Spore photoproduct within DNA is a surprisingly poor substrate for its designated repair enzyme—The spore photoproduct lyase

DNA repair enzymes typically recognize their substrate lesions with high affinity to ensure efficient lesion repair. In UV irradiated endospores, a special thymine dimer, 5-thyminyl-5,6-dihydrothymine, termed the spore photoproduct (SP), is the dominant DNA photolesion, which is rapidly repaired during spore outgrowth mainly by spore photoproduct lyase (SPL) using an unprecedented protein-harbored radical transfer process. Surprisingly, our in vitro studies using SP-containing short oligonucleotides, pUC 18 plasmid DNA, and E. coli genomic DNA found that they are all poor substrates for SPL in general, exhibiting turnover numbers of 0.01–0.2 min⁻¹. The faster turnover numbers are reached under single turnover conditions, and SPL activity is low with oligonucleotide substrates at higher concentrations. Moreover, SP-containing oligonucleotides do not go past one turnover. In contrast, the dinucleotide SP TpT exhibits a turnover number of 0.3–0.4 min⁻¹, and the reaction may reach up to 10 turnovers. These observations distinguish SPL from other specialized DNA repair enzymes. To the best of our knowledge, SPL represents an unprecedented example of a major DNA repair enzyme that cannot effectively repair its substrate lesion within the normal DNA conformation adopted in growing cells. Factors such as other DNA binding proteins, helicases or an altered DNA conformation may cooperate with SPL to enable efficient SP repair in germinating spores. Therefore, both SP formation and SP repair are likely to be tightly controlled by the unique cellular environment in dormant and outgrowing spore-forming bacteria, and thus SP repair may be extremely slow in non-spore-forming organisms.     

Characterization of an active spore photoproduct lyase, a DNA repair enzyme in the radical S-adenosylmethionine superfamily

The major photoproduct in UV-irradiated Bacillus spore DNA is a unique thymine dimer called spore photoproduct (SP, 5-thyminyl-5,6-dihydrothymine). The enzyme spore photoproduct lyase (SP lyase) has been found to catalyze the repair of SP dimers to thymine monomers in a reaction that requires S-adenosylmethionine. We present here the first detailed characterization of catalytically active SP lyase, which has been anaerobically purified from overexpressing Escherichia coli. Anaerobically purified SP lyase is monomeric and is red-brown in color. The purified enzyme contains approximately 3.1 iron and 3.0 acid-labile S(2-) per protein and has a UV-visible spectrum characteristic of iron-sulfur proteins (410 nm (11.9 mM(-1) cm(-1)) and 450 nm (10.5 mM(-1) cm(-1))). The X-band EPR spectrum of the purified enzyme shows a nearly isotropic signal (g = 2.02) characteristic of a [3Fe-4S]1+ cluster; reduction of SP lyase with dithionite results in the appearance of a new EPR signal (g = 2.03, 1.93, and 1.89) with temperature dependence and g values consistent with its assignment to a [4Fe-4S]1+ cluster. The reduced purified enzyme is active in SP repair, with a specific activity of 0.33 micromol/min/mg. Only a catalytic amount of S-adenosylmethionine is required for DNA repair, and no irreversible cleavage of S-adenosylmethionine into methionine and 5'-deoxyadenosine is observed during the reaction. Label transfer from [5'-3H]S-adenosylmethionine to repaired thymine is observed, providing evidence to support a mechanism in which a 5'-deoxyadenosyl radical intermediate directly abstracts a hydrogen from SP C-6 to generate a substrate radical, and subsequent to radical-mediated beta-scission, a product thymine radical abstracts a hydrogen from 5'-deoxyadenosine to regenerate the 5'-deoxyadenosyl radical. Together, our results support a mechanism in which S-adenosylmethionine acts as a catalytic cofactor, not a substrate, in the DNA repair reaction.     

