Photochemistry and Photobiology, 2015, 91: 1263–1290

Invited Review Photochemistry and Photobiology of the Spore Photoproduct: A 50-Year Journey

Peter Setlow*1 and Lei Li*2,3 1Department of Molecular Biology and Biophysics, UConn Health, Farmington, CT 2Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis (IUPUI), Indianapolis, IN 3Department of Biochemistry and Molecular Biology, Department of Dermatology, Indiana University School of Medicine, Indianapolis, IN Received 5 May 2015, accepted 21 July 2015, DOI: 10.1111/php.12506

ABSTRACT contribute to spore death (3), and with high UV doses, it may involve up to 28% of total thymine residues in the spore genome Fifty years ago, a new thymine dimer was discovered as the (4). Although the spore lesion was also isolated from UV irradi- dominant DNA photolesion in UV-irradiated bacterial spores ated dry (4,5) or frozen DNA (6), the yield was never as great as Science, – [Donnellan, J. E. & Setlow R. B. (1965) 149, 308 that in irradiated spores. Therefore, this photolesion was com- 310], which was later named the spore photoproduct (SP). monly referred to as the spore photoproduct (SP). Using DNA Formation of SP is due to the unique environment in the irradiated in ice at 78°C and then DNA hydrolysis in strong spore core that features low hydration levels favoring an A- acid at 165°C, Varghese was able to purify a significant amount DNA conformation, high levels of calcium dipicolinate that of SP base adduct (SPBASE (1), Fig. 2) by paper and column acts as a photosensitizer, and DNA saturation with small, chromatography (7). The UV–visible spectrum of the purified acid-soluble proteins that alters DNA structure and reduces SPBASE was also determined and SP was found to possess an side reactions. In vitro studies reveal that any of these factors 1 1 extinction coefficient of 8200 M cm at 265 nm, almost iden- alone can promote SP formation; however, SP formation is tical to that of a single thymine residue. Such an absorption usually accompanied by the production of other DNA pho- spectrum agrees with the SP structure shown in Fig. 1, since the tolesions. Therefore, the nearly exclusive SP formation in 50-thymine ring does not absorb at 265 nm due to the loss of this spores is due to the combined effects of these three factors. ring’s aromaticity. Using not only UV–visible spectroscopy, but via Spore photoproduct photoreaction is proved to occur a also IR, NMR and mass spectrometry, Varghese then correctly unique H-atom transfer mechanism between the two involved suggested that 5-thyminyl-5,6-dihydrothymine is the most proba- thymine residues. Successful incorporation of SP into an ble structure for SP after both glycosidic bonds were hydro- via BASE oligonucleotide has been achieved organic synthesis, lyzed (7). Later, using NMR spectroscopy coupled with density which enables structural studies that reveal minor conforma- functional theory (DFT) to examine the dinucleotide SP TpT tional changes in the SP-containing DNA. Here, we review (SPTIDE (2), Fig. 2) obtained from the dinucleotide TpT photore- the progress on SP photochemistry and photobiology in the action, Mantel et al. (8) determined that the dihydropyrimidine past 50 years, which indicates a very rich SP photobiology moiety was located at the 50-end of the dinucleotide and the C5 that may exist beyond endospores. chiral center adopts an R configuration. The SPTIDE 2 was found to be identical to SPs obtained from UV-irradiated spores after DNA digestion (9), proving that the SPs formed in spores con- INTRODUCTION tain a 5R-chiral center (Fig. 1). The chirality is ascribed to the In July 1965, Donnellan and Setlow published a short communi- right-handed DNA helical structure, which prevents any 5S-SP cation in the journal Science, reporting that in Bacillus mega- being formed and enables generation of 5R-SP as the dominant terium spores irradiated with monochromatic ultraviolet (UV) DNA photoproduct in UV-irradiated spores. light at 265 nm, a new thymine lesion was produced as the dom- Shortly after SP was discovered in UV-irradiated spores, Don- inant DNA photodamage product (1). This lesion is different nellan and Stafford (3) established that when spores come back from the cyclobutane pyridimine dimer (CPD, Fig. 1), the nature to life in the process of germination, SP was eliminated from of which was relatively well established at that time. It was also genomic DNA by a dark-repair mechanism somewhat different different from the pyrimidine (6-4) pyrimidone photoproduct from that responsible for CPD repair in vegetative cells. Genetic (6-4PP) discovered by Varghese and Wang 2 years later in UV- analysis indicated that at least two genes contribute to the irradiated DNA (2). The new spore lesion was soon shown to observed high UV resistance of Bacillus subtilis spores (10), with these two genes determining distinct processes for SP fi *Corresponding authors’ emails: [email protected] (Peter Setlow), lilei@iupui. removal from DNA during spore germination (11,12). The rst edu (Lei Li) is a spore-specific repair mechanism by which SP is removed © 2015 The American Society of Photobiology

1263 1264 Peter Setlow and Lei Li

O O O O O HN NH HN OH O HN NH N N O N N O O N N O N O O O O - O - O O O 3' O 3' 5' O 3' 5' OR 5' OR OR O P RO O P O RO O P O RO - O O O O CPD 6-4PP SP Figure 1. Chemical structure of the three thymine dimers.

O O O O O O

HN (R) NH HN NH HN NH O O (R) (S) O N N O HO HO O N N O HO O N N O HN (R) NH O O O HO O O O N N O O O O HO H H P O OH OH - OH OH O OH SPBASE (1) dinucleoside 5R-SP TT dinucleoside 5S-SP TT dinucleotide SP TpT (5R-SPSIDE)(3) (5S-SPSIDE)(4) (SPTIDE) (2) Figure 2. Structures of spore photoproduct (SP) and SP derivatives discussed in this review. In the literature, dinucleoside SP TT and dinucleotide SP TpT were used, respectively, for SP dimers with and without the phosphodiester linker; while Desnous et al. used SPSIDE and SPTIDE to refer to these two species (40). Although we used SPSIDE and SPTIDE in our review, we wish to show both nomenclatures. rapidly without appearing in an acid-soluble form or released via chemical and photochemical means. The reviews of Desnous from the germinating spores (13). The second mechanism, now et al. (40) and of Yoshimura et al. (41) have covered the SP known as the general nucleotide-excision repair (NER) pathway, chemistry and photochemistry till 2010 and 2012 respectively; removes SP relatively slowly. In addition to SP, the NER path- here we will focus more on progress since 2012. For a number way is also responsible for removing CPDs and 6-4PPs and is of years, except for SPL-mediated repair, very little was learned regarded as the major DNA-repair process involved in the recog- about SP photobiology owing to the difficulty in obtaining nition and removal of UV-mediated DNA damage in growing SP-containing oligonucleotides for biological studies. However, bacteria as well as in eukaryotes (14–17). However, blocking progress in synthetic chemistry in the past several years has either pathway only slightly increases spore UV sensitivity, while largely solved this issue, and opened the door for the understand- blocking both pathways prevents the efficient removal of SP, ing of general SP photobiology. resulting in very UV-sensitive spores (11,12). fi The spore-speci c SP repair mechanism is now known to be SP FORMATION IN BACTERIAL SPORES mediated by a metalloenzyme—the spore photoproduct lyase (SPL) encoded by the splB gene (18). In the 1970s, it was found Spore photoproduct is isolated as the dominant DNA photopro- that the radioactive tritium label disappearing from SP was duct in UV-irradiated spores. This unique photochemistry is recovered stoichiometrically in thymine residues in germinated largely ascribed to the unique environment inside a spore spores, indicating that SPL directly reverts SP to TpT (3,13). (40,42–45). As indicated in Fig. 3, underneath the outer glyco- Bioinformatics studies have established that SPL is a so-called protein layer called the exosporium is a proteinaceous coat pro- radical S-adenosylmethionine enzyme, which utilizes a [4Fe-4S] viding much of spores’ resistance to enzymes and toxic cluster coupled with an S-adenosylmethionine cofactor to gener- chemicals. Beneath this coat there is an outer membrane, which ate a 50-deoxyadenosyl radical for catalysis (19). Research from may not be a functional membrane in spores, and then a thick various groups has further established that the 50-deoxyadenosyl layer of specialized peptidoglycan called the cortex, which plays radical initiates the SP repair process by abstracting an H-atom an important role in maintaining the low water content in spores’ from the C6 position on SP, which triggers a cascade radical central core. A germ cell wall composed of peptidoglycan with a transfer process (9,18–33). The most recent results suggest that structure different from that of the cortex lies under the cortex, SPL uses an unprecedented radical transfer pathway composed followed by the inner membrane, the major permeability barrier of one cysteine and two tyrosine residues to facilitate radical against damaging chemicals. The center of the spore, the core, transfer (33). This hypothesis has been fully supported by analy- has a relatively low water content, and houses the genomic DNA sis of the recently solved SPL structure (34). saturated with spore-specific DNA-binding proteins, ribosomes Progress in SPL mechanistic studies has been extensively and a large amount (see below) of pyridine-2,6-dicarboxylic acid reviewed in the past several years (35–39). In this review, we (dipicolinic acid (DPA)), most of which is chelated to divalent choose to cover SP photochemistry and photobiology revealed cations, primarily Ca2+ (CaDPA). This unique environment for Photochemistry and Photobiology, 2015, 91 1265

INNER OUTER formed in TpC and TpT steps were also detected, but in small MEMBRANE MEMBRANE amounts (50). COAT In contrast to results with UVC, with UVB radiation, SP for- mation efficiency is reduced by ~3 orders of magnitude (51). Moreover, although SP still accounts for the majority of the DNA photolesions formed, appreciable quantities of CPDs are generated. As a consequence, the NER and the SPL repair path- ways appear to contribute approximately equally to the survival CORTEX of UVB-irradiated spores (49,52). No DNA strand breaks were detected in UVB-irradiated spores; however, they are the major DNA damaging events in UVA-irradiated spores, as CPD and EXOSPORIUM SP formation are undetectable in UVA-irradiated spores (49,52). GERM CELL WALL If full spectrum sunlight is used to irradiate spores, the DNA damage pattern resembles the combined effects of UVA and Figure 3. Structure of a bacterial endospore (5). The relative sizes of various layers are not drawn to scale, and the large exosporium is not UVB. Therefore, in addition to the NER and SPL pathways to present in spores of all species. Note that there can be several sublayers repair DNA photodimers, the homologous recombination and in individual layers, in particular the coat and probably in the exospo- nonhomologous end joining pathways that repair DNA double- rium. strand breaks may also play important roles in DNA repair in spore germination (53,54). Indeed, proteins involved in these the spores’ genome is responsible for efficient SP formation in latter repair processes are present in dormant spores to allow for spores. At the same time, formation of other DNA photodamage rapid DNA repair when spores germinate (55). as well as DNA damage induced by agents such as heat, desicca- Spore photoproduct formation is the main DNA photochemical tion and genotoxic chemicals is largely suppressed (46). event occurring in UVB/UVC-irradiated spores. In contrast, in vitro irradiation of spore genomic DNA and/or UV exposure of vegetative B. subtilis cells results in CPDs as the dominant SP formation as a function of UV wavelength products, with minimal SP formation. Three factors have been The electromagnetic spectrum of UV irradiation, defined as 100– suggested to contribute to the unique DNA photochemistry in 400 nm in wavelength is generally divided into three categories, spores: (1) action of CaDPA as a photosensitizer; (2) the low UVC (100–280 nm), UVB (280–315 nm) and UVA (315– hydration level in the spore core and (3) the saturation of spore 400 nm). Stratospheric ozone effectively blocks wavelengths genomic DNA by a group of DNA-binding proteins termed small, shorter than 290 nm from reaching the earth’s surface, filtering acid-soluble spore proteins (SASPs). As described below, the out all the UVC and most of the UVB light (47). Consequently, functions of these factors seem to be intertwined, such that their about 95% of the UV radiation from the sun that reaches the combined effects enable SP formation as the dominant DNA pho- earth is UVA, and the remaining 5% is UVB. However, owing tochemical event in UV-irradiated spores. However, when spores to the high energy in UVC photons, UVC has been widely used germinate, CaDPA is excreted, spore core water content rises to in germicidal lamps for killing microorganisms in sterilization that in growing cells, and SASPs are all degraded (56–58). After applications. Indeed, with UVC radiation, SP photochemistry in all these germination events are completed, spore UV resistance spores proceeds with slightly higher efficiency than does CPD or and DNA photochemistry revert to that of growing cells. 6-4PP formation in growing cells (44,48). Although formed as the dominant species in spores, SP formation is accompanied by Function of CaDPA formation of other minor DNA photoproducts, such as CPDs as well as single- and double-strand DNA breaks. The extent of As noted above, the spore core contains an extremely high level these “side” photoreactions appears to be very dependent on the of CaDPA (Fig. 4). This complex comprises 5–15% of the dry wavelength of UV light. weight of spores in both Bacillus and Clostridium species. Given Although UVC radiation is unlikely to play a significant role that the core occupies ~50% of the overall spore volume, in SP photochemistry in nature, UVC has been used for the vast CaDPA accounts for ~20% of spore core weight (59). Assuming majority of spore photobiology studies owing to the high photo- that the rest of the core is water, the concentration of CaDPA 2 efficiency of this radiation. With a dose of 16 kJ m of UVC (M.W. = 205 Da) in the core is estimated to be ~1.2 M. Using light predominantly at 254 nm, SP is the principal DNA photo- microfluidic Raman tweezers, the CaDPA concentration in the 5 product in Bacillus subtilis endospores (~4.2 9 10 per chromo- spore core was found to be between 800 mM and 1 M (59), very some); CPDs and single-/double-strand breaks are also found, close to the estimate given above. but the levels of these lesions are only 0.16% and 0.3% of SP Much work has strongly implicated CaDPA in playing impor- respectively (49). Therefore, it was concluded that SP generation tant roles in: (1) spore resistance; (2) spore germination and (3) is the dominant factor determining spore survival after UVC irra- spore stability (60,61). (1) CaDPA accumulation in the spore diation, and physiological consequences of CPDs and strand core decreases the core water content (60), and spores with lower breaks may be negligible. Such a conclusion is further supported core water content are more resistant to wet heat (60–62). by a later study, which used high-performance liquid chromatog- CaDPA is also suggested to interact with DNA in spores, raphy coupled with tandem mass spectrometry (HPLC-MS/MS) preventing DNA damage from dry heat and desiccation (60,61). to reanalyze the DNA lesions in UVC-irradiated spores, finding In addition, CaDPA is thought to serve as a photosensitizer, that SP was the predominant DNA photoproduct (93 5%); facilitating energy transfer from photoexcited DPA to thymine CPDs formed in TpT, TpC and CpT steps as well as 6-4PPs residues to promote SP formation (63). Hyperresistance to UVC 1266 Peter Setlow and Lei Li

