main photocatalytic agent. FAD needs to be in fully reduced state, FADH-, to be active for photorepair of DNA. The fully oxidized (FADox) and semi-reduced (FADH°) states can be converted to FADH- by a secondary photoreaction, that does not involve DNA, the so called photoactivation. In CPD photolyase, it proceeds by ultrafast electron transfer to the flavin through a chain of three tryptophan residues, and has become a paradigm system for the understanding of long-range intra-protein electron transfer. Yet, a tyrosine appears to be involved in (6-4) photolyase (Weber et al., PNAS 99 (2002) 1319).
For the mechanism of CPD repair by CPD photolyase, it has been established by time-resolved spectroscopic studies that, upon excitation of FADH-, an electron is transferred in ~100 ps to a CPD present in the enzyme’s substrate binding pocket. The two intra-dimer bonds split rapidly (<300 ps) and the excess electron is returned to the flavin (that was transiently oxidized in the FADH° state), thus restoring two intact pyrimidines in ~1 ns.
Compared to the repair of CPD, that of the 6-4PP lesion is chemically more challenging, because it requires the transfer of a functional group, in addition to intra-dimer bond splitting. Furthermore, experimental study of the reaction mechanism of 6-4PP repair by (6-4) photolyase is also technically more challenging because of a low repair quantum yield (~10% compared to 50-100% for CPD repair). The only time-resolved study published to date (Li et al., Nature 466 (2010) 887) provided evidence for electron transfer from excited FADH- to the 6–4PP in 225 ps and back electron transfer with a time constant of 50 ps without repair in ~90% of the excited photolyases. In the remaining ~10%, the electron was not returned to FADH° within the experimental time window of 2.5 ns, suggesting that the repair reaction takes considerably longer.
The present project aims at advancing the understanding of the reaction mechanisms of DNA repair by (6-4) photolyase, including photoactivation. In a pluridisciplinary approach, biochemistry, molecular biology and organic synthesis for the preparation of enzymes and substrates are combined with advanced time-resolved optical spectroscopic techniques for functional studies.
For the photorepair reaction, essential innovations like developing a special synthetic substrate for (6-4) photolyase, covering all relevant time scales (from 200 fs to seconds) and inclusion of the difficult, but crucial 265 nm band of intact nucleobases in the transient absorption studies, should contribute to resolve mechanism and dynamics of photorepair of the 6-4PP.
For the photoactivation reaction, an all timescales approach will be combined with site-directed mutagenesis to establish the electron transfer pathway and kinetics. We in particular envisage to understand the coupling between electron and proton transfer that probably plays a crucial role in oxidation of tyrosine by a tryptophanyl radical, and in the reduction of FADox to FADH°.