Mechanistic studies of the radical SAM enzyme spore photoproduct lyase (SPL)

Spore photoproduct lyase (SPL) repairs a special thymine dimer 5-thyminyl-5,6-dihydrothymine, which is commonly called spore photoproduct or SP at the bacterial early germination phase. SP is the exclusive DNA photo-damage product in bacterial endospores; its generation and swift repair by SPL are responsible for the spores' extremely high UV resistance. The early in vivo studies suggested that SPL utilizes a direct reversal strategy to repair the SP in the absence of light. The research in the past decade further established SPL as a radical SAM enzyme, which utilizes a tri-cysteine CXXXCXXC motif to harbor a [4Fe-4S] cluster. At the 1+ oxidation state, the cluster provides an electron to the S-adenosylmethionine (SAM), which binds to the cluster in a bidentate manner as the fourth and fifth ligands, to reductively cleave the CS bond associated with the sulfonium ion in SAM, generating a reactive 5'-deoxyadenosyl (5'-dA) radical. This 5'-dA radical abstracts the proR hydrogen atom from the C6 carbon of SP to initiate the repair process; the resulting SP radical subsequently fragments to generate a putative thymine methyl radical, which accepts a back-donated H atom to yield the repaired TpT. SAM is suggested to be regenerated at the end of each catalytic cycle; and only a catalytic amount of SAM is needed in the SPL reaction. The H atom source for the back donation step is suggested to be a cysteine residue (C141 in Bacillus subtilis SPL), and the H-atom transfer reaction leaves a thiyl radical behind on the protein. This thiyl radical thus must participate in the SAM regeneration process; however how the thiyl radical abstracts an H atom from the 5'-dA to regenerate SAM is unknown. This paper reviews and discusses the history and the latest progress in the mechanistic elucidation of SPL. Despite some recent breakthroughs, more questions are raised in the mechanistic understanding of this intriguing DNA repair enzyme. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.     

Dinucleotide spore photoproduct, a minimal substrate of the DNA repair spore photoproduct lyase enzyme from Bacillus subtilis

The overwhelming majority of DNA photoproducts in UV-irradiated spores is a unique thymine dimer called spore photoproduct (SP, 5-thymine-5,6-dihydrothymine). This lesion is repaired by the spore photoproduct lyase (SP lyase) enzyme that directly reverts SP to two unmodified thymines. The SP lyase is an S-adenosylmethionine-dependent iron-sulfur protein that belongs to the radical S-adenosylmethionine superfamily. In this study, by using a well characterized preparation of the SP lyase enzyme from Bacillus subtilis, we show that SP in the form of a dinucleoside monophosphate (spore photoproduct of thymidilyl-(3'-5')-thymidine) is efficiently repaired, allowing a kinetic characterization of the enzyme. The preparation of this new substrate is described, and its identity is confirmed by mass spectrometry and comparison with authentic spore photoproduct. The fact that the spore photoproduct of thymidilyl-(3'-5')-thymidine dimer is repaired by SP lyase may indicate that the SP lesion does not absolutely need to be contained within a single- or double-stranded DNA for recognition and repaired by the SP lyase enzyme.     

Spore Photoproduct Lyase from Bacillus subtilis Spores Is a Novel Iron-Sulfur DNA Repair Enzyme Which Shares Features with Proteins such as Class III Anaerobic Ribonucleotide Reductases and Pyruvate-Formate Lyases

The major photoproduct in UV-irradiated spore DNA is the unique thymine dimer 5-thyminyl-5,6-dihydrothymine, commonly referred to as spore photoproduct (SP). An important determinant of the high UV resistance of Bacillus subtilis spores is the accurate in situ reversal of SP during spore germination by the DNA repair enzyme SP lyase. To study the molecular aspects of SP lyase-mediated SP repair, the cloned B. subtilis splB gene was engineered to encode SP lyase with a molecular tag of six histidine residues at its amino terminus. The engineered six-His-tagged SP lyase expressed from the amyE locus restored UV resistance to spores of a UV-sensitive mutant B. subtilis strain carrying a deletion-insertion mutation which removed the entire splAB operon at its natural locus and was shown to repair SP in vivo during spore germination. The engineered SP lyase was purified both from dormant B. subtilis spores and from an Escherichia coli overexpression system by nickel-nitrilotriacetic acid (NTA) agarose affinity chromatography and was shown by Western blotting, UV-visible spectroscopy, and iron and acid-labile sulfide analysis to be a 41-kDa iron-sulfur (Fe-S) protein, consistent with its amino acid sequence homology to the 4Fe-4S clusters in anaerobic ribonucleotide reductases and pyruvate-formate lyases. SP lyase was capable of reversing SP from purified SP-containing DNA in an in vitro reaction either when present in a cell-free extract prepared from dormant spores or after purification on nickel-NTA agarose. SP lyase activity was dependent upon reducing conditions and addition of S-adenosylmethionine as a cofactor.     