(A) (B)

O Ca (H2O)4 - O O - H O O 2 2+ Ca N Ca H2O N Ca2+ - Ca O - O O

(H2O)4 H2O O H2O (5) (C)

Figure 4. (A) Structure of the CaDPA dimer as the basic assembly unit for the inorganic polymer (75). The dimer adopts a planar structure. Each Ca2+ is coordinated to eight atoms. Among them, three are from a DPA, one is from a carboxylate moiety of another DPA in the dimer and the rest are from two pairs of water molecules, with one pair above and the other pair beneath the dimer plane. Both water molecules within the pair coordinate to another Ca2+ from another dimer, thus serving as bridging ligands to link the dimers into an inorganic polymer. (B) The coordination sphere around the calcium ion. The Ca2+ ion is shown in green, nitrogen in blue, and oxygen atoms in red. The four bridging water ligands are also labeled in the structure. (C) The structure of the CaDPA polymer linked by bridging water molecules.

develops upon spore germination following CaDPA release but The ~1 M CaDPA concentration found in the spore core is before SASPs degradation (64–67). This observation is ascribed roughly 10 times above its known solubility in water (59). to the fact that SP is still formed as the dominant DNA photo- Therefore, the complex is likely to adopt a three-dimensional product early in spore germination due to the DNA conformation structure as a solid matrix rather than existing as a mononuclear generated by SASP binding; however, much fewer SPs are pro- complex dissolved in the central protoplast. Indeed, it has been duced under the same UV dose after the loss of photosensitizing suggested that the spore core adopts a glass-like state, with CaDPA. (2) CaDPA release early in the germination of an CaDPA being a major contributor to the matrix structure (70,71), individual Bacillus spore is an important part of the signal trans- although this idea has been disputed (71–74). CaDPA has been duction process in germination (56–58), and spores can even be crystallized as the trihydrate in both H2O (75) and D2O (76), triggered to germinate by CaDPA (58,68,69). As a consequence, and exhibits the same structure, where the essential unit is a it is possible that CaDPA released from one spore may stimulate CaDPA dimer 5 (Fig. 4A). Around each Ca2+ cation, eight the germination of neighboring spores, especially if spores are at ligands, seven oxygen-based and one nitrogen-based, can be extremely high concentrations (68). (3) Spores containing no or found. Three of these ligands come from the two carboxylates low levels of CaDPA are unstable and germinate spontaneously and the pyridine nitrogen from one DPA, the fourth ligand is (60,61,68). Although the mechanism behind this latter behavior from the carboxylate of the other DPA in the dimer unit is not completely understood, it is likely that the absence of (Fig. 4B). The dimer unit adopts a planar structure in which the CaDPA results in activation of enzymes that specifically degrade Ca2+ cations in the dimer are coordinated by two pairs of water the spore cortex, thus initiating the germination process. molecules, with a pair on each side of the plane, to finish the Photochemistry and Photobiology, 2015, 91 1267 eight coordinated states. Each pair of water molecules also coor- the fixation of water molecules. Two theories have been pro- dinates to the same Ca2+ center in another dimer. Consequently, posed to describe the physical property of the spore core: a the dimer units are linked together by these di-water bridges into glass-like state where the entire core including the water mole- a polymer-like chain structure (Fig. 4C). cules are immobilized (70), and a gel-like state where the macro- It was suggested more than 40 years ago that a crystalline molecules are immobilized in the matrix while the water matrix may exist in the spore core (75). By comparing the signa- molecules remain mobile (72). Measurements of the 2H and 17O ture resonance Raman signals from DPA, recent spectroscopic spin relaxation rates in solid-state NMR spectroscopy in D2Oor 17 studies revealed that Raman signals from CaDPA are similar to H2O -exchanged Bacillus subtilis spores indicated that there those from crystalline CaDPA, and these signals clearly differ was high water mobility throughout the spore, which is compara- from those due to DPA or CaDPA dissolved in water (77,78). ble with binary protein water systems at similar hydration levels This result supports the idea that a CaDPA matrix may con- (73,88), supporting the idea that the spore core has a gel-like tribute to a glass-like state in spore core. However, although structure. However, another study using deuterium solid-state somewhat dehydrated, the water content in the spore core is still NMR found both bound and unbound water in the spore core more than in a CaDPA crystal. Also, the presence of DNA, ribo- and the core water exchanged only slowly with water in the somes, RNA and proteins could interfere with CaDPA packing, medium (76). While the latter observation can be better making the structure of the CaDPA matrix in spores likely to be explained by the glass-like state of the spore core, the low per- more complicated than that shown in Fig. 4 (77). meability of the spores’ inner membrane may well contribute to Resonance Raman data also indicated that CaDPA may inter- slow water exchange between the core and the medium. calate into the DNA bases (78), and in vitro spectroscopic stud- While the exact state of water in the spore core is still not ies found that CaDPA directly interacts with DNA (79,80). The completely clear, it is most likely that the low core water content yield of SP as a function of UV fluence was 10- to 20-fold lower contributes to SP formation. In UVC-irradiated wild-type B. sub- in spores of mutants which do not make DPA than in wild-type tilis spores, SP is the major (>98%) DNA photolesion formed by spores (81). In dry calf thymus DNA films exposed to UVC radi- UVC radiation (88). However, while SP was still a major pro- ation, the yield of SP was greatly increased by the presence of duct in UVC-irradiated spores lacking either CaDPA or SASPs, CaDPA in the DNA film (63). Moreover, it was suggested that significant amounts of CPDs and 6-4PPs were also observed the thymine triplet excited state supports SP formation (82), and (44,63,88). Most notably, in spores lacking both CaDPA and CaDPA was therefore suggested to function as a photosensitizer, SASPs, only ~2% of the pyrimidine dimers formed were SP facilitating energy transfer from the triplet excited state of (88). However, compared with UVC-irradiated vegetative cells CaDPA to thymine residues in DNA (63). Thus, the involvement where no detectable SP was formed, the 2% yield implies that of CaDPA in SP photochemistry has been supported by both these spores’ relatively low water content may still be important in vivo and in vitro observations. in facilitating SP photochemistry. Indeed, while the core of Among the four nucleobases, thymine possesses the lowest tri- B. subtilis spores lacking CaDPA has ~30% higher core water plet excited state while cytosine has the highest energy (83). If the content than the wild-type spore core (45% vs 35%), the energy of the lowest triplet excited state of CaDPA is lower than CaDPA-less spore core still has a much lower water content than that of the nucleobase, energy transfer to the nucleobase is not a growing cell (50). As described below, an A-like DNA confor- favored resulting in the quenching of certain photoreactions. mation, which is favored at low water content, is suggested to be Given the large amount of CaDPA in the spore core, if its triplet critical for SP photochemistry. Therefore, the reduced SP forma- excited state is only higher than that of a thymine residue, photore- tion in DPA-less, SASP-less spores could be due at least in part actions involving other nucleobases will largely be suppressed. to the increased water content in these spores’ core. Since in spores thymine dimerization into SP is the dominant reac- The immobility of core ions and protein noted above indicates tion and reactions associated with cytosine are negligible, this that DNA is also likely to be immobilized in the spore core, and reaction pattern may be due to the excited state energy of CaDPA, such DNA immobilization may also contribute to SP photochem- and this is certainly a matter for future experiments. istry. In aqueous solution, DNA can freely undergo conforma- tional changes; these conformations largely belong to the B-form and support CPD formation upon photoexcitation. However, due Low water content in the spore core to the glass- or gel-like state in the spore core and the binding of Another function of the high CaDPA concentration in the spore SASPs (see below), the conformational changes in the immobile is to lower the water content in the core, and this has been genomic DNA are expected to be greatly slowed. The A-like strongly implicated as playing a major role in spores’ resistance genomic DNA in spores favored by the low hydration level is to wet heat (62). While water comprises 75–80% of the wet further stiffened by the DNA immobility and binding of SASPs weight of the protoplast of a growing cell, water makes up only (88–90), supporting SP formation as the nearly exclusive pho- 27–55% of the spore core wet weight depending on the species toreaction upon UV irradiation and largely suppressing all other (44), and this low water content may result in unique physical dimerization reactions. properties of the core. Indeed, ions in the spore core were shown long ago to be immobile (84,85), lipids in the inner membrane DNA binding by SASPs of dormant spores of Bacillus species are also largely immobile (86), and at least one normally soluble core protein is immobile In comparison with the presence of CaDPA and low water con- in B. subtilis spores (87). These findings further suggested that tent in the spore core, the binding of SASPs to spore DNA is core water itself may also be immobile. generally considered as the most important factor in the occur- However, recent results challenge the assumption that the rence of SP photochemistry (44,88,91). Such a conclusion is immobility of core macromolecules and ions is directly related to ascribed to the fact that SASP binding alone is effective in 1268 Peter Setlow and Lei Li promoting SP photochemistry and quenching the formation of The impact of binding of a/b-type SASPs to spore DNA was other dimers, while the presence of CaDPA nonselectively examined by immunofluorescence microscopy in developing increases the yields of both SP and CPDs (63,92–94). While dor- spores (100,101). The results indicate that the chromatin adopts a mant spores are ~10-fold more UVC resistant than growing cells, tightly packed ring-shaped morphology as a consequence of spore UVC resistance actually rises dramatically after CaDPA SASP binding. To reveal more details of this protein–DNA inter- release and before SASPs degradation that takes place minutes action, a variety of studies were conducted in vitro. Circular after CaDPA release (64–67). Spore photoproduct is still the dichroism and Fourier-transform infrared spectroscopy showed major UVC photoproduct in these highly UV-resistant spores that binding of SspC to DNA alters the DNA conformation from early in germination, but the efficiency of SP formation drops the B-form to an A-like form (89). The relative affinity of SspC significantly, likely because of the loss of the photosensitizing to various DNA sequences was determined to be poly(dG)•poly action of CaDPA on SP photochemistry. However, as a/b-type (dC) > poly(dG-dC)•poly(dG-dC) > plasmid pUC19 > poly(dA- SASP are degraded, spore UV resistance decreases dramatically dT)•poly(dA-dT) poly(dA) •poly(dT); such an order parallels to that in growing cells, and SP formation ends. As expected, the ease with which these DNAs adopt an A-like conformation significant slowing of SASP degradation by loss of the germina- (102). Measured by both electron microscopic and cyclization tion protease specific for SASPs greatly prolongs the length of assays, it was found that SspC binding greatly increases DNA the high UV-resistant state following spore germination as well stiffness, but does not alter the rise per base pair (bp) (90). This as continued SP formation. In addition, spores lacking the major- latter finding is inconsistent with the DNA–SspC complex ity of their a/b-type SASP do not exhibit this period of elevated having the classical structure of A-DNA found in dry DNA UV resistance soon after spore germination. These latter results fibers. Using cryoelectron microscopy techniques, the SspC bind- are consistent with the importance of a/b-type SASP in causing ing to DNA was indicated to induce only minor conformational SP photochemistry in spores. changes, and the DNA pitch (3.18 nm) remains close to that of The SASPs are synthesized only in the developing spore late canonical B-form DNA (103). A tight packing of the DNA– in sporulation, and account for 8–20% of total spore core protein protein filaments was also implied, suggesting that the spore gen- depending on the species analyzed (91). The SASPs in spores can ome may possess a toroidal conformation as a result of SASP– be divided into two types: the a/b-type and the c-type. There are DNA interaction (103). multiple a/b-type SASPs in spores of Bacillus and Clostridium Although a/b-type SASPs are designed to bind and protect species, and one c-type SASP in Bacillus spores although c-type spore DNA, they are not intended to bind permanently. Indeed, SASP is not present in spores of Clostridium species (91). The if they do not dissociate quickly from DNA soon after spore a/b-type SASPs have molecular weights of 5–9 kDa, and account germination is initiated, then subsequent transcription and spore for 5–10% of total protein in the spore core. They have a signifi- outgrowth will be dramatically affected leading to spore death cant percentage of hydrophobic amino acids, share remarkable (104). Thus, their intrinsic binding affinity to DNA is rather low sequence conservation both within and across species and genera, and this low affinity becomes a major obstacle for in vitro and their functions are largely interchangeable in spores DNA–protein interaction studies. To increase SASP binding (91,95,96). Therefore, one B. subtilis a/b-type SASP, SspC, has affinity for DNA, an SspC mutant, SspCDN11-D13K-C3, was con- been used in most in vitro studies of SASPs’ properties, even structed, which has a ~20-fold higher affinity for DNA than though this protein is not the most abundant a /b-type SASP in wild-type SspC (105). This SspC variant promotes a similar spores (91). The c-type SASP has a very different amino acid DNA photochemistry as the wild-type protein, suggesting that sequence than the a/b-type and has an extremely low content of both wild-type and variant proteins bind to DNA in a similar large hydrophobic amino acids. There is much evidence that a/b- manner (105). type SASPs bind to and saturate the spore genomic DNA (88). The SspC variant noted above was used to solve the crystal However, c-type SASP does not bind to DNA, even though this structure of an oligo(dG)•oligo(dC) 10-mer complexed with protein is often the most abundant SASP in spores. All SASPs SspCDN11-D13K-C3 (106,107). The structure suggests that the a/b- are digested into amino acids in a process initiated by a protease type SASPs adopt a helix–turn–helix motif on DNA (Fig. 5A), that recognizes a highly conserved amino acid sequence in although the SASPs are largely random coil alone in solution. SASPs, and the amino acids generated in this process are used for SASPs interact with DNA through minor groove contacts and energy generation and protein synthesis (91). bind to ~6 bp of DNA as a dimer, agreeing with biochemical The binding of SASPs to spore DNA is suggested to modify data obtained previously (108). More importantly, the DNA in the DNA conformation favoring SP formation upon photoexcita- the nucleoprotein complex adopts an A-B type conformation, tion, and quenching other “side” reactions such as CPD forma- where the sugar puckering adopts a C30-endo conformation, a tion (93,94). In wild-type B. subtilis spores, UVC irradiation feature of A-form DNA, while the base planes are essentially generates almost exclusively SP with little if any CPD and 6- parallel to each other and normal to the helix axis, which is char- 4PP (88). In contrast, in spores lacking ~85% of their a/b type acteristic of B-DNA (Fig. 5B). In addition, molecular simulation SASPs, while SP is still the major (~70%) DNA photoproduct, studies replacing the 6th and 7th GC pairs with AT pairs in the cis-syn CPDs formed in TpT, TpC, CpT and CpC steps are also 10-mer indicated that the C5 of one T is only 3.4 A away from observed (94). Decreased levels of a/b-type SASPs also decrease the –CH3 of another T (106,107). This distance is shorter than the resistance of Clostridium perfringens spores to UV radiation, the 3.9 A between the two C5 positions which is required to possibly due to the increased formation of CPDs (97,98). More- connect in CPD formation (106). The corresponding moieties over, UV irradiation of Escherichia coli engineered to have high involved in 6-4PP formation are even further apart. These results levels of an a/b-type SASP also yields less CPDs and some SPs, offer a rationale for how the DNA conformation in the SASP- in contrast to the undetectable SP formation in E. coli lacking DNA complex promotes SP photochemistry and quenches other such a protein (99). “side” photoreactions. This structure is also consistent with Photochemistry and Photobiology, 2015, 91 1269 spectroscopic studies, indicating that the SP photochemistry in photochemistry in detail. For instance, in vitro studies have estab- spores cannot be simply ascribed to a pure A-like DNA confor- lished that the formation of CPDs at a dipyrimidine site is rever- mation, but at least partially to DNA conformational changes sible; the “equilibrium” of the reaction appears to be controlled induced by dehydration (109). Although the A-like DNA confor- by the wavelength employed (111). In contrast, formation of 6- mation favors SP formation, the B-A transitional conformations 4PP and SP are irreversible (112). Because of the latter property, as a consequence of reduced DNA hydration level already sup- with 254 nm UVC irradiation, formation of CPDs quickly port the formation of SP, although such a process is likely reaches a maximum yield of ~20% while yields of 6-4PP keep accompanied by the formation of other thymine dimers. increasing. In vitro studies of CPDs and 6-4PPs in aqueous solu- tion also make the elucidation of their formation mechanism, i.e. SP FORMATION IN VITRO the excited states involved and the reaction kinetics, possible (113–120). UV irradiation of DNA in aqueous solution results in Besides investigations in spores, SP photochemistry has also no SP, which can be ascribed to the normal DNA hydration level been extensively studied in vitro. By irradiating single- or preventing DNA from adopting appropriate reactive conforma- double-strand oligonucleotides in aqueous solution, CPDs and 6- tions to form SP. Consequently, generation of SP in in vitro 4PPs can be readily formed at a given dipyrimidine site (110), DNA photochemistry is very inefficient, since reactions have to and further studies have allowed elucidation of CPD and 6-4PP be conducted under unique environmental conditions.

