Characterization of an Active Spore Photoproduct Lyase, a DNA Repair Enzyme in the Radical S-Adenosylmethionine Superfamily

Abstract

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 ϳ3.1 iron and 3.0 acid-labile S 2؊ per protein and has a UVvisible spectrum characteristic of iron-sulfur proteins (410 nm (11.9 mM H]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 ␤-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.The unusual resistance of bacterial spores to UV light and ionizing radiation has been of long-standing interest. In 1965, Donnellan and Setlow (1) showed that UV-irradiated bacterial spores did not contain the thymine dimers typically found in other irradiated cells but instead contained another type of thymine photoproduct. This dimer was later determined to be spore photoproduct (SP, 3 5-thyminyl-5,6-dihydrothymine; see Fig. 1), a thymine dimer that appears to be unique to Bacillus subtilis and other spore-forming microorganisms (2). The generation of these unique thymine dimers upon UV irradiation is thought to result from the binding of small, acid-soluble proteins to the spore DNA (3-7). During dormancy spores are unable to repair this damage to their DNA from UV exposure. Early in the germination cycle, however, two distinct mechanisms of DNA repair are active, presumably giving rise to the high level of UV resistance of bacterial spores. One of these mechanisms is the nucleotide excision repair pathway that detects and removes lesions from the DNA (including SP and cyclobutane-type pyrimidine dimers) in a manner similar to that characterized in Escherichia coli (8, 9). The second repair mechanism involves the specific reversal of SP to two thymines catalyzed by the enzyme SP lyase in B. subtilis (10,11).Pyrimidine dimers such as the spore photoproduct of Bacillus are a major component of UV-induced DNA damage. These dimers can block replication and transcription or can result in mutations if transcription does proceed past the region of the pyrimidine dimer. Repair of these dimers, therefore, is critical in order to avoid mutations. The only well characterized means of pyrimidine dimer repair is photoreactivation, which is catalyzed by the enzyme DNA photolyase (12, 13). The photolyase famil...

Identification and Characterization of a Novel Member of the Radical AdoMet Enzyme Superfamily and Implications for the Biosynthesis of the Hmd Hydrogenase Active Site Cofactor

“…3B). The decrease in signal, which is similar to that observed previously when the radical AdoMet enzyme spore photoproduct lyase was incubated in the presence of AdoMet (3,25), could be the result of reductive cleavage of AdoMet (which would be accompanied by cluster oxidation); alternatively, a change in the spin state of the [4Fe-4S] 1ϩ cluster could give rise to a decrease in the spin ϭ 1/2 EPR signal. In addition to the decrease in signal intensity, the change in G values observed with the addition of AdoMet may be indicative of interaction with the cluster.…”

Rescuing DNA repair activity by rewiring the H-atom transfer pathway in the radical SAM enzyme, spore photoproduct lyase

“…Several SP lyases from Bacilli 7,12,15,16 and one from Clostridia 9 have been biochemically characterized. However, it was not clear so far how the repair activity takes place in Clostridia as the crucial cysteine residue found in Bacilli (C140 in Gt) is not conserved among clostridial SP lyases.…”

Role of DNA Repair by Nonhomologous-End Joining in Bacillus subtilis Spore Resistance to Extreme Dryness, Mono- and Polychromatic UV, and Ionizing Radiation

“…Binding of ␣/␤-type SASP to spore DNA, coupled with spore core dehydration, appears to change the helical conformation of spore DNA from the B form to an A-like form (34, 48), which in turn alters its UV photochemistry to favor the production of 5-thyminyl-5,6-dihydrothymine, the unique spore-specific spore photoproduct (SP) (8,32,35,50). For the removal of the SP, spores possess an SP-specific repair enzyme called SP lyase, encoded by the splB gene, that monomerizes the SP dimer back to two thymine residues in an adenosyl-radical-dependent reaction (4,28,42).While the UV photochemistry of spore DNA and the repair of UV damage to DNA during germination are well described (12,32,33,47,50), there has been relatively little work on the nature of DNA damage in spores caused by ionizing radiation or extreme dryness and on the occurrence of a specific DNA repair system(s) for repair of this damage. It is assumed that DNA double-strand breaks (DSB), which are the most critical damage caused by ionizing radiation (57) and desiccation (9, 10, 39) in vegetative cells, are also induced in bacterial spores.…”

“…Binding of ␣/␤-type SASP to spore DNA, coupled with spore core dehydration, appears to change the helical conformation of spore DNA from the B form to an A-like form (34, 48), which in turn alters its UV photochemistry to favor the production of 5-thyminyl-5,6-dihydrothymine, the unique spore-specific spore photoproduct (SP) (8,32,35,50). For the removal of the SP, spores possess an SP-specific repair enzyme called SP lyase, encoded by the splB gene, that monomerizes the SP dimer back to two thymine residues in an adenosyl-radical-dependent reaction (4,28,42).…”