UV-radiation-induced formation of DNA bipyrimidine photoproducts in Bacillus subtilis endospores and their repair during germination.

The spore photoproduct (SP) is the main DNA lesion after UV-C irradiation, and its repair is crucial for the resistance of spores to UV. The aims of the present study were to assess the formation and repair of bipyrimidine photoproducts in spore DNA of various Bacillus subtilis strains using a sensitive HPLC tandem mass spectrometry assay. Strains deficient in nucleotide excision repair, spore photoproduct lyase, homologous recombination (recA), and with wild-type repair capability were investigated. Additionally, one strain deficient in the formation of major small, acid-soluble spore proteins (SASPs) was tested. In all SASP wild-type strains, UV-C irradiation generated almost exclusively SP (>95 %) but also a few by-photoproducts. In the major SASP-deficient strain, SP and by-photoproducts were generated in equal quantities. The status time of 60 min, >75% of the SP was repaired in wild-type strains and in the SASP-deficient strain, while half of the photoinduced SP was removed in the recA-deficient strain. SP-lyase-deficient spores repaired 20% of the SP produced. Thus, SP lyase, with respect to nucleotide excision repair, has a remarkable impact on the removal of SP upon spore germination.     

Photosensitization of DNA by dipicolinic acid, a major component of spores of Bacillus species.

The DNA in spores of Bacillus species exhibits a relatively novel photochemistry, as 5-thyminyl-5,6-dihydrothymine (spore photoproduct (SP)) is by far the major UV photoproduct whereas cyclobutane dimers (CPDs) and (6-4) photoproducts (6-4PPs) are the major photoproducts in growing cells. Dehydration and more importantly complexation of DNA by alpha/beta-type small, acid-soluble spore proteins (SASP) have been shown to partly explain the photochemistry of spore DNA. The large amount ( approximately 10% of dry weight) of the spore's dipicolinic acid (DPA) also has been shown to play a role in spore DNA photochemistry. In the present work we showed by exposing spores of various strains of B. subtilis to UVC radiation that DPA photosensitizes spore DNA to damage and favors the formation of SP. The same result was obtained in either the presence or absence of the alpha/beta-type SASP that saturate the spore chromosome. Addition of DPA to dry films of isolated DNA or to frozen solutions of thymidine also led to a higher yield of SP and increased ratio of CPDs to 6-4PPs; DPA also significantly increased the yield of CPDs in thymidine exposed to UVC in liquid solution. These observations strongly support a triplet energy transfer between excited DPA and thymine residues. We further conclude that the combined effects of alpha/beta-type SASP and DPA explain the novel photochemistry of DNA in spores of Bacillus species.     

Spore Photoproduct Lyase: The Known,the Controversial,and the Unknown

Spore photoproduct lyase (SPL) repairs 5-thyminyl-5,6-dihydrothymine, a thymine dimer that is also called the spore photoproduct (SP), in germinating endospores. SPL is a radical S-adenosylmethionine (SAM) enzyme, utilizing the 5′-deoxyadenosyl radical generated by SAM reductive cleavage reaction to revert SP to two thymine residues. Here we review the current progress in SPL mechanistic studies. Protein radicals are known to be involved in SPL catalysis; however, how these radicals are quenched to close the catalytic cycle is under debate.     

The two major spore DNA repair pathways, nucleotide excision repair and spore photoproduct lyase, are sufficient for the resistance of Bacillus subtilis spores to artificial UV-C and UV-B but not to solar radiation.