SP formation in ice (A) It was discovered by Rahn et al. that DNA conformation at low temperatures favors SP photochemistry and quenches formation of other dimers (6,121). DNA is less hydrated at low tempera- tures than at room temperature (122), which may cause a DNA conformational change toward the A-form, providing a rationale for the observed SP formation in ice. In contrast, no SP forma- tion was observed at 25°C and CPDs were found as the major DNA lesions. However, the percentage of CPDs in all thymine photolesions gradually decreased as temperature decreased to 196°C while the percentage of SP gradually increased at the very beginning of the temperature decrease, reached a maximum at 100°C, and then decreased as the temperature was lowered further (121). At a given temperature, the SP yield increased with increasing UV doses. Indeed, as noted earlier, Varghese uti- lized DNA irradiation in ice to generate a relatively large amount of SP and prove that SP is a thymine dimer, with a structure of 5-thyminyl-5,6-dihydrothymine (7). This chemical structure also allowed Varghese to propose that SP is formed via formation (B) and combination of 5-a-thyminyl and 5,6-dihydrothymin-5-yl radicals, which is now known to be largely correct (see discus- sion in Section III). Besides isolated genomic DNA, irradiation of thymidine in ice also produces dinucleoside SP (123). The yield of SPSIDE was not quantified; however, it is likely to have been very low (likely <1%). Because of the right-handed helical structure of duplex DNA and of TpT, the SP formed can only adopt an R configuration at the C5 chiral center (Figs. 1 and 2). Such a restriction no longer exists in monomeric thymidine residues. As a consequence, a pair of SPSIDE diastereoisomers (Fig. 2) adopt- ing 5R (3) and 5S (4) configurations, respectively, are produced from the thymidine photoreaction (124) (see discussion below). Although only the 5R-SP was predicted to form in spores and be subsequently repaired by the SP repair enzyme SPL during spore germination, some earlier studies suggested that SPL – Figure 5. Crystal structure of the a/b-type SASP (SspC) oligo(dG) repairs the 5S-isomer (125,126). The discrepancy was corrected •oligo(dC)10-mer complex (pdb entry 2Z3X) (106). (A) Stereo diagram of the complex indicating that SspC adopts a helix–turn–helix motif by later SPL enzymology studies (127). In a recent SPL structure when binding to DNA. The positions of the N and C termini of each pro- solved by Benjdia et al., the enzyme contains a 5R-SPSIDE, fur- tomer are labeled: orange, SspC1; magenta, SspC2; cyan, SspC3. (B) ther confirming that the 5R-isomer is the SPL substrate (34). DNA conformation in the SspC-DNA complex. This conformation is a Moreover, NMR spectroscopy confirmed that the SP con- combination of A- and B-conformations, and is different from the typical TIDE A- or B-form DNA. Analyses by molecular simulation indicated that tains a 5R chiral center (8); this chirality was further established such a DNA conformation favors formation of spore photoproduct over by a dinucleotide SP structure containing a formacetal linker, cyclobutane pyridimine dimer and 6-4PP. which is also an SPL substrate (128). After incorporating 1270 Peter Setlow and Lei Li

5R-SPSIDE and 5S-SPSIDE into a dodecamer sequence, respec- and subsequent SP formation upon UV irradiation. Inclusion of tively, via solid-phase DNA synthesis, subsequent structural CaDPA in the dry film increased the SP yield by ~15-fold, but studies found that the 5R-SPSIDE fits in the topology of the right- only increased the CPD yield by ~two-fold. As a consequence, handed helix well, while the 5S-SPSIDE results in a severe strand comparable amounts of CPDs and SPs were generated. distortion (129). Therefore, although the thymidine photoreaction Interestingly, a careful analysis of UVC-irradiated calf thymus in ice produces both 5R- and 5S-SPSIDE, only the 5R-isomer can DNA as a dry film indicated that 30% of thymine dimers (mainly be formed in the spore genomic DNA. CPD and SP) are formed between thymine residues located on different DNA strands, with the major interstrand photoproduct being SP (135). The presence of interstrand dimers was revealed SP formation in DNA or thymidine films after DNA digestion using a combination of nuclease P1, alka- Besides photoreaction in ice, SP may also be formed in dry line phosphatase and phosphodiesterases I and II, with product DNA films, where DNA molecules exist as fibers. The A ⇔ B analysis via HPLC-MS/MS. As shown in Fig. 6, the exonucle- conformational change in DNA fibers as a function of DNA ases do not cleave the phosphodiester bonds in the intrastrand hydration level is well documented (130,131). Thus, in the pres- dimer, but readily cleave these bonds in interstrand dimers. How- ence of sodium ions, DNA fibers adopt the A-form at 40–88% ever, if the DNA is hydrolyzed with strong acids at elevated relative humidity (RH) and the B-form at >88% RH (130). As temperatures, such as was used in previous photoproduct analysis shown by Rahn et al., when irradiating an E. coli genomic DNA on UV-irradiated spores, both inter- and intrastrand dimers are film prepared via evaporation of an aqueous DNA solution on converted to the same SPBASE and become indistinguishable. quartz plates under high RH, CPDs were the dominant DNA Some DNA in dry films may also exist in denatured states, espe- photoproduct (132). As RH decreased, the DNA underwent con- cially at low RH (130,131), and the formation of interstrand SPs formational changes to a potential A-form, formation of CPDs may come from denatured DNA. However, DNA in spores is was suppressed and formation of SP increased dramatically saturated with SASPs, which likely stiffen the duplex DNA and (132). It was also found that at a very low RH level, DNA likely prevent the occurrence of photoreactions other than the formation adopts a denatured conformation leading to suppression of for- of intrastrand SP. Notably, yields of interstrand SP were <1% of mation of both thymine photoproducts, although compared with intrastrand SP in UVC-irradiated hydrated spores or a dry or SP, the reduction in CPD formation was much larger (132). hydrated SspC–DNA complex (5). Spore photoproduct formation in dry films was also observed Besides thymidines in duplex DNA, dry films of monomeric with short oligonucleotides, such as a 35-mer duplex (133). Air thymidine can also give rise to SP photochemistry (5). The drying of the aqueous solution of the 35-mer on Saran wrap thymidine can be dissolved in methanol or ethanol (135), since resulted in a single DNA layer. UV irradiation of this layer at solvent evaporation gives much better films than are prepared via 10% RH produced SP-containing strands; formation of other thy- lyophilization, perhaps because better thymidine stacking interac- mine dimers was not investigated. By preparing the film via tions are obtained with solvent evaporated films, resulting in freeze-drying an aqueous buffer containing calf thymus DNA, much cleaner SP formation. In photoreactions conducted in Douki et al. found that irradiation of the dry film produced CPDs frozen aqueous solution (135), SP accounts for ~38% of the thy- at a yield 5- to 10-fold higher than that of SP (63,134). The SP midine dimers formed, with CPDs being the dominant products. yield is much lower than that found by Rahn et al. in the earlier In photoreactions with a dry film prepared via lyophilization, the work (132), suggesting that the method for DNA film preparation percentage of SP in all thymine dimers was lower at ~27%. In may play a major role in determining the thymine stacking pattern contrast, with a thymidine film prepared via ethanol evaporation,

dR P dR P dR P dR P dR B acid B B T B B hydrolysis (normal bases) + B T B T T TT dR P dR P dR P dR P dR (Inter- + intra-strand thymine dimers) enzymatic digestion

