Thymine Photoproducts but not Thymine Dimers Found in Ultraviolet-Irradiated Bacterial Spores

Donnellan, J. Edward; Setlow, R. B.

doi:10.1126/science.149.3681.308

Cited by 247 publications

(172 citation statements)

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“…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)(6)(7)(8).…”

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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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“…3 Supported by a postdoctoral fellowship grant from the Commissariat à l'Energie Atomique. 4 To whom correspondence should be addressed. Tel.…”

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confidence: 99%

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 major photoproduct in DNA of UV-irradiated dormant spores is the unique thymine dimer 5-thyminyl-5,6-dihydrothymine, informally called spore photoproduct or SP (4,5). SP can be removed from DNA during spore germination by the general nucleotide excision repair (NER) pathway (6,7).…”

mentioning

confidence: 99%

The subunit structure and catalytic mechanism of the Bacillus subtilis DNA repair enzyme spore photoproduct lyase

Rebeil

Nicholson

2001

Proc. Natl. Acad. Sci. U.S.A.

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A n important property of bacterial endospores is their ability to survive high doses of UV radiation, which is due to the packaging of DNA and the unique photochemistry that this packaging imparts on dormant spores (1-3). The major photoproduct in DNA of UV-irradiated dormant spores is the unique thymine dimer 5-thyminyl-5,6-dihydrothymine, informally called spore photoproduct or SP (4, 5). SP can be removed from DNA during spore germination by the general nucleotide excision repair (NER) pathway (6, 7). In addition to NER, spores possess an SP-specific enzyme called SP lyase that can directly reverse SP to two thymines in situ without excision from DNA (6,8,9). Analysis of the deduced amino acid sequence encoded by the SP lyase gene (splB) cloned from Bacillus subtilis revealed a limited region with similarity to DNA photolyases (10), although early indirect experiments indicated that SP lyase caused direct reversal of SP back to two thymines in situ in a light-independent process (8, 9). These observations led to the question as to the mechanism SP lyase utilizes to reverse SP to two thymines. Inspection of the 342-aa deduced SplB sequence (10) revealed that the protein contains four cysteines, three of which are clustered at residues 91, 95, and 98, and the fourth at residue 141. The region from C91 to C98 was similar to the signature cysteine clusters present in the recently dubbed ''radical SAM'' protein superfamily (11), which includes enzymes such as anaerobic ribonucleotide reductase (RNR), pyruvate formate-lyase (PFL), lysine-2,3-aminomutase (KAM), and biotin synthase (BioB) (12). In radical SAM enzymes, the clustered cysteines serve as ligands in the formation of iron-sulfur ([Fe-S]) centers, and these enzymes use S-adenosylmethionine (S-AdoMet) as a cofactor to generate an adenosyl radical (13-16). We constructed a version of SplB protein containing an N-terminal tag of 10 histidines [(10His)SplB] and subsequently determined that (10His)SplB (i) contained both iron and acid-labile sulfur in a stoichiometric ratio of 1-2 atoms of iron or sulfur per (10His)SplB subunit (17); (ii) exhibited a UV-visible absorption spectrum consistent with other [Fe-S] proteins (17); (iii) required both anaerobic reducing conditions and S-AdoMet for activity in vitro (17, 18); and (iv) was inactive for SP cleavage unless its (10His) tag was first removed proteolytically (17, 18). The above evidence led us to the hypothesis that SP lyase carries out catalysis using an [Fe-S] center to cleave S-AdoMet, thus generating a 5Ј-adenosyl radical that could participate either directly or indirectly in SP cleavage. Support for this hypothesis has recently been obtained by Mehl and Begley (19). Using synthetic analogues of SP, they proposed a mechanistic scenario for SP reversal to two thymines in which the radical generated from S-AdoMet cleavage by electron donation from the [4Fe-4S] center can abstract a proton from C6Ј of SP, effectively generating an SP radical that fragments readily back to two thymines (Fig. 6) (19). The...

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“…For example, Fourrey and coworkers used T (6-4)s 5 T, produced by irradiation of 5 0 -O-thymidylyl-4-thiothymidine, for mechanistic studies, which indicated that repair by (6-4) photolyases proceeds via a oxetane such as azetidine. 35 Furthermore, a synthetic route to the phosphoramidite of this thio analogue is available.…”

Section: Pyrimidine-pyrimidone-(6-4)-photoproducts and Dewar Valence mentioning

confidence: 99%

Chemical investigation of light induced DNA bipyrimidine damage and repair

2011

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In all organisms, genetic information is stored in DNA and RNA. Both of these macromolecules are damaged by many exogenous and endogenous events, with UV irradiation being one of the major sources of damage. The major photolesions formed are the cyclobutane pyrimidine dimers (CPD), pyrimidine-pyrimidone-(6-4)-photoproducts, Dewar valence isomers and, for dehydrated spore DNA, 5-(a-thyminyl)-5,6-dihydrothymine (SP). In order to be able to investigate how nature's repair and tolerance mechanisms protect the integrity of genetic information, oligonucleotides containing sequence and site-specific UV lesions are essential. This tutorial review provides an overview of synthetic procedures by which these oligonucleotides can be generated, either through phosphoramidite chemistry or direct irradiation of DNA. Moreover, a brief summary on their usage in analysing repair and tolerance processes as well as their biological effects is provided.

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Thymine Photoproducts but not Thymine Dimers Found in Ultraviolet-Irradiated Bacterial Spores

Cited by 247 publications

References 10 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

The subunit structure and catalytic mechanism of the Bacillus subtilis DNA repair enzyme spore photoproduct lyase

Chemical investigation of light induced DNA bipyrimidine damage and repair

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