Bacterial endospores are 1 to 2 orders of magnitude more resistant to 254-nm UV (UV-C) radiation than are exponentially growing cells of the same strain. This high UV resistance is due to two related phenomena: (i) DNA of dormant spores irradiated with 254-nm UV accumulates mainly a unique thymine dimer called the spore photoproduct (SP), and (ii) SP is corrected during spore germination by two major DNA repair pathways, nucleotide excision repair (NER) and an SP-specific enzyme called SP lyase. To date, it has been assumed that these two factors also account for resistance of bacterial spores to solar UV in the environment, despite the fact that sunlight at the Earth's surface consists of UV-B, UV-A, visible, and infrared wavelengths of approximately 290 nm and longer. To test this assumption, isogenic strains of Bacillus subtilis lacking either the NER or SP lyase DNA repair pathway were assayed for their relative resistance to radiation at a number of UV wavelengths, including UV-C (254 nm), UV-B (290 to 320 nm), full-spectrum sunlight, and sunlight from which the UV-B portion had been removed. For purposes of direct comparison, spore UV resistance levels were determined with respect to a calibrated biological dosimeter consisting of a mixture of wild-type spores and spores lacking both DNA repair systems. It was observed that the relative contributions of the two pathways to spore UV resistance change depending on the UV wavelengths used in a manner suggesting that spores irradiated with light at environmentally relevant UV wavelengths may accumulate significant amounts of one or more DNA photoproducts in addition to SP. Furthermore, it was noted that upon exposure to increasing wavelengths, wild-type spores decreased in their UV resistance from 33-fold (UV-C) to 12-fold (UV-B plus UV-A sunlight) to 6-fold (UV-A sunlight alone) more resistant than mutants lacking both DNA repair systems, suggesting that at increasing solar UV wavelengths, spores are inactivated either by DNA damage not reparable by the NER or SP lyase system, damage caused to photosensitive molecules other than DNA, or both.     

Molecular cloning and characterization of the Bacillus subtilis spore photoproduct lyase (spl) gene, which is involved in repair of UV radiation-induced DNA damage during spore germination.

Upon UV irradiation, Bacillus subtilis spore DNA accumulates the novel thymine dimer 5-thyminyl-5,6-dihydrothymine. Spores can repair this "spore photoproduct" (SP) upon germination either by the uvr-mediated general excision repair pathway or by the SP-specific spl pathway, which involves in situ monomerization of SP to two thymines by an enzyme named SP lyase. Mutants lacking both repair pathways produce spores that are extremely sensitive to UV. For cloning DNA that can repair a mutation in the spl pathway called spl-1, a library of EcoRI fragments of chromosomal DNA from B. subtilis 168 was constructed in integrative plasmid pJH101 and introduced by transformation into a mutant B. subtilis strain that carries both the uvrA42 and spl-1 mutations, and transformants whose spores exhibited UV resistance were selected by UV irradiation. With a combination of genetic and physical mapping techniques, the DNA responsible for the restoration of UV resistance was shown to be present on a 2.3-kb EcoRI-HindIII fragment that was mapped to a new locus in the metC-pyrD region of the B. subtilis chromosome immediately downstream from the pstI gene. The spl coding sequence was localized on the cloned fragment by analysis of in vitro-generated deletions and by nucleotide sequencing. The spl nucleotide sequence contains an open reading frame capable of encoding a 40-kDa polypeptide that shows regional amino acid sequence homology to DNA photolyases from a number of bacteria and fungi.     

Effects of DNA-polymerase-defective and recombination-deficient mutations on the ultraviolet sensitivity of Bacillus subtilis spores.

The DNA of UV-irradiated Bacillus subtilis spores, which contains 5-thyminyl-5,6-dihydrothymine (TDHT) as the major thymine photoproduct, is known to be repaired during germination by two complementary mechanisms: (I) the well-known excision repair, and (2) a special process, "spore repair", which destroys TDHT in situ without rendering it acid-soluble. In the absence of both mechanisms TDHT is not removed, and spores are highly UV-sensitive. When either of two mutations (pol-59 and pol-151) giving defective DNA polymerase, or one (rec-A1) giving a recombination deficiency are introduced into strains defective in one of these known TDHT removal processes, the chemically measured elimination of TDHT from spore DNA is unaltered, but spore UV-sensitivity is increased. The pol mutations produce their greatest sensitivity increase in spores of strains already deficient for the in situ destruction of TDHT, while the rec mutation gives its maximum sensitivity increase to spores of strains lacking excision. These facts argue that the pol mutations interfere mostly with excision repair (presumably its later resynthesis step), shile the rec mutation impairs "spore repair" in some step occurring subsequent to the TDHT destruction in situ. With either of these impairments of the later repair steps, DNA of UV-irradiated and germinated spores is considerably degraded, unless germination is carried out in the presence of chloramphenicol.     