T T T T B ++ dR dR dR dR dR P (normal (intra-strand (inter-strand nucleosides) thymine dimers) thymine dimers) Figure 6. Enzymatic and acid hydrolysis of duplex DNA containing inter- and intrastrand photoproducts (135). B, normal base; dR, 20-deoxyribose; P, phosphate. The phosphate in intrastrand dimers cannot be cleaved by enzymatic digestion while all phosphates are hydrolyzed by acid, providing an assay to distinguish these two types of DNA photoproducts. Photochemistry and Photobiology, 2015, 91 1271

SP is the dominant product, accounting for ~80% of the dimers residues, and it may not be practical to employ large genomic or formed (135). plasmid DNAs which contain thousands of nucleotides for SP As described above, the thymidine photoreaction generates a mechanistic studies in a reaction that features atom movements pair of SPSIDE diastereoisomers, with the C5 chiral center adopt- and C-C/C-H bond formation. Rather, the ideal system for such ing an R or S configuration. With thymidine in ice or in films studies seems likely to be thymidine monomers or the dinu- prepared via lyophilization, roughly equal amounts of the 5R- cleotide TpT. and 5S- SPSIDE isomers are produced (124). In contrast, using films prepared via methanol or ethanol evaporation, the 5S- Earlier deuterium labeling studies SPSIDE isomer becomes the dominant product and the 5R-isomer is a very minor species (~20:1 ratio) (135). Such a product distri- After determining the structure of SP by NMR spectroscopy, bution pattern indicates that the thymidine stacking conformation Varghese et al. rationalized that SP is likely formed through a is different in these two types of films, resulting in different consecutive mechanism via combination of a 5-a-thyminyl and a SPSIDE diastereoisomers upon UV irradiation. 5,6-dihydrothymin-5-yl radical (7,123). To verify this intriguing hypothesis, it is important to know how these radicals are gener- ated. Therefore, Cadet and co-workers utilized a clever approach SP formation in aqueous solutions employing a labeled d3-thymidine residue containing a –CD3 Successful SP photochemistry requires DNA to possess a moiety and followed the deuterium during the SP photoreaction. reduced hydration level, and such a condition is difficult to fulfill After conducting the thymidine photoreaction on ice to generate in aqueous solution. Consequently, UV irradiation of DNA in SP, they purified the SPSIDE product probably as a mixture of common buffers generates no detectable SP. However, SP photo- the 5S and 5R diastereoisomers. Analyses of the SPSIDE structure chemistry in aqueous solution can be achieved by binding a/b- via NMR spectroscopy found that only two-thirds of the gener- type SASP to DNA to achieve the DNA conformation required ated SPs possessed a deuterium on the C6 carbon. Although this for SP photochemistry. At moderate or low concentrations of salt finding indicated that a deuterium from the –CD3 moiety and with an ~3:1 weight ratio of SASPs/DNA, a DNA/SASP migrated to the C6 position during SP formation, such a conclu- complex can be generated that mimics the DNA environment in sion was undermined by the incomplete deuterium incorporation a germinated spore when the spore core has been rehydrated and observed, leading to a conclusion that the origin of the two H6 CaDPA has been released (92). In this nucleoprotein complex, atoms was unclear (138). Based on these data, a concerted reac- the DNA is still in an A-like conformation (89). Consequently, tion mechanism was proposed, where formation of the C6-H and UVC irradiation of this SASP-DNA complex at pH 7.0 in buffer C5-C bonds in SP occur simultaneously, possibly via a four- produces SP as the dominant product, with a ratio between SP member ring transition state (Fig. 7A), and the 5-a-thyminyl and and CPD formed that is close to 5:1. Such a reaction pattern is 5,6-dihydrothymin-5-yl radicals are not involved as reaction close to that found in dormant spores, although the yield of SP intermediates (40,138). as a function of UV fluence is more than an order of magnitude lower, probably due to the absence of CaDPA. When the nucleo- In the dinucleotide TpT protein complex was irradiated with UVC in the dry state, CPD formation was further suppressed and a SP/CPD ratio of >40/1 To further investigate the mechanism of SP formation, Lin et al. was observed (92). repeated the deuterium labeling experiments in a dinucleotide con- Another means to obtain A-like DNA conformation support- text (139). Two dinucleotides were used in their studies: a d3-TpT ing SP photochemistry is to add ethanol to an aqueous solution, (6) containing a –CD3 moiety at the 3ʹ-thymine and a d4-TpT (8) since DNA adopts an A-form in 80% ethanol (136,137). Early with all four carbon-based hydrogen atoms on the 50-thymine base studies by Patrick and Gray indicated that even in 80% ethanol, being replaced by deuteriums (Fig. 8). UVC irradiation of dry only a tiny amount of SP was produced with CPDs being the films prepared by methanol evaporation led to deuterium-labeled major thymine dimer formed (109). More recent analysis of SPTIDEs 7 and 9, which were purified by HPLC and analyzed by DNA’s photoreaction in 80% ethanol by Douki et al. using the NMR spectroscopy. Unlike the previous thymidine labeling stud- HPLC-MS/MS assay found that the intrastrand SP accounted for ies, LC-MS analysis of the resulting SPTIDEs did not reveal any ~30% of all thymine dimers formed, as well as other dimers deuterium loss in either reaction. The NMR data reveal that in the including three CPD isomers adopting cis-syn, cis-anti and trans- d3-TpT photoreaction, a deuterium is stoichiometrically trans- anti configurations (135). Interstrand SP was also obtained at a ferred from the 3ʹ-CD3 group of TpT to the H6proS position in the yield of ~10% of that for the intrastrand SP. These observations d3-SPTIDE 7. In the d4-TpT photoreaction, a protium transfer to indicate the presence of a wide-range of DNA conformations in the H6proS was observed, with the D6 at the 5ʹ-thymine occupying the ethanol solution, highlighting the importance of the precise the H6proR position of the resulting d4-SPTIDE 9. Such observa- state of the spore core and/or binding interactions between tions indicate that UV irradiation likely excites the 5ʹ-C5=C6 bond SASPs and DNA to ensure that intrastrand SP photochemistry is into a diradical, which then abstracts an H atom from the methyl the dominant DNA photoreaction in spores. moiety of the adjacent 30-thymine residue, resulting in 5-a-thymi- nyl and 5,6-dihydrothymin-5-yl radicals which then combine to REACTION MECHANISM FOR SP yield SP (route (i), Fig. 7B). Moreover, the protium transfer in the FORMATION d4-TpT photoreaction was found to be 3.5 times faster than the deuterium transfer in the d3-TpT-mediated SP formation. Such a The progress made on in vivo and in vitro SP studies has paved primary deuterium isotope effect indicates that the H-atom the way for detailed mechanistic investigations of SP photochem- abstraction step before the two radical intermediates are formed is istry. Spore photoproduct formation only involves two thymine involved in the rate-determining process. 1272 Peter Setlow and Lei Li

(A)

O O O O O H H O HN HN NH HN hv HN (R) NH O N H N O O N O N O N H proS N O HproR 5' 5' 3' 5' 3' 3' SP

(B)

O O H H O HN HN H NH O N hv H O N + N O

(i) (ii)

O H O O H NH HN HN H N O O N 6proS N H6proS H + + O 6proR H6proR

O O O O HN NH HN NH (R) (S) H N N O N 6proS N O O H6proS O H6proR H6proR

5R-SP 5S-SP Figure 7. (A) The concerted mechanism for spore photoproduct (SP) formation (40,138). The breakage of the C-H bond in the methyl moiety and the formation of the two new bonds in SP are suggested to occur simultaneously, potentially via a four-member ring transition state. (B) The consecutive mechanism for SP formation (7,139). The C6 radical abstracts an H-atom from the methyl group, generating a 5-a-thyminyl radical, which combines with the 5,6-dihydrothymin-5-yl radical to generate SP. The right-handed helical structure of duplex DNA ensures that only route (i) is possible in spores, resulting in the 5R-SP. In a solid state photoreaction using monomeric thymidine, route (ii) also occurs, resulting in dinucleoside 5S-SP.

In addition, when SP photochemistry is examined in dry films Re-examination of earlier deuterium-labeling studies prepared by methanol evaporation, the deuterium discrimination Why does the same labeling strategy result in the two different still exists, albeit to a much smaller extent. Analysis of the outcomes described above? To determine the reasons for this dis- resulting 5R- and 5S-SPSIDEs formed in d3-thymidine photoreac- crepancy, the Li and the Cadet groups reinvestigated the thymi- tion by NMR spectroscopy confirms that a deuterium is trans- dine photoreaction in both ice and a dry film (124). Their data ferred exclusively from the methyl group to the H6proR position exclude the possibility that deuterium was washed away by in 5R-SPSIDE, but to the H6proS position in 5S-SPSIDE. Therefore, exchanging with protium from the environment. Instead, they observations from both dinucleotide TpT and thymidine photore- found that deuterium is largely discriminated against during actions support the same reaction mechanism (Fig. 7B). It is also SPSIDE formation in ice, as in the formation of 5R-SPSIDE, a 17- worth mentioning that to the best of our knowledge, the SP fold deuterium discrimination over protium was observed. Gener- photoreaction is the only example known to date in which a pho- ally, the commercially available deuterium reagents are ~98% toexcited nucleotide residue exhibits a diradical feature. pure and the ~2% protiated impurities usually do not cause sig- Although the reaction mechanism for SP formation is largely nificant problems. However, with the 17-fold discrimination established as noted above, some questions still remain. One of against deuterium exhibited in the SP photochemistry, the 2% them concerns the kinetic isotope effects (KIEs) observed in SP protiated thymidine residues in d3-thymidine would become 34% formation. It was shown that the 5R-SPTIDE formation in the din- in the SPSIDE generated. Such an unusually large protium enrich- ucleotide TpT photoreaction in a dry film exhibits a KIE of 3.5, ment offers a reasonable explanation of the previous observation the KIE becomes 17 1.5 in thymidine photoreaction in ice that only two-thirds of SPs formed contained a deuterium at the and 6.5 1.5 in thymidine photoreaction in a dry film (124). C6 position. Moreover, although the 5R- and 5S-SPSIDEs are generated by the Photochemistry and Photobiology, 2015, 91 1273

(A) O O D2 O NH C 5' HN NH HO N O O 5' O D C HO O N DproS N O 3 NH 254 nm, 45 min O HproR O OH O N O OH O P dry film O O O P O 0.1% OH O 3' OH d3-TpT (6) 3' d3-SPTIDE (7)

O (B) O CD O D C 3 3 NH HN NH 5' O 5' HO D N O 254 nm, 45 min HO O N HproS N O O NH O DproR O OH dry film OH O O N O O O P O P 0.4% OH O O 3' OH 3' d4-TpT (8) d4-SPTIDE (9) Figure 8. H-atom transfer during spore photoproduct (SP) formation as revealed by the formation of deuterated dinucleotide SPs via solid-phase photoreaction using selectively labeled dinucleotide TpTs (139).

