Direct H Atom Abstraction from Spore Photoproduct C-6 Initiates DNA Repair in the Reaction Catalyzed by Spore Photoproduct Lyase:  Evidence for a Reversibly Generated Adenosyl Radical Intermediate

Cheek, Jennifer; Broderick, Joan B.

doi:10.1021/ja017784g

Cited by 115 publications

(206 citation statements)

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“…Preparation of SPTpT, the Dinucleoside Monophosphate SP Lesion-To date, SP lyase activity has been assayed using either SP-containing [methyl-3 H]DNA extracted from irradiated spores or tritiated pUC18 plasmid DNA exposed to UV radiation in the presence of a small acid-soluble protein from B. subtilis, as a substrate (17). The repair activity was defined in terms of remaining SP after a given reaction time, determined after hydrolysis of DNA, isolation of the released SP, and scintillation counting (10,17,20).…”

Section: Resultsmentioning

confidence: 99%

“…The repair activity was defined in terms of remaining SP after a given reaction time, determined after hydrolysis of DNA, isolation of the released SP, and scintillation counting (10,17,20). To study the repair reaction with a better defined substrate, available in larger quantities, we decided to use instead the modified dinucleoside monophosphate SPTpT in a pure form (Scheme 1B).…”

Section: Resultsmentioning

confidence: 99%

“…Samples (20 l from the initial 50 l) were injected onto an Uptisphere ODB (particle size 3 m, 150 ϫ 2 mm inner diameter) octadecylsilyl silica gel column (Interchim, Montluçon, France) connected to a series 1100 Agilent chromato-SCHEME 2. Proposed chemical mechanism for SP lyase-mediated DNA repair (17). SAM is AdoMet.…”

Section: Methodsmentioning

confidence: 99%

“…This CXXXCXXC sequence is indeed the signature for a superfamily of [4Fe-4S] iron-sulfur enzymes, named "radical SAM" (12), involved in a variety of biosynthetic pathways and metabolic reactions that proceed via radical mechanisms (13-15). Spectroscopic and biochemical studies from Nicholson and co-workers (16) and Broderick and co-workers (17) have shown the following: (i) the protein carries a single iron-sulfur cluster (16); (ii) the reaction is absolutely dependent on S-adenosylmethionine (AdoMet) (16,17); and (iii) the repair mechanism is likely to involve a 5Ј-deoxyadenosyl radical (Ado ⅐ ) generated through reductive cleavage of AdoMet (16,17). Labeling experiments indicate * The costs of publication of this article were defrayed in part by the payment of page charges.…”

mentioning

confidence: 99%

“…Spectroscopic and biochemical studies from Nicholson and co-workers (16) and Broderick and co-workers (17) have shown the following: (i) the protein carries a single iron-sulfur cluster (16); (ii) the reaction is absolutely dependent on S-adenosylmethionine (AdoMet) (16,17); and (iii) the repair mechanism is likely to involve a 5Ј-deoxyadenosyl radical (Ado ⅐ ) generated through reductive cleavage of AdoMet (16,17). Labeling experiments indicate that the reaction is initiated by direct C-6 hydrogen atom abstraction by Ado ⅐ , with the resulting substrate radical undergoing scission to re-generate the two initial thymines (Scheme 2) (17).…”

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confidence: 99%

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Dinucleotide Spore Photoproduct, a Minimal Substrate of the DNA Repair Spore Photoproduct Lyase Enzyme from Bacillus subtilis