Structural insights into recognition and repair of UV-DNA damage by Spore Photoproduct Lyase, a radical SAM enzyme

Bacterial spores possess an enormous resistance to ultraviolet (UV) radiation. This is largely due to a unique DNA repair enzyme, Spore Photoproduct Lyase (SP lyase) that repairs a specific UV-induced DNA lesion, the spore photoproduct (SP), through an unprecedented radical-based mechanism. Unlike DNA photolyases, SP lyase belongs to the emerging superfamily of radical S-adenosyl-l-methionine (SAM) enzymes and uses a [4Fe–4S]¹⁺ cluster and SAM to initiate the repair reaction. We report here the first crystal structure of this enigmatic enzyme in complex with its [4Fe–4S] cluster and its SAM cofactor, in the absence and presence of a DNA lesion, the dinucleoside SP. The high resolution structures provide fundamental insights into the active site, the DNA lesion recognition and binding which involve a β-hairpin structure. We show that SAM and a conserved cysteine residue are perfectly positioned in the active site for hydrogen atom abstraction from the dihydrothymine residue of the lesion and donation to the α-thyminyl radical moiety, respectively. Based on structural and biochemical characterizations of mutant proteins, we substantiate the role of this cysteine in the enzymatic mechanism. Our structure reveals how SP lyase combines specific features of radical SAM and DNA repair enzymes to enable a complex radical-based repair reaction to take place.     

The Ultraviolet Photochemistry and Photobiology of Vegetative Cells and Spores of Bacillus megaterium

The ultraviolet (UV) photochemistry and photobiology of spores and vegetative cells of Bacillus megaterium have been studied. The response of vegetative cells of B. megaterium appears qualitatively similar to those of Escherichia coli, Micrococcus radiodurans, and Bacillus subtilis with respect to photoproduct formation and repair mechanisms. UV irradiation, however, does not produce cyclobutane-type thymine dimers in the DNA of spores, although other thymine photo-products are produced. The photoproducts do not disappear after photoreactivation, but they are eliminated from the DNA by a dark-repair mechanism different from that found for dimers in vegetative cells. Irradiations performed at three wavelengths produce the same amounts of spore photoproduct and give the same survival curves. Variation of the sporulation medium before irradiation results in comparable alterations in the rate of spore photoproduct production and in survival.     

Spore photoproduct (SP) lyase from Bacillus subtilis specifically binds to and cleaves SP (5-thyminyl-5,6-dihydrothymine) but not cyclobutane pyrimidine dimers in UV-irradiated DNA