same mechanism, the KIEs exhibited by 5S-SPSIDE photochem- UVC light can only penetrate several nanometers into the film. istry are different from those found in 5R-SPSIDE formation Therefore, only the thymidine residues close to the surface can (124). Currently, these KIEs are tentatively ascribed to the pri- be excited sufficiently to react. mary isotope effect during the H-atom abstraction process, with To circumvent the problem of UVC penetration of dry films, the large effects exhibited in the thymidine photoreactions indi- Jian et al. examined SPSIDE formation in thymidine microcrys- cating that hydrogen atom tunneling effects may be involved in tals, where the thymidine residues adopt a homogeneous stacking SP photochemistry. As the tunneling effect is affected by the dis- conformation across the whole crystal lattice (Fig. 9A). To tance between the two reacting moieties (140), the different KIEs increase the reactive surface area, they suspended the thymidine observed may be ascribed to the various distances resulting from microcrystals in an organic solvent such as methyl t-butyl ether the different stacking interactions in free thymidine or in the din- and irradiated the suspension by unfiltered UVB light centered at ucleotide TpT (124). However, this hypothesis needs further 302 nm under vigorous stirring (144). The homogeneous thymi- experimental examination. dine stacking conformation in the crystal lattice inhibits the for- mation of other thymine dimers, resulting in 5S-SPSIDE as the dominant photoproduct. After a 32-h reaction, the yield of 5S- Reaction in thymidine microcrystals SPSIDE was found to be ~85%. Because thymidine residues likely The remaining KIE issue highlights the importance of DNA con- take a random order to dimerize, the reaction yield relies on the formation controlled by nucleobase stacking interactions on the dimerization order. If every adjacent thymidine pair takes turns outcome of DNA photoreaction. As pointed out by Schreier to dimerize, a 100% conversion to SP is expected. In contrast, if et al., the timescale for excited thymine residues to dimerize is dimerization occurs at the first two of every three thymidine resi- in the range of picoseconds (113), and it has been generally dues, the third thymidine will be left between two formed accepted that the original DNA conformation determines the SPSIDEs and remain intact, resulting in the lowest possible yield DNA photoproducts (113,141–143). In genomic DNA, the mil- at 67%. The actual yield of 5S-SPSIDE is expected to fall in lions of nucleobases may adopt an equally large number of between the maximum and minimum yields, with 83%, the aver- stacking conformations at any given time, with the vast majority age, the most probable choice. Therefore, the ~85% 5S-SPSIDE of these conformations being unreactive. Such a problem is even formation with 302 nm UV irradiation represents the optimal more significant in SP photoreaction in vitro which often occurs yield in the dimolecular dimerization reaction. in the solid state where conformational changes due to thermal Analysis of the thymidine crystal structure solved by Young motion are restricted. As a consequence, most in vitro SP pho- et al. (145) also reveals that the methyl group of one thymine toreactions exhibit extremely low yields, typically lower than 1% residue (a, Fig. 9B) is located right above the other thymine ring after a prolonged UVC irradiation. On the other hand, in some (b, Fig. 9B), and the shortest distance between an H-atom of the dry film reactions, in particular with film prepared by alcohol –CH3 moiety and the C6 of the adjacent thymidine is only evaporation, 5S-SPSIDE is the dominant product produced, sug- 3.178 A. As indicated by the SP mechanism in Fig. 7B, the C6 gesting that thymidines adopt a nearly homogeneous conforma- radical formed after photoexcitation of the C5=C6 bond is pro- tion in films prepared by alcohol evaporation. However, the jected to abstract an H-atom from the methyl moiety. Such an yield of SPSIDE is still lower than 1% (124,135), likely because abstraction is highly feasible in the thymidine crystal as shown 1274 Peter Setlow and Lei Li by the short distance between these moieties. In addition, the As mentioned above, a major role of CaDPA in spores is to C5=C6 bonds on adjacent thymidines are 4.86 A away from each serve as a photosensitizer. Analysis of SP formation in the dry other in the crystal lattice, making it very difficult for these bonds state at the presence of CaDPA reveals that this compound to move closer to allow for CPD formation. The C5=C6 bond is increases the yields of SP and cis-syn CPDs formed between two even further from the C4=O moiety of the adjacent thymidine, thymine residues or between a cytosine and a thymine, while and thus 6-4PP formation should also be very unfavorable. generation of 6-4PP and CPD between two cytosine residues is relatively unaffected (63). These observations indicate that for- mation of both CPD and SP involves the thymine triplet excited Triplet state photochemistry state, which likely possesses an energy level lower than the tri- The experimental data to date strongly support a reaction mecha- plet excited state of CaDPA so that the energy transfer from nism for SP formation in which H-atom abstraction by the CaDPA to thymine can readily occur. Moreover, cytosine pos- excited C5=C6 bond initiates SP photochemistry. As both the sesses the highest triplet excited state energy among the four singlet and the triplet excited states can be involved in thymine nucleobases (83). This energy state may be higher than that of photoreaction, the remaining question is which excited state CaDPA, resulting in the suppression of cytosine dimer formation plays a major role in SP photochemistry. in UV-radiated spores. The involvement of the thymine triplet excited state in SP for- mation is further supported by results of a dry state thymidine photoreaction in the presence of the pyridopsoralen derivatives, (A) 7-methylpyrido[3,4-c]psoralen (MePyPs), pyrido[4,3-c]psoralen (H-PyPs), and 7-methylpyrido[4,3-c]psoralen (2N-MePyPs), under 365 nm UVA irradiation (146). These reactions produce four CPD isomers possessing the cis-syn, trans-syn, cis-anti, and trans-anti configurations and the 5R- and 5S-SPSIDE isomers. Yields of these dimers depend on the nature of the photosensi- tizer employed, with H-PyPs being the most efficient, followed by MePyPs and 2N-MePyPs. Cyclobutane pyridimine dimer and SPSIDE formation was not observed when photosensitizers, such as 3-carbethoxypsoralen, 5-methoxypsoralen and 8-methoxypso- ralen, which possess a triplet excited state energy lower than that of the thymine, were used. The involvement of the thymine triplet excited state in SP formation was further supported by a recent DFT calculation by (B) Du et al. (82). This calculation shows that any reaction route involving the singlet excited state S1, the triplet excited state T1 or the singlet ground state S0 alone is energetically unfavorable. The favorable reaction route involves a triplet excited state T1 which enables H-atom abstraction from the thymine methyl group as the rate-limiting process to form the 5-a-thyminyl and 5,6-dihydrothymin-5-yl radicals. The resulting diradical interme- diate at the triplet state is very unstable, and interacts with the singlet ground state to undergo triplet–singlet surface crossing to reach the singlet ground state S0 before radical combination occurs, yielding SP. Therefore, the reaction features a consecu- tive reaction mechanism, as originally proposed by Varghese (7). Moreover, the calculation also shows that with the concerted mechanism shown in Fig. 7A, no triplet excited states can be located. Singlet excited states may be involved, but then the reaction has to go over a hefty energy barrier of 70.4 kcal mol1, which is in sharp contrast to the barrier of 14.2 kcal mol1 in the consecutive mechanism involving the triplet excited state. Taken together, in the presence of CaDPA, the triplet excited state is likely to be solely responsible for SP formation in bacterial spores.

PROGRESS ON SP SYNTHETIC CHEMISTRY Figure 9. (A) Molecular packing in the thymidine single crystal (145). The distance between two thymidine rings is ~3.1 A, close to the 3.36 A As discussed earlier, yields of SP in DNA photoreactions in vitro average rise found in the SASP-oligo(dG)•oligo(dC) nucleoprotein (106). are generally very low. In most cases, not only is SP always a (B) The shortest distance between an H atom in –CH3 and the C6 of ~ minor product, but other thymine dimers are also formed. It is another thymine is 3.2 A (blue dotted line), which is close to the 3.4 A fi found in the molecular simulation (106), and supports the role of the key also very dif cult to separate an SP-containing oligonucleotide H-abstraction step in initiating spore photoproduct formation (124,139). from oligonucleotides containing other lesions by HPLC. There- Photochemistry and Photobiology, 2015, 91 1275 fore, unlike preparation of oligonucleotides containing CPD or 6- protected by other reagents (126,127,149,150). Separation of the 4PP via UV irradiation in aqueous solution, it is impractical to protected R and S-diastereomers by flash chromatography fol- utilize photochemistry to prepare large amounts of homogeneous lowed by deprotection of the resulting 5R-isomer readily yields SP-containing oligonucleotides. The latter difficulty may explain the 5R-SPSIDE (route (a), Fig. 10), which proved useful in the the lack of studies to reveal the SP repair efficiency by the NER elucidation of the mechanism of the SP repair enzyme SPL pathway and the mutagenic effects of SP in germinated spores. (24,127). Alternatively, selective deprotection of the hydroxyl Consequently, an effective method for SP incorporation into groups on the deoxyribose allows the functionalization of the an oligonucleotide is essential in order to understand intimate 30-OH at the 30-side of the dinucleoside into the 2-cyanoethyl-N, details of SP photochemistry and photobiology. N-diisopropyl phosphoramidite, yielding the SPSIDE phospho- ramidite as shown in route (b) (129). Moreover, by using 2- chlorophenyl dichlorophosphate to react with the selectively General strategy for SP synthesis deprotected dinucleoside SP, the phosphodiester moiety can be Synthesis of the SPBASE was achieved by Bergstrom et al. inserted producing the protected SPTIDE (route (c), Fig. 10). shortly after the chemical structure of SP was determined SPTIDE and SPTIDE phosphoramidites can be subsequently pre- (147). Begley and coworkers later synthesized SPBASE deriva- pared from this protected SPTIDE precursor. tives via different strategies (21,148). Two routes were adopted in these studies: connection of two pyrimidine base derivatives Synthesis of dinucleoside and dinucleotide SP or building the 5,6-dihydropyrimidine skeleton onto a pre-exist- ing pyrimidine base. The first route developed in these early The first SPTIDE was synthesized by Kim et al. (150), which also studies provided the basis for later syntheses of SPSIDE and provided a general strategy for later SPTIDE syntheses. In Kim’s SPTIDE. approach, the 5R- and 5S-SPs were not separated until the The SPSIDE and SPTIDE as well as their phosphoramidites very last step. However, most recent syntheses separated these were prepared via similar strategies. The scheme used by most isomers via flash chromatography right after the coupling step of these syntheses is summarized in Fig. 10, which features cou- (127,139,149). A reaction scheme for SPTIDE synthesis is shown pling the enolate of dihydrothymidine with bromomethyl deox- in Fig. 11 which is modified from Kim’s procedure to reflect the yuridine to give a pair of dinucleoside SP diastereomers with latest trend. The N3 atoms on the thymine ring are protected by both N3 atoms protected by [2-(trimethylsilyl) ethoxy]methyl SEM. Both the 3ʹ-OH and 5ʹ-OH at the bromomethyl deoxyuri- acetal (SEM) and the hydroxyl groups on deoxyriboses being dine were protected by tert-butyldimethylsilyl ether (TBS), while

O O O SEM N SEM SEM N N Rh/Al O , H (S) O 2 3 2 R O N O 1 O N N O O R1O OR3 NH LDA, THF, O O OR2 -78 oC HO N O OR2 OR4 O O + SEM OH Bromination by Br N O O Br2 or NBS SEM SEM N N N R3O O (R) O O N N O R1O OR3 O O OR4

OR2 OR4

Separation Cl O O O O O O O P Cl Cl HN (R) NH HN NH O O O (R) HN (R) NH SEM SEM OH O N N O N N (c1) N N OH O O OR5 O N N O (R) Deprotection O O O O N N O HO O O R1O OR3 OH O O O O O Route (c) O O Route (a) P O P O - OH O OR OR OR6 2 4 SP (3) OH OR4 SIDE SPTIDE (2) O O O O SEM SEM (c2) N (R) N HN (R) NH Route (b) R O O N N O OR N N 5 3 OR5 O O O O O O OR O O O O 2 P P CN i-Pr) N OR O ( 2 7 P CN

SPSIDE phosphoramidite N(i-Pr)2

SPTIDE phosphoramidite Figure 10. Summary of a synthetic strategy commonly adopted for the syntheses of dinucleoside and dinucleotide spore photoproduct as well as their phosphoramidite. Here, both N3 atoms on the thymine ring are protected by [2-(trimethylsilyl) ethoxy]methyl acetal (SEM). 1276 Peter Setlow and Lei Li both hydroxyl groups were protected by triethylsilyl ether (TES) used for the 5R-CH2SP synthesis was similar to the SPTIDE syn- at the dihydrothymidine before they are coupled to form a pair thesis shown in Fig. 11, the key protection/deprotection strate- of protected dinucleoside SP diastereomers 14a and 14b. Only gies were different due to the instability of the formacetal the 5R isomer 14a was indicated to be formed in vivo due to the linkage (128). Consequently, SEM was no longer used as the right-handed helical structure of DNA, which prohibits the for- protecting reagent for the thymine N3 atom as the SnCl4-medi- mation of the 5S-SP (150). After column chromatography to sep- ated SEM deprotection also detaches the formacetal linker. The arate these two isomers (127,149), deprotection of the resulting benzyloxymethyl group (CH2OBn) was therefore chosen for N3 14a readily affords the 5R-SPSIDE as indicated by the route (a) protection, which increased the diastereoselectivity of the 5R in Fig. 10 (127). Alternatively, following the route (c1)in (24a) over the 5S isomer (24b) to 2.6: 1, in contrast to the Fig. 10, both TES groups of 14a were removed before the 5ʹ- nonselective 1:1 mixture (13a vs 13b) obtained in the SPTIDE OH was protected by dimethoxytrityl (DMTr) (150). The 3ʹ-OH synthesis (150). After removing the CH2OBn moiety via oxida- was then allowed to react with 2-chlorophenyl dichlorophosphate tion using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) to introduce the phosphodiester moiety into the protected dinu- followed by deprotection, the 5R-CH2SP was readily obtained. cleotide SP 17. Deprotection of the resulting compound yields SP 2. TIDE Synthesis of dinucleoside SP phosphoramidite The synthesis of SPTIDE analog 5R-CH2SP (31) containing a formacetal linker adopted by Lin et al. is shown in Fig. 12. A The synthesis described above provides the foundation for the formacetal is the simplest and smallest nonchiral isostere for a synthesis of SPSIDE phosphoramidite, making its incorporation phosphate moiety and has proved very useful in oligonucleotide into an oligonucleotide possible. As shown in Fig. 11, the syn- biochemical studies (151–156). Although the general strategy thesis of SPTIDE is lengthy and tedious, and preparation of large