Chandor

Berteau

Douki

et al. 2006

Journal of Biological Chemistry

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The overwhelming majority of DNA photoproducts in UVirradiated 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 singleor double-stranded DNA for recognition and repaired by the SP lyase enzyme.The DNA of all organisms is subject to modifications upon exposure to a wide variety of chemical and physical agents. Among them, solar ultraviolet radiation is known to induce dimerization reactions between adjacent pyrimidines (1). In the vast majority of living systems, the resulting photoproducts are cyclobutane pyrimidine dimers (CPDs) 5 and pyrimidine (6-4) pyrimidone photoproducts (Scheme 1A) that can be generated at any of the four bipyrimidine doublets (TT, CT, TC and CC), although the yields of the lesions depend on the bases involved (2). These lesions induce mutations and can be lethal because of blocking of the replication machinery. The photochemistry in bacterial spores is quite different. Indeed, in this dormant form produced by some bacteria such as Bacillus subtilis, the only photoproduct produced upon exposure to UV light corresponds to two thymines linked by the methyl group of one of the bases (3, 4). The formation of this specific lesion, 5-thyminyl-5,6-dihydrothymine (spore photoproduct, SP) (Scheme 1A), is explained by specific features of the spores, including DNA conformation (A form), dehydration, the presence of dipicolinic acid in the core, and binding of small acid-soluble proteins to DNA (5-8). The formation of SP as the unique DNA lesion in irradiated spores is proposed to account for their extreme resistance to UV radiation. Indeed, spores express a specific repair enzyme, the spore photoproduct lyase (SP lyase) that directly reverts SP to two unmodified thymines upon germination (9, 10), much more efficiently than dimeric photoproducts are removed from other cell types by the classical nucleotide excision repair pathway. The specific photochemistry of DNA in spores combined with the action of SP lyase appears to be a major evolutionary advantage for spore-forming bacteria in resistance to UV radiation.In their N-terminal half, all SP lyase enzymes contain a strictly conserved a...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Dinucleotide Spore Photoproduct, a Minimal Substrate of the DNA Repair Spore Photoproduct Lyase Enzyme from Bacillus subtilis

Chandor

Berteau

Douki

et al. 2006

Journal of Biological Chemistry

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Direct observation of a deoxyadenosyl radical in an active enzyme environment

et al. 2016

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5'-deoxyadenosyl radicals have been proposed as the first common intermediate in the molecular reaction mechanism of the family of radical S-adenosyl-l-methionine (SAM) enzymes. However, this radical species has not yet been directly observed in a catalytically active enzyme environment. In a reduced and SAM-containing C140A mutant of the spore photoproduct lyase from Geobacillus thermodenitrificans, a mutant with altered catalytic activity, we were able to identify an organic radical with pronounced hyperfine structure using electron paramagnetic resonance spectroscopy. Guided by quantum-chemical computations at the density functional theory level of theory, this radical could be tentatively assigned to a deoxyadenosyl radical, which provides first experimental evidence for this intermediate in the reaction mechanism of radical SAM enzymes.

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Radical S ‐Adenosylmethionine ( SAM ) Superfamily

Jarrett

Farrar

2014

Encyclopedia of Life Sciences

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A large superfamily of enzymes uses S ‐adenosyl‐ l ‐methionine (SAM) to generate high‐energy carbon radicals as intermediates in a variety of metabolic and biosynthetic reactions. All radical SAM enzymes contain one or more iron‐sulphur clusters, with SAM binding directly to an iron‐sulphur cluster in a manner that facilitates reduction of SAM and generation of a highly reactive 5′‐deoxyadenosyl radical intermediate. This potent intermediate typically initiates the subsequent radical transformations by abstracting a hydrogen atom from the substrate or from a nearby protein residue. Despite the diverse array of reactions catalysed, members of the Radical SAM superfamily share a similar structural topology that facilitates this interaction between SAM and an iron‐sulphur cluster and allows restricted access of substrates to the site of radical generation. Key Concepts: The Radical SAM superfamily comprises a diverse array of enzymes that contain a common core structural fold and use S ‐adenosylmethionine to generate organic radicals. Radical SAM enzymes contain one or more iron‐sulphur clusters that are essential for catalysis. S ‐Adenosylmethionine is reduced and cleaved at the sulfonium centre to generate a 5′‐deoxyadenosyl radical. Radical SAM enzymes typically use a 5′‐deoxyadenosyl radical to abstract a hydrogen atom from the substrate. Reaction types catalysed by radical SAM enzymes include oxidation, reduction, methylation, methylthiolation, sulfurylation and complex rearrangement reactions. Radical SAM enzymes are use to introduce several posttranslational and posttranscriptional modifications into proteins and RNA. Radical SAM enzymes are involved in thousands of uncharacterised bacterial natural product biosynthesis pathways.

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Direct H Atom Abstraction from Spore Photoproduct C-6 Initiates DNA Repair in the Reaction Catalyzed by Spore Photoproduct Lyase: Evidence for a Reversibly Generated Adenosyl Radical Intermediate

Cited by 115 publications

References 18 publications

Dinucleotide Spore Photoproduct, a Minimal Substrate of the DNA Repair Spore Photoproduct Lyase Enzyme from Bacillus subtilis

Dinucleotide Spore Photoproduct, a Minimal Substrate of the DNA Repair Spore Photoproduct Lyase Enzyme from Bacillus subtilis

Direct observation of a deoxyadenosyl radical in an active enzyme environment

Radical S ‐Adenosylmethionine ( SAM ) Superfamily

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