The predominant photolesion in the DNA of UV-irradiated dormant bacterial spores is the thymine dimer 5-thyminyl-5,6-dihydrothymine, commonly referred to as spore photoproduct (SP). A major determinant of SP repair during spore germination is its direct reversal by the enzyme SP lyase, encoded by the splB gene in Bacillus subtilis. SplB protein containing an N-terminal tag of six histidine residues [(6His)SplB] was purified from dormant B. subtilis spores and shown to efficiently cleave SP but not cyclobutane cis,syn thymine-thymine dimers in vitro. In contrast, SplB protein containing an N-terminal 10-histidine tag [(10His)SplB] purified from an Escherichia coli overexpression system was incompetent to cleave SP unless the 10-His tag was first removed by proteolysis at an engineered factor Xa site. To assay the parameters of binding of SplB protein to UV-damaged DNA, a 35-bp double-stranded oligonucleotide was constructed which carried a single pair of adjacent thymines on one strand. Irradiation of the oligonucleotide in aqueous solution or at 10% relative humidity resulted in formation of cyclobutane pyrimidine dimers (Py lozengePy) or SP, respectively. (10His)SplB was assayed for oligonucleotide binding using a DNase I protection assay. In the presence of (10His)SplB, the SP-containing oligonucleotide was selectively protected from DNase I digestion (half-life, >60 min), while the Py lozengePy-containing oligonucleotide and the unirradiated oligonucleotide were rapidly digested by DNase I (half-lives, 6 and 9 min, respectively). DNase I footprinting of (10His)SplB bound to the artificial substrate was carried out utilizing the (32)P end-labeled 35-bp oligonucleotide containing SP. DNase I footprinting showed that SplB protected at least a 9-bp region surrounding SP from digestion with DNase I with the exception of two DNase I-hypersensitive sites within the protected region. (10His)SplB also caused significant enhancement of DNase I digestion of the SP-containing oligonucleotide for at least a full helical turn 3' to the protected region. The data suggest that binding of SP lyase to SP causes significant bending or distortion of the DNA helix in the vicinity of the lesion.     

Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals

A number of mechanisms are responsible for the resistance of spores of Bacillus species to heat, radiation and chemicals and for spore killing by these agents. Spore resistance to wet heat is determined largely by the water content of spore core, which is much lower than that in the growing cell protoplast. A lower core water content generally gives more wet heat-resistant spores. The level and type of spore core mineral ions and the intrinsic stability of total spore proteins also play a role in spore wet heat resistance, and the saturation of spore DNA with alpha/beta-type small, acid-soluble spore proteins (SASP) protects DNA against wet heat damage. However, how wet heat kills spores is not clear, although it is not through DNA damage. The alpha/beta-type SASP are also important in spore resistance to dry heat, as is DNA repair in spore outgrowth, as Bacillus subtilis spores are killed by dry heat via DNA damage. Both UV and gamma-radiation also kill spores via DNA damage. The mechanism of spore resistance to gamma-radiation is not well understood, although the alpha/beta-type SASP are not involved. In contrast, spore UV resistance is due largely to an alteration in spore DNA photochemistry caused by the binding of alpha/beta-type SASP to the DNA, and to a lesser extent to the photosensitizing action of the spore core's large pool of dipicolinic acid. UV irradiation of spores at 254 nm does not generate the cyclobutane dimers (CPDs) and (6-4)-photoproducts (64PPs) formed between adjacent pyrimidines in growing cells, but rather a thymidyl-thymidine adduct termed spore photoproduct (SP). While SP is formed in spores with approximately the same quantum efficiency as that for generation of CPDs and 64PPs in growing cells, SP is repaired rapidly and efficiently in spore outgrowth by a number of repair systems, at least one of which is specific for SP. Some chemicals (e.g. nitrous acid, formaldehyde) again kill spores by DNA damage, while others, in particular oxidizing agents, appear to damage the spore's inner membrane so that this membrane ruptures upon spore germination and outgrowth. There are also other agents such as glutaraldehyde for which the mechanism of spore killing is unclear. Factors important in spore chemical resistance vary with the chemical, but include: (i) the spore coat proteins that likely react with and detoxify chemical agents; (ii) the relative impermeability of the spore's inner membrane that restricts access of exogenous chemicals to the spore core; (iii) the protection of spore DNA by its saturation with alpha/beta-type SASP; and (iv) DNA repair for agents that kill spores via DNA damage. Given the importance of the killing of spores of Bacillus species in the food and medical products industry, a deeper understanding of the mechanisms of spore resistance and killing may lead to improved methods for spore destruction.     

Action of T4 endonuclease V on DNA containing various photoproducts

The action of T4 endonuclease V on DNA containing various photoproducts was investigated. (1) The enzyme introduced strand breaks in DNA from ultraviolet-irradiated vegetative cells of Bacillus subtilis but not in DNA from irradiated spores of the same organism. DNA irradiated with long wavelength (360 nm peak) ultraviolet light in the presence of 4,5',8-trimethylpsoralen was not attacked by the enzyme. These results indicate that 5-thyminyl 5,6-dihydrothymine (spore photoproduct) and psoralen mediated cross-links in DNA are not recognized by T4 endonuclease V. (2) DNA of phage PBS1, containing uracil in place of thymine, and DNA of phage SPO1, containing hydroxymethyluracil in place of thymine, were fragmented by the enzyme when the DNA's had been irradiated with ultraviolet light. T4 endonuclease V seems to act on DNA with pyrimidine dimers whether the dimers contain thymine residues or not.     