O O O 1) TBSCl, imidazole, DMF SEM N Br2, CCl4 SEM NH 2) i Br N SEMCl, Pr2NEt, CH2Cl2 light O O N O SEM N O N O SEM N N 84% (S) TBSO O 53% HO O TBSO O TESO O N N O OTBS O OH OTBS OTBS (10) (11) (12) OTES O (14b) OTBS 1) H , Rh-Al O O 2 2 3 1) LDA, THF, -78 °C + (2) TESCl, imidazole, DMF SEM Separation by flash N 2) 12, -78 °C-> r.t. chromatography O O 2) SEMCl, iPr NEt, CH Cl 2 2 2 SEM TESO O N SEM N N 53% (R) 4% HF. 93% O TESO O N N O in acetonitrile OTES O OTBS 53% (13) OTES O

(14a) OTBS O O O O O O SEM SEM N (R) N SEM SEM SEM SEM N (R) N 2-Chlorophenyl N (R) N HO O N N O DMTrCl dichlorophosphate O N N O O OTBS Pyridine DMTrO 1,2,4-triazole, THF DMTrO O N N O O OTBS O OTBS OH O 81% 93% OH O O O O P OH OTBS OTBS O Cl OTBS (15) (16) (17)

O O O O O O SEM SEM N (R) N HN (R) NH HN (R) NH DMTrO O N N O HO O N N O 1) Bu NF, THF HO O N N O 4 O O NH OH 2) MSNT, Pyridine SnCl4, CCl4 4 O O O O O O P O O P O 87% O O 99% 85% P O O - O OH O OH O Cl Cl OH

SPTIDE (2) (18) (19)

Figure 11. Synthesis of the SPTIDE starting from thymidine, which was originally reported by Kim and Begley (150) and later modified by other groups (139,149). The 5S- and 5R-SP diastereomers were not separated until the last step of Kim’s synthesis, while most recent syntheses separated these iso- mers via flash chromatography right after the coupling step. The synthesis shown here was thus modified accordingly. Photochemistry and Photobiology, 2015, 91 1277 amounts of the SPTIDE phosphoramidite to enable the preparation were generated via hybrid approaches where the corresponding of SP-containing oligonucleotides is challenging. Therefore, dimers were first produced by photochemistry using partially pro- although 5R-SPSIDE 3 is not considered to be truly biologically tected dinucleotide TpT before the phosphoramidite moiety was relevant, it provides a proof-of-concept example for the prepara- introduced on the 30-OH group at the 30-end of the dimer via tion of SP phosphoramidite. Moreover, the SPSIDE-containing organic synthesis. Such an approach, however, proves futile in oligonucleotide is a substrate of SPL, the repair of which releases preparing the SPTIDE phosphoramidite, since UV irradiation of the two smaller oligonucleotide fragments, facilitating the examina- unprotected dinucleotide TpT in the solid state generates SP in tion of the SPL activity (26,157). ~1% yield (139). In addition, once the phosphodiester moiety is The strategy for the synthesis of SPSIDE phosphoramidite has protected by esterization with either –CH3 or –CH2CH2CN, the been summarized in route (b), Fig. 10. Compared with SPTIDE resulting dinucleotide no longer supports SP formation (149), sug- synthesis, the major difference in the preparation of SPSIDE phos- gesting that the negative charge carried by the phosphodiester lin- phoramidite 35 is the selective protection of the hydroxyl groups ker is essential for the two thymine residues to adopt the “right” at the 2ʹ-deoxyriboses (129). The 3ʹ-OH group of the bro- stacking conformation to enable SP photochemistry. Therefore, momethyl deoxyuridine and the 5ʹ-OH group of the dihy- the hybrid strategy cannot be applied to the preparation of SPTIDE drothymidine were protected by TBS, while the other two phosphoramidite. hydroxyl groups were protected by tert-butyldiphenylsilyl ether To prepare SPTIDE phosphoramidite in a relatively large scale, (TBDPS) before the protected dimer 32 was allowed to form Jian et al. built from the synthesis of the dinucleotide SP (149), (Fig. 13). After removal of the TBS moieties, the 5ʹ-OH group as shown in route (c2), Fig. 10. Importantly, they found that the of the dihydrothymidine was protected by DMTr before the bulky 2-chlorophenyl moiety as the phosphate protecting group phosphoramidite was introduced on the 3ʹ-OH group to give the used in the SPTIDE synthesis may create steric hindrance, pre- dinucleoside SP phosphoramidite 35. This phosphoramidite read- venting an efficient coupling reaction from occurring during the ily supports solid-phase DNA synthesis for SPSIDE incorporation, solid-phase DNA synthesis. By exchanging the 2-chlorophenyl although the yield for the SP coupling step was probably low as for a much small methyl moiety, the resulting phosphoramidite reflected by the overall 15% yield in the synthesis of a dode- 37 improved the coupling efficiency from ~15% to >90% camer oligonucleotide (129). (Fig. 14). This high coupling efficiency enables the large scale preparation of SPTIDE-containing oligonucleotides. Synthesis of dinucleotide SP phosphoramidite SP STRUCTURAL STUDIES The CPD TpT (158–165) and 6-4PP TpT (166) phosphoramidites are available, enabling their incorporation into oligonucleotides As noted above, progress on SP synthetic chemistry and photo- via standard solid-phase DNA synthesis. These phosphoramidites chemistry has now made SP photobiology studies possible.

O O O O BnO N N OBn BnOCH2Cl Br N OBn Br NH i O N N O ( -Pr)2EtN TESO DMF O TBSO N O TBSO N O TBSCl, 2 CH2Cl2 O TBSO 1. O THF, LDA TESO imidazole(BzO) Separation by flash O O 36% o chromatography NBS, , reflux OTBS -78 C (24b) 2. 4 OTBS NH CCl (20) (21) 15% OTBS 1. + N O Rh/Al HO MeOH, O O O O O 2 O 2. 3 , • TESCl, H H NH BnOCH2Cl BnO N N OBn 2 O 2 i N OBn HF Py OH imidazole ( -Pr)2EtN DMF CH3CN N O O N N O (10) TESO CH2Cl2 TESO N O TESO O O O 78% 77% TBSO OTES OTES TESO O (22) (23) (24a) OTBS O O 39% O O O O O O BnO N N OBn BnO N N OBn BnO N Ac BnO N N OBn TBDPSCl N OBn 2O, HO O N N O AcOH ppts O N N O imidazole O N TBDPSO O TBDPSO O N N O DMSO TBDPSO N O MeOH O DMF O O TBSO 62% HO HO O 87% TBSO S O 75% TBSO S O O HO O O (25) OTBS (28) OTBS (26)(OTBS 27) OTBS

O O O O O O 1. DDQ, HN NH HN NH BnO N N OBn ClCH2CH2Cl NIS, TfOH 55 oC O N N O TBAF, THF ClCH CH Cl O N N O TBDPSO O N N O 2 2 TBDPSO HO O rt O o O i 0 C 2. ( -Pr)2EtN, CH Cl 90% 42% 2 2 O O O O O O reflux O O O 84% OTBS OTBS OH (29) (30) 5R-CH2SP (31)

Figure 12. Synthesis of SPTIDE analog 5R-CH2SP (30) containing a formacetal linker (128). 1278 Peter Setlow and Lei Li

O O O O SEM SEM N (R) N SEM SEM toluene N (R) N sulfonic acid N N O DMTrCl, DMAP TBSO O O N N O O HO OTBDPS 8% O OTBDPS 66% OTBDPS O OTBDPS O OTBS OH (32) (33) O O SEM SEM N N O O (R) 2-cyanoethyl N,N- SEM SEM DMTrO O N N O N (R) N diisopropylchlorophosphoramidite O OTBDPS DMTrO O N N O 66% OTBDPS O O OTBDPS O OTBDPS O P CN i OH ( -Pr)2N

(34) SPSIDE phosphoramidite (35)

Figure 13. Key steps for the synthesis of SPSIDE phosphoramidite from the protected SPSIDE (129).

These studies have led to breakthroughs in analyses of SP struc- by DFT calculations that found ten favorable correlations for the R ture, offering much-needed insight into the impact of SP on isomer but only seven for the S isomer. Taken together, these stud- genomic DNA. ies strongly indicate that the C5a chiral center in SP adopts an R configuration, agreeing with the assumption proposed by Kim et al. based on the right-handed DNA helical structure (150). NMR structure of dinucleotide SP Starting from the dinucleotide TpT and conducting DNA photo- Structure of the formacetal analog of the dinucleotide SP chemistry in dry films formed via lyophilization, Mantel et al. purified sufficient SPTIDE to elucidate its solution structure via 2D- Although the NMR spectroscopic studies indicated that 5R-SP is NMR spectroscopy coupled with DFT calculations (8). As shown the biologically relevant species, a crystal structure allowing in Fig. 15A, using 1H-13C and 1H-31P Heteronuclear Multiple- direct observation of the atom arrangements in SP would clearly Bond Correlation (HMBC) spectroscopy, the through-bond be significant. Given that the dinucleotide SP possesses the size correlations in SP TpT were determined, revealing the enchain- of a small organic molecule, it may be crystalized via solvent ment of different chemical groups (thymine, deoxyribose and evaporation, a method widely used for small molecule crystal- phosphate). The results unambiguously demonstrate that SP is lization. However, the negative charge associated with the phos- formed via addition of the 30-methyl moiety to the C5=C6 bond at phate moiety may prevent crystal growth as reflected by the fact the 50-thymine residue, excluding the involvement of the 50-methyl that all CPD dimers crystallized to date contain either no or a group in SP photochemistry. In addition, after analyzing the neutral linker between the two T residues (155,167). Formacetal through-space 1H-1H interaction via Rotating frame Overhauser is a nonchiral isostere for a phosphate moiety which was used to Effect SpectroscopY (ROESY) experiments, it was found that allow for crystallization of a dinucleotide CPD formed between 0 H6aproS displays a weaker coupling to the methyl group at the 5 - two uracil residues (155). After synthesizing the 5R-SP analog dihydrothymine moiety than does H6aproR, indicating that the C6a containing a formacetal linker with its steric configuration indi- position is located above the sugar ring (Fig. 15B). In addition, cated by 2D-NMR spectroscopy, Lin et al. dissolved the result- the strong interaction between H6aproR and the methyl group ing CH2SP in water, and obtained a single crystal via solvent unequivocally proves that the configuration of the C5a carbon is R evaporation (128). The solved structure of 5R-CH2SP (Fig. 16) because if an S configuration is encountered, the proton H6aproR reveals that the two thymine rings are connected by a methylene could not display strong coupling signals both to H30A and to pro- bridge. The C5 carbon at the 5ʹ-thymine moiety clearly adopts tons of the methyl group. Such an observation is further supported an R configuration, confirming the assignment by previous NMR

O O O O O SEM SEM O N SEM CN N SEM N Cl N DNA HN P O NH NaOMe O N DMTrO N O i DMTrO O N DNA MeOH N( -Pr)2 N O synthesis O O N O O N O 18 DIPEA, DCM O 90% >90% O O O 85% O O O O P O P O coupling yield O O P O MeO OH MeO O HO P O O (36) i CN N( -Pr)2 DNA SP TIDE SP TpT containing oligomer phosphoramidite (37)

Figure 14. Synthesis of dinucleotide spore photoproduct phosphoramidite from the penultimate intermediate during the synthesis of SPTIDE shown in Figure 11 (149). Photochemistry and Photobiology, 2015, 91 1279

(A) (now Geobacillus stearothermophilus) DNA polymerase I (B. st. Pol I; Fig. 17) (129). The structure shows that the 5R-SPSIDE 3 O H O fits quite well into the DNA duplex framework, forming two nearly perfect Watson–Crick base pairs with the apposed adenine CH3 H residues. As a control, they also obtained the structure of the HN NH strand with identical sequence except replacing SP by undam- H aged TpT. These two structures can be almost perfectly superim- posed, suggesting that the 5R-SPSIDE does not induce much local distortion. Such an observation may partially reflect the property N H O H N O of SPTIDE in duplex DNA. However, due to the lack of phospho- HO ' " ' diester bonds in the dinucleoside, the two thymine residues may H3 H H O 5 5 freely rotate, releasing any distortion resulting from the presence ' of SP. Also, much of the oligonucleotide was embedded in the H O B. st. Pol I protein. As indicated by the analysis using a software O 1 O O package named 3DNA (168,169), the duplexes exhibit an A-con- P formation near the polymerase active site. The unusual groove OH H1' width and helical forms indicate that the conformation may be - controlled by the protein framework, but not by the nucleic acid O sequence. Therefore, the structure revealed by the SPSIDE-con- taining DNA may differ somewhat from that in the SPTIDE-con- (B) taining genomic DNA in germinated spores.

