The spore photoproduct lyase repairs the 5S- and not the 5R-configured spore photoproduct DNA lesion

Friedel, Marcus G.; Berteau, Olivier; Pieck, J. Carsten; Atta, Mohamed; Choudens, Sandrine Ollagnier de; Fontecave, Marc; Carell, Thomas

doi:10.1039/b514103f

Cited by 40 publications

(57 citation statements)

References 27 publications

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“…PFL-AE, BioB, and LipA) in the radical AdoMet superfamily. Previously published reports indicated that SP lyase uses AdoMet as a cosubstrate (35,48); however, results from our laboratory demonstrate that SP lyase can repair SP using only catalytic amounts of AdoMet. The data in Table 1 illustrate this point.…”

Section: Overexpression and Purification Of Sp Lyasecontrasting

confidence: 56%

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

Buis

Cheek

Kalliri

et al. 2006

Journal of Biological Chemistry

103

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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...

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Section: Overexpression and Purification Of Sp Lyasecontrasting

confidence: 56%

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

Buis

Cheek

Kalliri

et al. 2006

Journal of Biological Chemistry

103

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“…Enzymatic activity with SPTpT can also be compared with that obtained with an interstrand synthetic SP dinucleoside. With the same enzyme preparations and comparable assay conditions as those used in this study, we reported a specific activity of about 0.004 mol/mol/min using the dinucleoside substrate (18). This 60-fold difference in repair activity demonstrates the importance of the phosphodiester bridge, probably because it limits the conformational freedom of the substrate dimer and suggests that the enzyme is not effective in repairing interstrand lesions.…”

Section: Discussionmentioning

confidence: 50%

“…This new substrate provides a convenient assay for further enzymatic studies. Combined with a previous study addressing the question of the stereoselectivity of the enzyme (18), this work provides new insights into the chemistry of SP lyase.…”

mentioning

confidence: 79%

“…(iii) They are repaired with the same efficiency by SP lyase. The latter result is of importance because, by using separated 5R and 5S isomers of the synthetically prepared SP thymidine derivative, we recently demonstrated that only the 5S-configured lesion was repaired, showing that SP lyase is highly stereoselective (18). The present series of observations thus strongly suggest that the same diastereoisomer was produced upon irradiation of TpT and DNA, even though further spectroscopic data are needed to definitively establish its absolute configuration (see Scheme 1A for the structure of the two stereoisomers).…”

Section: Discussionmentioning

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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“…[51][52][53] Indeed, a mechanism has been proposed and is supported by experiments such as mutation studies on SP lyase, but structural information such as crystal structures is still lacking. 54,55 …”

Section: The Spore Photoproductmentioning

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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The spore photoproduct lyase repairs the 5S- and not the 5R-configured spore photoproduct DNA lesion

Cited by 40 publications

References 27 publications

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

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

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

Chemical investigation of light induced DNA bipyrimidine damage and repair

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