Transitory germinative excision repair in Bacillus subtilis.

Bacillus subtilis strains UVSSP-42-1 (hcr42 ssp1) and UVSSP-1-1 (hcr1 ssp1) are ultraviolet (UV) radiation sensitive both as dormant spores and as vegetative cells. These strains are unable to excise cyclobutane-type dimers from the deoxyribonucleic acid (DNA) of irradiated vegetative cells and fail to remove spore photoproduct from the DNA of irradiated spores either by excision (controlled by gene hcr) or by spore repair (controlled by gene ssp1). When irradiated soon after spore germination, these strains excise dimers, but not spore photoproduct, from their DNA. This process, termed germinative excision repair, functions only transiently in the germination phase and is responsible for the high UV resistance of germinated spores and for their temporary capacity to host cell reactivate irradiated phages infecting them. The recA1 mutation confers higher UV sensitivity to the germinated spores, but does not interfere with dimer removal by germinative excision repair.     

Inter-strand photoproducts are produced in high yield within A-DNA exposed to UVC radiation

Far-UV irradiation of DNA leads to the dimerization of pyrimidine bases, resulting in the formation of cyclobutane type dimers and (6–4) photoproducts. In the dry state, an additional thymine dimeric photolesion, the spore photoproduct, is also generated. While most photoproducts are expected to be produced between adjacent pyrimidines, little attention has been paid to lesions involving bases located on different DNA strands. Using HPLC– mass spectrometry analysis of enzymatically digested DNA, we observed that, in the dry state, inter-strand dimeric photoproducts represented 30% of the total yield of dimeric thymine lesions. The major inter-strand damage was found to be the spore photoproduct. Formation of inter-strand lesions in significant yield could be obtained in solution upon modification of the DNA conformation as the result of the addition of large amounts of ethanol. In both cases, DNA is in the A-form, which is characterized by a high compaction, likely to favor inter-strand photoreactions. Since the latter DNA conformation is also predominant in bacterial spores, the formation and repair of dimeric photoproducts involving thymine bases located on different DNA strands may thus be relevant in terms of deleterious effects of UV radiation to the latter microorganisms.     

Binding of DNA to alpha/beta-type small, acid-soluble proteins from spores of Bacillus or Clostridium species prevents formation of cytosine dimers, cytosine-thymine dimers, and bipyrimidine photoadducts after UV irradiation.

Small, acid-soluble proteins (SASP) of the alpha/beta-type from spores of Bacillus and Clostridium species bind to DNA; this binding prevents formation of cyclobutane-type thymine dimers upon UV irradiation, but promotes formation of the spore photoproduct, an adduct between adjacent thymine residues. alpha/beta-Type SASP also bound to poly(dG).poly(dC) and poly(dA-dG).poly(dC-dT). While UV irradiation of poly(dG).poly(dC) produced cyclobutane-type cytosine dimers as well as fluorescent bipyrimidine adducts, the yields of both types of photoproduct were greatly reduced upon irradiation of alpha/beta-type SASP-poly(dG).poly(dC) complexes. UV irradiation of poly(dA-dG).poly(dC-dT) produced a significant amount of a cyclobutane dimer between cytosine and thymine, as well as a 6-4 bipyrimidine adduct. Again, binding of alpha/beta-type SASP to poly(dA-dG).poly(dC-dT) greatly reduced formation of these two photoproducts, although formation of the cytosine-thymine analog of the spore photoproduct was not observed. These data provide further evidence for the dramatic change in DNA structure and photoreactivity which takes place on binding of alpha/beta-type SASP and suggest that binding of these proteins to DNA in vivo prevents formation of most deleterious photoproducts upon UV irradiation.