O H O S HR – H3C Structure of the dinucleotide SP in a duplex strand protein complex HN 5a 5b NH H6aproS 6a 6b Employing a 16-mer oligonucleotide prepared via solid-phase H DNA synthesis using SP phosphoramidite, the duplex was 5'/5" O N N O TIDE HO H6aproR H3' H2" crystalized as a complex with the N-terminal fragment of Molo- O 4' A ney murine leukemia virus reverse transcriptase (MMLV RT) H ' 1 (170). In the 16-mer sequence used, the trimers at the ends are O O O recognized by the MMLV RT fragment and the internal 10-mer P 5'/5" O sequence can be variable. The central 10 base pairs in this 16- - 4' B 1' O 3' 2'/2" mer is thus free of interactions with the protein, allowing the OH nucleic acid to adopt a structure dictated by the sequence. The 5R-SPTIDE sequence used also needs to be symmetric against the middle

1 13 1 31 point of the 16-mer sequence, as the 8 base pairs are a part of Figure 15. (A) Structure of SPTIDE determined from H- C and H- P Heteronuclear Multiple-Bond Correlation (HMBC) spectroscopic studies (8). From the through-bond correlations revealed by HMBC, the enchain- ment of different chemical groups (thymine, sugar, and phosphate) in spore photoproduct was determined. The groups exhibiting strong HMBC signals are indicated by the same color. (B) Summary of key NOESY cross-peaks determined from the NMR spectra of SPTIDE (8,128). spectroscopic studies (8). The 30-thymine ring in SP is planar, and the 50-dihydrothymine is distorted owing to the loss of C5=C6 bond and ring aromaticity. Moreover, the distance between the H6proS and the bridging methylene carbon was found to be 2.63 A in the SP structure, which is 0.7 A longer than that between the H6proR and the methylene carbon. This observation supports the conclusion in previous labeling studies that a H atom from the methyl group on the 3ʹ-thymine migrates to the H6proS position during 5R-SP formation and that SP is formed via an intramolecular H-atom transfer process (139).

Structure of a dinucleoside SP in a duplex strand–protein Figure 16. X-ray structure of the 5R-CH2SP with the nitrogen and oxy- gen atoms labeled (128). The structure clearly shows that the C5 chiral complex fi center adopts an R con guration. The distance between the H6proS and the methylene carbon (blue dotted line) is 2.63 A and that between the The synthesis of the dinucleoside SP phosphoramidite enables its incorporation into an oligonucleotide. Using a duplex dodecamer H6proR and the methylene carbon (red dotted line) is 3.36 A, which sup- ports the reaction mechanism in which the H6proS of spore photoproduct with SP at positions 6 and 7 of one strand, Heil et al. obtained (SP) originates from the methyl group in 30-thymine of the TpT step its structure in complex with the Bacillus stearothermophilus before SP formation. 1280 Peter Setlow and Lei Li

(A) trol, the 16-mer duplex with SPs replaced by undamaged TpTs was also crystalized to facilitate structural comparisons. As shown in Fig. 18, the SPTIDE-containing 16-mer duplex adopts a right-handed helical conformation with continuous base stacking (170). However, significant unwinding at the SP step followed by overwinding at the next three nucleotides was observed, preventing the duplex from adopting a perfect B-con- formation. In contrast, in the undamaged oligonucleotide 16-mer duplex complexed with the MMLV RT crystallized as a control, the DNA adopts a perfect B-conformation throughout the entire sequence (170). Surprisingly, all hydrogen bonding interactions between SP and apposed adenines are maintained; the bonding distances are even ~0.5 A shorter than those with undamaged TpT. This result is in contrast to the drastically weakened hydro- gen bonds between the 5ʹ-thymine residue in CPD and its com- plementary adenine as revealed by an X-ray crystallographic study (172). On the other hand, the unwinding at the 5ʹ-T of SP followed by overwinding results in the widening of the minor groove by ~3 A, from 9.7 A in the undamaged DNA strand to 12.5 A in the SP-containing strand. These local structural changes may explain the thermal destabi- lization induced by the presence of SP as observed by a DNA denaturation study (149). By comparing the thermal denaturation curves of SPTIDE-containing oligonucleotide duplexes with those of the corresponding undamaged duplexes, Jian et al. found that 1 SPTIDE destabilizes the duplex by 10–20 kJ mol , with the pre- cise extent of destabilization dependent on the oligonucleotide sequence examined (149). A cyclobutane pyridimine dimer with a (B) cis-syn conformation is known to destabilize an oligonucleotide duplex by 5.7–8.4 kJ mol1 (173,174). Therefore, barring effects of the sequence difference between oligonucleotides used in these different studies, the larger Gibbs free energy change induced by SP indicates that even though SPTIDE maintains all hydrogen bonding interactions with opposite adenine residues, it collec- tively disturbs the duplex local structure more than cis-syn CPD.

IMPACTS OF SP ON THE GENOMIC DNA

Mutagenesis potential indicated by the thymine dimer structures The X-ray crystallographic structure of a cis-syn CPD-contain- ing oligonucleotide has been solved in a duplex decamer sequence (172); the solution-state NMR structure of a dodecamer duplex with a cis-syn CPD is also available (174). Although 6-4PP-containing oligonucleotides have been crystal- lized when complexed with a 6-4PP antibody (175), the 6-4PP photolyase (176) and the nucleotide-excision repair protein DDB1–DDB2 (177), respectively, the 6-4PP lesion was flipped out of the duplex context into the protein binding pocket in all Figure 17. (A) Crystal structure of the dinucleoside 5R-SP-containing structures. Therefore, how 6-4PP alters the local structure of duplex complexed with B. st. Pol I (pdb entry 2YLJ) (129). (B) Superim- the duplex DNA was not revealed. Luckily, the solution-state posed structures of the undamaged DNA (pdb entry 1NJY) and the dinu- cleoside SP-containing DNA bound to B. st. Pol I (129). The DNA NMR structures of two 6-4PP-containing decamer duplexes backbone in the latter case is broken due to the lack of phosphodiester with ApA and GpA steps opposite to the 6-4PP, respectively, group in the dinucleoside spore photoproduct (SP). Except for the broken are available (178,179). Together with the SP-containing 16- backbone, the two DNA structures overlay nearly perfectly. mer duplex structure solved by Singh (170), a side-by-side comparison of the structural changes induced by these three the asymmetric component, the unique repeating unit within the thymine dimers becomes possible (Fig. 19). crystal (171). Therefore, two SPTIDEs, one in each strand, were The crystal structure shows that the cis-syn CPD bends the incorporated into the 16-mer duplex oligonucleotide. As a con- overall helical axis by 30° toward the major groove and unwinds Photochemistry and Photobiology, 2015, 91 1281

Figure 18. (A) Crystal structure of the nucleoprotein complex including two MMLV RT molecules and one 16-bp oligonucleotide duplex (170). The asymmetric unit of the crystal includes one protein molecule and eight base pairs of the duplex oligomer. (B) Structure of the 16-mer duplex strand con- taining two spore photoproduct (SP) dimers (shown in cyan) (170), where all H-bonding interactions in the duplex are maintained. The two 8-mer asym- metric units are disconnected in the figure although an integral 16-mer strand was used for crystallization. Such a fragmentation is ascribed to the artificial effect of the crystallographic data; it does not imply cleavage of the 16-mer strand. (C) Superimposed structures of the 8-bp asymmetric unit of the undamaged oligomer (in blue, pdb entry 4M95) and the SPTIDE-containing oligomer (in magenta, pdb entry 4M94) bound to MMLV RT. The minor groove is widened from 9.7 A in the undamaged strand to 12.5 A in the SP-containing strand. the helix by 9° (Fig. 19A). However, the solution-state NMR and a 30° helix unwinding at the lesion site (178). Replacing the structure reveals only minor structural changes from undamaged AA by the GA step opposite to the 6-4PP removes the local duplex DNA, including a slightly narrower minor groove, a helix unwinding; however, an overall helix bend of 27.4 1.5° small helix bend toward the major groove, and a slight unwind- was still observed (Fig. 19C) (179). Although the solution-state ing of the duplex at the lesion site (Fig. 19B) (174). Although NMR structure for SP-containing duplex oligonucleotides is these two structures vary significantly, the larger bend observed currently unavailable, the crystal structure shown in Fig. 19D in the crystal structure was indicated to result from crystal pack- indicates that SP only induces minor DNA structural changes. ing forces and the structure likely represents one of many confor- The extent of changes is probably comparable to that caused by mational states of the duplex in solution (180). Therefore, it was cis-syn CPD but much smaller than caused by 6-4PP. concluded that cis-syn CPD induces only minor DNA conforma- Such a hypothesis is further supported by the hydrogen bond- tional changes in cells. In contrast, the solution-state NMR struc- ing pattern observed within the dimer sites. As indicated by the ture reveals that the two pyrimidine rings in 6-4PP become key distances between nonhydrogen atoms, the H-bonding inter- almost perpendicular, making it impossible for simultaneous base actions associated with the 50-T are largely maintained in the cis- pairing and stacking inside the duplex (178,179). Such a drastic syn CPD crystal structure, while the H-bonds associated with the local structural alteration led to a 44° bending in the overall helix 30-T are much weakened (Fig. 20A) (172). In contrast, in the 1282 Peter Setlow and Lei Li solution-state NMR structure, the H-bonds associated with interactions with the opposing adenine residues, and the H-bonds the 50-T are weakened while those with the 30-T were main- are even shortened relative to those found between TT and AA tained. Moreover, examination of the weakened H-bonds reveals in undamaged oligonucleotides (170). that the H-bond vectors deviate from the planes of the T and A Pyrimidine photodimers are mainly recognized and repaired bases. The H-bonding changes are even more drastic with the 6- by the NER pathway in prokaryotes and eukaryotes (14–17). 4PP. Although the 50-residue, i.e. the 5,6-dihydro-5-hydrox- Nucleotide-excision repair can be divided into two subpathways: ythymine, maintains hydrogen bonds with its partner adenine, no global genomic NER (GG-NER) and transcription coupled NER hydrogen bond is present with the 30-residue of the 6-4PP. Inter- (TC-NER). Local conformational changes are thought to be the estingly, when inserting a GA step opposite to the 6-4PP lesion, major recognition elements for the GG-NER machinery (14,15). the C2=O of the 30-T form two H-bonds with the G residue 6-4PP drastically distorts the local TpT structure, which is (Fig. 20C) (179). Such an H-bonding pattern may explain the responsible for the sharp helix kink and significant local distor- A?G mutation resulting from the 6-4PP bypass event during tions at the 6-4PP site in duplex DNA (180). As a consequence, translesion synthesis. In contrast, SP maintains all H-bonding 6-4PP is readily recognized and repaired by GG-NER in live

(A) (B)

(C) (D)

Figure 19. X-ray crystallographic or solution-state NMR structures of duplex oligonucleotides containing the three thymine dimers, cis-syn cyclobutane pyridimine dimer (CPD), 6-4PP and spore photoproduct (SP) respectively. (A) Crystal structure of a decamer duplex containing a cis-syn CPD lesion (pdb entry 1N4E) (172). (B) Solution-state NMR structure of a dodecamer duplex containing a cis-syn CPD lesion (pdb entry 1TTD) (174). (C) Solu- tion-state NMR structure of a decamer duplex containing a stable GA•6-4PP mismatch (pdb entry 1CFL)(179). (D) Structure of a 16-mer duplex strand containing two SPTIDEs deduced from a nucleoprotein complex (pdb entry 4M94) (170). The crystal structure shown in Panel A was indicated to only represent one of many solution structures that was captured during the crystallization process; the solution-state NMR structure shown in Panel B thus may better reflect the local structure of a cis-syn CPD-containing DNA (180). It is also evident that 6-4PP drastically changes the duplex oligonucleotide structure; while CPD and SP only induce minor changes. Photochemistry and Photobiology, 2015, 91 1283

(A) (B)

(C) (D)

Figure 20. Zoom-in view of the two base pairs containing the thymine dimers in a duplex oligonucleotide. The distances between the nonhydrogen atoms involved in hydrogen bond formation are also shown. From Panel A (from the crystal structure) and Panel B (from the solution-state NMR struc- ture), it is clear that the hydrogen bonding interactions in one of the base pairs associated with the cis-syn cyclobutane pyridimine dimer are weakened. The 6-4PP maintains weaker hydrogen bonds with its 50-5,6-dihydro-5-hydroxythymine residue; its 30-residue does not form any hydrogen bond with the opposite adenine. However, two hydrogen bonds were found if the opposite nucleobase becomes a guanine as shown in Panel C, explaining its ten- dency to induce an A?G mutation during the translesion synthesis event. In contrast, spore photoproduct maintains all four hydrogen bonds (Panel D); these bonds may be even stronger than those formed between undamaged nucleobases as indicated by the shortened distances between the involving atoms. Again, due to the restriction of the 8-mer asymmetric units in the crystallographic data, the 16-mer strand was shown as two fragments. This “artificial” fragmentation, however, does not reflect the integrity of the 16-mer oligonucleotide and does not alter the DNA conformation.

