DNA photolyases use light energy to repair DNA that comprises ultraviolet-induced lesions such as the cis-syn cyclobutane pyrimidine dimers (CPDs). Here we report the crystal structure of a DNA photolyase bound to duplex DNA that is bent by 50 degrees and comprises a synthetic CPD lesion. This CPD lesion is flipped into the active site and split there into two thymines by synchrotron radiation at 100 K. Although photolyases catalyze blue light-driven CPD cleavage only above 200 K, this structure apparently mimics a structural substate during light-driven DNA repair in which back-flipping of the thymines into duplex DNA has not yet taken place.

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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...

UV-light irradiation induces the formation of highly mutagenic lesions in DNA, such as cis-syn cyclobutane pyrimidine dimers (CPD photoproducts), pyrimidine(6-4)pyrimidone photoproducts ((6-4) photoproducts) and their Dewar valence isomers ((Dew) photoproducts). Here we describe the synthesis of defined DNA strands containing these lesions by direct irradiation. We show that all lesions are efficiently repaired except for the T(Dew)T lesion, which cannot be cleaved by the repair enzyme under our conditions. A crystal structure of a T(6-4)C lesion containing DNA duplex in complex with the (6-4) photolyase from Drosophila melanogaster provides insight into the molecular recognition event of a cytosine derived photolesion for the first time. In light of the previously postulated repair mechanism, which involves rearrangement of the (6-4) lesions into strained four-membered ring repair intermediates, it is surprising that the not rearranged T(6-4)C lesion is observed in the active site. The structure, therefore, provides additional support for the newly postulated repair mechanism that avoids this rearrangement step and argues for a direct electron injection into the lesion as the first step of the repair reaction performed by (6-4) DNA photolyases.

The Geobacillus stearothermophilus splG gene encodes a thermophilic spore photoproduct lyase (SplG) that belongs to the family of radical S-adenosylmethionine (AdoMet) enzymes. The aerobically purified apo-SplG forms a homodimer, which contains one [4Fe-4S] cluster per monomer unit after reconstitution to the holoform. Formation of the [4Fe-4S] cluster was proven by quantification of the amount of iron and sulfur per homodimer and by UV and EPR spectroscopy. The UV spectrum features a characteristic absorbance at 420 nm typical for [4Fe-4S] clusters, and the EPR data were found to be identical to those of other proteins containing an [4Fe-4S] ؉ center. Probing of the activity of the holo-SplG with oligonucleotides containing one spore photoproduct lesion at a defined site proved that the enzyme is able to turn over substrate. In addition to repair, we observed cleavage of AdoMet to generate 5-deoxyadenosine. In the presence of aza-AdoMet the SplG is completely inhibited, which provides direct support for the repair mechanism.Spores of various Bacillus and Clostridium species are extremely resistant to harsh physical, chemical, and biological conditions allowing them to survive even under extreme conditions (1, 2). The oldest known viable spore was discovered from a Bacillus species, designated 2-9-3, in a 250 million-yearold salt crystal from the Permian Salado Formation (3). The resistance of spores from Geobacillus stearothermophilus toward heat is even so high that the survival of the organism during heat sterilization is used as a bioindicator for insufficient heat treatment (4).Particularly noteworthy is the unusually high stability of spores in the presence of UV light. For example under typical UV sterilization conditions, only about 70% of thermophilic G. stearothermophilus spores are inactivated. Under the same conditions, typical pathogens such as herpes simplex or polio viruses are fully destroyed (5, 6). In addition, UV irradiation of spores gives rise to different DNA lesions (7). Although in normal cells mostly cyclobutane pyrimidine dimers and (6-4) lesions (8) are formed, in spores the unusual photoproduct 5-thyminyl-5,6-dihydrothymine (SP), 2 depicted in Scheme 1 (7, 9), is exclusively generated (10). These differences in the photoreactivity may be because of an unusual packing of the DNA in spores (1, 11, 12) and the high amounts of dipicolinic acid (DPA) present in spores (13).During germination, the SP lesion is repaired either by the general nucleotide excision repair pathway (14, 15) or by a single enzyme, called spore photoproduct lyase, which is able to split the SP lesions directly back into two thymidines. Recent studies by Nicholsen et al. (16) and Broderick and co-workers (17) performed with the SP-lyase from Bacillus subtilis showed that the enzyme requires S-adenosylmethionine (AdoMet) as a cofactor for repair. A detailed sequence comparison, spectroscopic studies (18,19), and a recent labeling experiment (17) all provide evidence that the SplG is a member of the radical AdoMet enz...

Pseudomonas aeruginosa produces a number of alkylquinolone-type secondary metabolites best known for their antimicrobial effects and involvement in cell-cell communication. In the alkylquinolone biosynthetic pathway, the β-ketoacyl-(acyl carrier protein) synthase III (FabH)-like enzyme PqsBC catalyzes the condensation of octanoyl-coenzyme A and 2-aminobenzoylacetate (2-ABA) to form the signal molecule 2-heptyl-4(1H)-quinolone. PqsBC, a potential drug target, is unique for its heterodimeric arrangement and an active site different from that of canonical FabH-like enzymes. Considering the sequence dissimilarity between the subunits, a key question was how the two subunits are organized with respect to the active site. In this study, the PqsBC structure was determined to a 2 Å resolution, revealing that PqsB and PqsC have a pseudo-2-fold symmetry that unexpectedly mimics the FabH homodimer. PqsC has an active site composed of Cys-129 and His-269, and the surrounding active site cleft is hydrophobic in character and approximately twice the volume of related FabH enzymes that may be a requirement to accommodate the aromatic substrate 2-ABA. From physiological and kinetic studies, we identified 2-aminoacetophenone as a pathway-inherent competitive inhibitor of PqsBC, whose fluorescence properties could be used for in vitro binding studies. In a time-resolved setup, we demonstrated that the catalytic histidine is not involved in acyl-enzyme formation, but contributes to an acylation-dependent increase in affinity for the second substrate 2-ABA. Introduction of Asn into the PqsC active site led to significant activity toward the desamino substrate analog benzoylacetate, suggesting that the substrate 2-ABA itself supplies the asparagine-equivalent amino function that assists in catalysis.

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Asymmetric bromolactonizations of alkynes are possible using a desymmetrization approach. The commercially available catalyst (DHQD)2PHAL promotes these cyclizations in combination with cheap NBS as a bromine source to give bromoenol lactones in high yield and with high enantioselectivity. The bromoenol lactone products, containing a tetrasubstituted alkene and a quaternary stereocenter, are valuable building blocks for synthetic chemistry.