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.     

Molecular cloning of a Bacillus subtilis gene involved in spore outgrowth

A lambda Charon 4A derivative carrying the outB gene of Bacillus subtilis has been identified by transformation of a B. subtilis mutant temperature-sensitive in spore outgrowth. The cloned region is a single EcoRI fragment 14 kb in length. In addition to outB, the cloned DNA includes at least part of the amyE and aroI loci.     

Molecular cloning of Bacillus subtilis genes involved in DNA metabolism

Summary Different clones carrying a chromosomal DNA fragment able to transform Bacillus subtilis mutants dnaA13, dnaB19, dnaG5, recG40 and polA42 to a wild-type phenotype were isolated from a library constructed in plasmid pJH101. A recombinant clone carrying a chromosomal fragment able to transform dnaC mutants was obtained from a Charon 4A library. A restriction map of the cloned DNA fragments was constructed. The 11.3 kb cloned DNA fragment of plasmid pMP60-13 containing the wild-type sequence of dnaG5 was shown to transform a recF33 mutant as well.     

Role of DNA repair in Bacillus subtilis spore resistance.

Wet-heat or hydrogen peroxide treatment of wild-type Bacillus subtilis spores did not result in induction of lacZ fusions to three DNA repair-related genes (dinR, recA, and uvrC) during spore outgrowth. However, these genes were induced during outgrowth of wild-type spores treated with dry heat or UV. Wet-heat, desiccation, dry-heat, or UV treatment of spores lacking major DNA-binding proteins (termed alpha-beta- spores) also resulted in induction of the three DNA repair genes during spore outgrowth. Hydrogen peroxide treatment of alpha-beta-spores did not result in induction of dinR- and rerA-lacZ but did cause induction of uvrC-lacZ during spore outgrowth. Spores of a recA mutant were approximately twofold more UV sensitive and approximately ninefold more sensitive to dry heat than were wild-type spores but were no more sensitive to wet heat and hydrogen peroxide. In contrast, alpha-beta- recA spores were significantly more sensitive than were alpha-beta- spores to all four treatments, as well as to desiccation. Surprisingly, RecA levels were quite low in dormant spores, but RecA was synthesized during spore outgrowth. Taken together, these data (i) are consistent with previous suggestions that some treatments (dry heat and UV with wild-type spores; desiccation, dry and wet heat, hydrogen peroxide, and UV with alpha-beta- spores) that kill spores do so in large part by causing DNA damage and (ii) indicate that repair of DNA damage during spore outgrowth is an important component of spore resistance to a number of treatments, as has been shown previously for UV.     

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.     

Properties of Bacillus megaterium and Bacillus subtilis mutants which lack the protease that degrades small, acid-soluble proteins during spore germination.

During germination of spores of Bacillus species the degradation of the spore's pool of small, acid-soluble proteins (SASP) is initiated by a protease termed GPR, the product of the gpr gene. Bacillus megaterium and B. subtilis mutants with an inactivated gpr gene grew, sporulated, and triggered spore germination as did gpr+ strains. However, SASP degradation was very slow during germination of gpr mutant spores, and in rich media the time taken for spores to return to vegetative growth (defined as outgrowth) was much longer in gpr than in gpr+ spores. Not surprisingly, gpr spores had much lower rates of RNA and protein synthesis during outgrowth than did gpr+ spores, although both types of spores had similar levels of ATP. The rapid decrease in the number of negative supertwists in plasmid DNA seen during germination of gpr+ spores was also much slower in gpr spores. Additionally, UV irradiation of gpr B. subtilis spores early in germination generated significant amounts of spore photoproduct and only small amounts of thymine dimers (TT); in contrast UV irradiation of germinated gpr+ spores generated almost no spore photoproduct and three to four times more TT. Consequently, germinated gpr spores were more UV resistant than germinated gpr+ spores. Strikingly, the slow outgrowth phenotype of B. subtilis gpr spores was suppressed by the absence of major alpha/beta-type SASP. These data suggest that (i) alpha/beta-type SASP remain bound to much, although not all, of the chromosome in germinated gpr spores; (ii) the alpha/beta-type SASP bound to the chromosome in gpr spores alter this DNA's topology and UV photochemistry; and (iii) the presence of alpha/beta-type SASP on the chromosome is detrimental to normal spore outgrowth.     

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.     

Molecular cloning, genetic characterization and DNA sequence analysis of the recM region of Bacillus subtilis.

In Bacillus subtilis the recM gene, whose product is associated with DNA repair and recombination, has been located between the dnaX and rrnA genes. The recM gene has been cloned and analyzed. Analysis of the nucleotide sequence (3.741-kilobase) around recM revealed five open reading frames (orf). We have assigned recM and dnaX to two of this orf, given the gene order dnaX-orf107-recM-orf74-orf87. The organization of genes of the dnaX-orf107-recM region resembles the organization of genes in the dnaX-orf12-recR region of the Escherichia coli chromosome. Proteins of 24.2 and 17.0 kDa would result from translation of the wild type and in vitro truncated recM genes, and radioactive bands of proteins of molecular weights of 24.5 and 17.0 kDa were detected by the use of the T7promoter-expression system. The RecM protein contains a potential zinc finger domain for nucleic acid binding and a putative nucleotide binding sequence that is present in many proteins that bind and hydrolyze ATP. Strains, in which the recM gene has been insertionally inactivated, were generated and show a phenotype essentially the same as previously described recM mutants.     

Molecular cloning of a Bacillus subtilis gene involved in cell division, sporulation, and exoenzyme secretion

The wild type div-341+ gene of Bacillus subtilis was cloned in a temperate phage rho 11, and was recloned in a smaller temperate phage phi 105. The resulting Div+ transducing phage carried a 3 kilobase Cfr13I digested chromosomal fragment which showed Div+ transforming activity and contained the whole div-341+ gene which is involved in cell division, sporulation, exoenzyme secretion, competent cell formation, and autolysis. A partial restriction map of the fragment was established. The merodiploid system of the div-341+ gene, wild type gene on the phage genome and mutant gene on the chromosome, resulted in the suppression of mutant phenotypes and indicated that the wild type div-341+ gene is dominant over mutant gene.