cells (half-life of 6-4PP = 2.3 h) (181). Analysis of CPD and SP Possible DNA secondary lesions induced by SP suggests that both lesions may only result in minor local struc- tural changes in DNA, as the involved thymine residues still Besides mutagenesis, the presence of SP may physically destabi- retain the stacking conformation. Repair of CPD by the GG- lize the genomic DNA and induce secondary lesions such as NER pathway is slow; many CPDs remain unrepaired unless strand breaks. Recently, by treating the SPTIDE 2 with a strong they are located in a transcriptionally active region and are base such as 200 mM KOH, Lin et al. found that a tetrahedral repaired by the TC-NER pathway (15,16,182–184). By the same hemiaminal intermediate (38) is generated at the C4=O at the token, the smaller duplex structural changes induced by SP (170) saturated 50-thymine residue of SP. The hemiaminal species sub- indicate that SP repair by the GG-NER pathway is also likely to sequently converts to a ring-opened hydrolysis product 39 be slow. through cleavage of the pyrimidine N3–C4 bond (Fig. 21). Alter- The slow repair of CPDs indicates that these lesions are more natively, during the hemiaminal decay process, one of the C4-O likely to encounter a replicative polymerase and induce muta- bonds can be cleaved, reverting the hemiaminal species back to tions when being bypassed during translesion synthesis (184). SP. As a consequence, the original oxygen atom attached with When a CPD TpT is bypassed by human Y-polymerase Pol g, the C4=O moiety only possesses a 50% chance to be retained two “A”s can be inserted in the opposite strand in a nearly error- with the regenerated SP after one reaction cycle, and it can even- free manner (<102) (185), although another study suggests that tually be replaced by an oxygen atom from water if the reaction the accuracy rate is only 83% for both positions (186). Thus, is allowed to proceed long enough. This possibility is supported although CPD is the dominant photolesion in DNA, the vast by 18O labeling experiments, where almost all remaining SP majority of these lesions may be harmless. In contrast, human molecules carried an 18O label after a 24-h reaction using 18O Pol g is seven-fold more likely to insert a G than an A at the labeled water at pH 12 (189). Such an oxygen exchange process position opposite the 30-residue of a 6-4PP (187), indicating that mediated by the hemiaminal intermediate is facilitated by basic 6-4PP is highly mutagenic. As the repair of SP by GG-NER is conditions; however, it also occurs at physiological pH albeit at likely to be slow, SP would have a good chance to encounter a slower rate (189). error-prone Y-polymerases and induce mutagenesis during Further analysis of the ring-opened SP hydrolysis product 39 translesion synthesis. Therefore, the unrepaired SP may be highly reveals that it readily deglycosylates to 41, which further decays mutagenic in germinating spores, as suggested by a previous via the intermediate 42 to eliminate the 20-deoxyribose at the 50- competent cell study (188). Confirmation of this hypothesis residue of SP, yielding 43 (Fig. 21). Should such a process occur and the nature of the mutations generated by SP await future within an SP-containing oligonucleotide, a DNA strand break investigations. would be generated (189). Taken together, these data suggest 1284 Peter Setlow and Lei Li that due to the loss of the ring aromaticity, the C4 position at the dimers, significant formation of SP requires DNA to be less 50-thymine residue of SP becomes a “hot spot” for the formation hydrated and/or adopt an A-like conformation, while CPD and 6- of a tetrahedral intermediate, the decay of which triggers a cas- 4PP formation requires B-DNA, the conformation a duplex DNA cade of elimination reactions that can convert the SP modifica- usually adopts in aqueous solution. As a consequence, although all tion into a DNA strand break. Even though the SP-induced DNA these dimers were discovered in the 1960s, the photochemistry strand cleavage is slow under physiological pH, the process is and photobiology of CPDs and 6-4PPs, which can be readily gen- irreversible. As dormant spores of Bacillus and Clostridium erated by UV irradiation of oligonucleotides in aqueous solution, strains can survive for hundreds of years and perhaps longer have been extensively studied in vivo and in vitro. In contrast, due (190–192), this SP-induced strand cleavage would gradually to the difficulty in maintaining an A-DNA conformation in vitro, accumulate in dormant spores during this period and potentially the SP photoreaction is always a minor event, and its occurrence is become a serious DNA damage event threating spore’s survival. accompanied by many other “side” reactions. Such photochemical behavior makes the preparation of SP-containing oligonucleotides SUMMARY AND PERSPECTIVES for subsequent biological studies problematic, thus hindering the development of SP photobiology. Spore photoproduct photobiol- The seminal finding by Donnellan and Setlow 50 years ago in ogy in spores is relatively well studied, because it is the nearly their studies of UV-irradiated bacterial spores initiated photochem- exclusive photolesion formed under UV irradiation. As described ical and photobiological studies of SP. Among the three thymine in this review, the combination of low water content, the presence

18 H2 O O O 18 OH O H O 18O O HN NH HN NH HN NH O N N O O N N O HO HO O N N O O O HO O O O O O O O O O O H2O O P P O O - OH - OH P O O - OH (38) O 18 Unlabeled SPTIDE (2) O-labeled SPTIDE (40)

O O HO2C HO C H2N NH 2 NH NH2 O N N O de- HO H O glycosylation O N N O O 2 HO H O OH O O O O O P O O - OH - P O O OH O (39) (41)

CONH2 O O O

HO C HO2C HO2C 2 NH NH NH

beta- H N N O H N N O H2N N O elimination HO 2 HO 2 O HO O OH O OH - P O O O O H O O O O O - P OH - P O OH (43) O OH O O (42) strand break

Figure 21. Formation of the hemiaminal intermediate 37 at the C4=O moiety of the 50-residue of spore photoproduct (SP). Either C-O bond associated with the hemiaminal species is cleavable. As a consequence, the oxygen atom at the C4=O moiety becomes exchangeable with the oxygen atom from water. Decomposition of the hemiaminal intermediate produces a SP hydrolysis product 38, whose further decomposition would induce a cascade of elimination reactions, eventually leading to DNA strand break. Photochemistry and Photobiology, 2015, 91 1285 of CaDPA and DNA binding by SASPs in spores ensures that SP combat these deadly diseases, improving human health as well photochemistry is the dominant photochemical event in dormant as national security. spores. However, SP photobiology beyond dormant spores, such as its repair and mutagenic impacts in germinating spores, is still Acknowledgements—We thank the reviewers for their insightful comments. an area where questions remain. This work has been supported in part by funds from NIH ES017177 and Spore photoproduct was first discovered in UV-irradiated bac- NSF CHE1506598 grants (LL). Work in the PS laboratory over the years terial spores, and the DNA photochemistry and photobiology was supported by grants from the NIH as well as the Army Research fi field generally ascribes SP formation to the unique environment Of ce. One of us (PS) is grateful to Richard (Dick) Setlow who died while this review article was in preparation. Dick’s contribution to DNA inside spores. However, is it truly just a spore photoproduct? It photobiology was immense, and his codiscovery with Ed Donnellan of SP is now known that in dry or desiccated microorganisms exposed in UV-irradiated Bacillus spores in 1965 initiated a new area of research, a to UVC or UVB photons, SP is formed as a major DNA pho- good bit of it in my laboratory, even though I will admit I never paid tolesion (193). Spore photoproduct was also formed in UV-irra- attention to Dick’s work in my younger years. However, I certainly did as diated dried yeast cells (194,195), and the yeast cells likely my research career progressed, and the work in my laboratory on SP was carrying SP exhibit greatly increased UV sensitivity. Spore pho- instrumental in enabling the funding of the college education of two of toproduct formation has also been suggested to occur in UV-irra- Dick’s grandchildren as well as paying the salary of one of Dick’s diated frozen bacteria, bacteriophage (196) and yeast (197), and daughter-in-laws for many years! the frozen cells appear to be more sensitive to UV irradiation possibly again due to the presence of SP. However, SP formation REFERENCES in these nonspore cells has not been verified and the molecular basis for the enhanced UV sensitivity for these microorganisms 1. Donnellan, J. E. Jr and R. B. Setlow (1965) Thymine photoprod- has not been studied. 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duced pyrimidine dimers in mouse and human cells. Proc. Natl AUTHOR BIOGRAPHIES Acad. Sci. 106, 18219–18224. 186. Taggart, D. J., T. L. Camerlengo, J. K. Harrison, S. M. Sherrer, A. Dr. Peter Setlow is K. Kshetry, J.-S. Taylor, K. Huang and Z. Suo (2013) A high- Board of Trustees Distin- throughput and quantitative method to assess the mutagenic poten- guished Professor of tial of translesion DNA synthesis. Nucleic Acids Res. 41, e96. Molecular Biology and 187. Johnson, R. E., L. Haracska, S. Prakash and L. Prakash (2001) Biophysics at UConn Role of DNA polymerase g in the bypass of a (6-4) TT photopro- – Health in Farmington, CT, duct. Mol. Cell. Biol. 21, 3558 3563. USA. His research on the 188. Munoz-S~ anchez, J. and E. Cabrera-Juarez (1991) In vitro mutation fl formation, biochemistry, of Haemophilus in uenzae transforming deoxyribonucleic acid by resistance, killing and ger- ultraviolet radiation at 70°C. Mutat. Res.-Fund. Mol. M. 251, – mination of spores of bac- 21 29. teria of Bacillus and 189. Lin, G., Y. Jian, K. J. Dria, E. C. Long and L. Li (2014) Reactivity Clostridium species has of damaged pyrimidines: DNA cleavage via hemiaminal formation resulted in more than 460 at the C4 positions of the saturated thymine of spore photoproduct publications on these and dihydrouridine. J. Am. Chem. Soc. 136, 12938–12946. fi topics. Dr. Setlow is a 190. Cano, R. J. and M. K. Borucki (1995) Revival and identi cation Fellow of the American of bacterial spores in 25- to 40-million-year-old Dominican amber. – Academy of Microbiol- Science 268, 1060 1064. ogy, an Honorary Mem- 191. Vreeland, R. H., W. D. Rosenzweig and D. W. Powers (2000) Iso- ber of the Society for lation of a 250 million-year-old halotolerant bacterium from a pri- – Applied Microbiology, is mary salt crystal. Nature 407, 897 900. on the Editorial Boards of 192. Kennedy, M. J., S. L. Reader and L. M. Swierczynski (1994) Applied and Environmen- Preservation records of micro-organisms: Evidence of the tenacity – tal Microbiology and the Journal of Bacteriology, and is an editor of the of life. Microbiology 140(Pt 10), 2513 2529. Journal of Applied Microbiology. 193. Cadet, J., A. Grand and T. Douki (2015) Solar UV radiation-in- duced DNA bipyrimidine photoproducts: Formation and mechanis- tic insights. In Photoinduced Phenomena in Nucleic Acids II, Vol. 356. (Edited by M. Barbatti, A. C. Borin and S. Ullrich), pp. 249– 275. Springer International Publishing, Switzerland. 194. Hieda, K. (1971) Effects of dehydration on the sensitivity of yeast Dr. Lei Li is an associate to ultraviolet light. Mutat. Res.-Fund. Mol. M. 12, 365–380. Professor of Chemistry 195. Hieda, K. (1981) Induction of genetic changes in Saccharomyces and Chemical Biology at cerevisiae by partial drying in air of constant relative humidity and Indiana University-Purdue by UV. Mutat. Res. 84,17–27. University Indianapolis 196. Ashwood-Smith, M. J., B. A. Bridges and R. J. Munson (1965) (IUPUI), IN, USA. He Ultraviolet damage to bacteria and bacteriophage at low tempera- also holds adjunct appoint- tures. Science 149, 1103–1105. ment in Department of 197. Hieda, K. and T. Ito (1968) The enhancement of UV sensitivity of Biochemistry & Molecular yeast in frozen state. Mutat. Res. 6, 325–327. Biology and Department 198. DiMaio, F., X. Yu, E. Rensen, M. Krupovic, D. Prangishvili and of Dermatology, Indiana E. H. Egelman (2015) A virus that infects a hyperthermophile University School of Med- encapsidates A-form DNA. Science 348, 914–917. icine. His research mainly 199. Mori, T., T. Matsunaga, T. Hirose and O. Nikaido (1988) Estab- focuses on the mechanistic lishment of a monoclonal antibody recognizing ultraviolet light-in- elucidation of the forma- duced (6-4) photoproducts. Mutat. Res. 194, 263–270. tion and enzymatic repair 200. Mori, T., M. Nakane, T. Hattori, T. Matsunaga, M. Ihara and O. of the spore photoproduct. Nikaido (1991) Simultaneous establishment of monoclonal anti- The Honors and Awards bodies specific for either cyclobutane pyrimidine dimer or (6-4) Dr. Li received include photoproduct from the same mouse immunized with ultraviolet-ir- NIH Pathway to Indepen- radiated DNA. Photochem. Photobiol. 54, 225–232. dence Award (2008) and 201. Strickland, P. T. and J. M. Boyle (1981) Characterisation of two NSF CAREER Award monoclonal antibodies specific for dimerised and non-dimerised (2015). adjacent thymidines in single stranded DNA. Photochem. Photo- biol. 34, 595–601. 202. Strickland, P. T., O. Nikaido, T. Matsunaga and J. M. Boyle (1992) Further characterization of monoclonal antibody indicates specificity for (6-4)-dipyrimidine photoproducts. Photochem. Pho- tobiol. 55, 723–727. 203. Prescott, L. M., J. P. Harley and D. A. Klein (2005) Microbiology. McGraw-Hill Higher Education, Dubuque, IA.