Synthesis and properties of DNA containing a spore photoproduct analog

Bürckstümmer, Eva; Carell, Thomas

doi:10.1039/b810008j

Cited by 14 publications

(13 citation statements)

References 26 publications

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“…Together with previous synthetic results from our laboratory, which show incorporation of the backboneopened 3'!5' 5S and 5R lesion analogues, the whole set of the 4 possible spore lesions can now be prepared and inserted into DNA. [8] Repair studies show that the 5'!3' 5R analogue, incorporated in oligonucleotides, is efficiently repaired. Crystallization of a DNA duplex containing the 5'!…”

Section: Resultsmentioning

confidence: 99%

“…Recently, we reported the synthesis of 3'!5' lesion analogues with 5S and 5R configurations as phosphoramidite building blocks and showed the preparation of DNA duplexes containing both compounds. [8] The 5S isomer was indeed found to create the more stable duplex proving that the 3'!5' reaction would form preferentially the 5S-configured lesion. Initial studies with a dinucleotide building block provided evidence that the Spl is able to repair the 5S compound.…”

Section: Introductionmentioning

confidence: 96%

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Crystal Structures and Repair Studies Reveal the Identity and the Base‐Pairing Properties of the UV‐Induced Spore Photoproduct DNA Lesion

Heil

Kneuttinger

Schneider

et al. 2011

Chemistry A European J

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UV light is one of the major causes of DNA damage. In spore DNA, due to an unusual packing of the genetic material, a special spore photoproduct lesion (SP lesion) is formed, which is repaired by the enzyme spore photoproduct lyase (Spl), a radical S-adenosylmethionine (SAM) enzyme. We report here the synthesis and DNA incorporation of a DNA SP lesion analogue lacking the phosphodiester backbone. The oligonucleotides were used for repair studies and they were cocrystallized with a polymerase enzyme as a template to clarify the configuration of the SP lesion and to provide information about the base-pairing properties of the lesion. The structural analysis together with repair studies allowed us to clarify the identity of the preferentially repaired lesion diastereoisomer.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 96%

Crystal Structures and Repair Studies Reveal the Identity and the Base‐Pairing Properties of the UV‐Induced Spore Photoproduct DNA Lesion

Heil

Kneuttinger

Schneider

et al. 2011

Chemistry A European J

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“…The lower rate of turnover for synthetic dinucleoside SP relative to SP generated by UV irradiation of plasmid DNA is not surprising, as the synthetic SP is not constrained to the conformation found in a DNA strand, and also lacks the phosphodiester bridge and oligonucleotide strand that likely contribute considerably to substrate binding interactions in the SPL active site. We have incorporated the protected SP phosphotriester described in the Supporting Information into an oligo, however our initial assays on this indicated that the enzyme cannot repair the di-SEM protected SP analog, Carell has also incorporated a synthetic SP analog into an oligonucleotide strand, however the analog contains numerous protecting groups and no phosphodiester linkage; no assay data on the resulting oligos were presented 16. We are currently pursuing deprotection of the SP phosphotriesters (S.I.)…”

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confidence: 99%

Spore Photoproduct Lyase Catalyzes Specific Repair of the 5R but Not the 5S Spore Photoproduct

et al. 2009

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Bacterial spores are remarkable in their resistance to chemical and physical stresses, including exposure to UV radiation. The unusual UV resistance of bacterial spores is a result of the unique photochemistry of spore DNA coupled with the efficient repair of accumulated damage. Exposure of bacterial spores to UV radiation results in the formation of a methylene-bridged thymine dimer, 5-thyminyl-5,6-dihydrothymine (spore photoproduct, or SP), as the primary photoproduct. 1-3 SP accumulates in UV-irradiated spores, however it is rapidly repaired upon germination, thus giving rise to the extraordinary UV resistance of bacterial spores. 4,5 The repair of SP is catalyzed by the enzyme spore photoproduct lyase (SPL), and involves the direct reversal of SP to two thymines without base excision (Scheme 1).SPL is a member of the radical AdoMet superfamily, and utilizes a [4Fe-4S] cluster and Sadenosylmethionine (AdoMet) as essential cofactors in SP repair. 6-9 We have previously shown that SP repair is initiated by abstraction of H• from C6 of SP by an AdoMet-derived 5′-deoxyadenosyl radical; 10,11 this H-atom abstraction is thought to initiate a radical-mediated β-scission of the C5-C bridge bond in the photoproduct, as originally proposed by Mehl and Begley. 12 While two distinct diastereomers of SP (5R or 5S, Fig 1) could in principle be formed upon UV irradiation of bacterial spores, only the 5R configuration is possible for SP formed from adjacent thymines in double helical DNA, due to the constraints imposed by the DNA structure. 13 The 5S configuration, therefore, is possible only in less well-defined DNA structures or as an interstrand crosslink. It was thus quite surprising when two recent reports concluded that SPL repairs only the 5S, and not the 5R, isomer of a synthetic SP substrate. 14,15 We report here results from HPLC and MS analysis of in vitro enzymatic assays on stereochemicallydefined synthetic SP substrates demonstrating that SPL specifically repairs only the 5R isomer of SP. This stereospecific repair of 5R-SP by SPL is consistent with the longstanding hypothesis that SP is a result of UV-induced dimerization of adjacent thymines in double-helical DNA.SPL was cloned from Clostridium acetobutylicum, overexpressed in Escherichia coli, and purified using a method similar to published procedures. 11 The enzyme contained 2.9 (± 0.2) Fe per SPL, and had UV-visible and EPR spectroscopic properties characteristic of an ironsulfur enzyme. The 5R and 5S diastereomers of protected (N-SEM, O-TES, and O-TBDMS) SP were synthesized using modifications of published procedures, 13,15 and were subsequently deprotected (Supporting Information). The structures of the fully protected, the di-SEM protected, and fully deprotected dinucleoside spore products were confirmed by 1 H and 13 C NMR techniques, and NOESY and ROESY were used to assign the stereochemistry at C-5 (S.I.). In order to remove any potential ambiguity associated with the assignment of stereochemistry at C-5 in the open dinucleoside forms of SP, the...

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“…It was found that both isomers were destabilising, but the 5R isomer more so. 215 A study of short AT hairpin structures used for the formation of thymine dimers revealed that formation is highly sequence dependent as well as dependent on the position of the T-T step. 216 Pyrimidine dimer formation has been used as a method of chemical ligation of oligonucleotides.…”

Section: Oligonucleotides Containing Modified Sugarsmentioning

confidence: 99%

Nucleotides and Nucleic Acids; Oligo- and Polynucleotides

Loakes

2010

Organophosphorus Chemistry

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Synthesis and properties of DNA containing a spore photoproduct analog

Abstract: The spore photoproduct is a unique photolesion, formed in spores upon irradiation with UV light; to investigate the properties of spore photoproduct containing DNA we have synthesized 5S and 5R lesion analogs and incorporated them into DNA.

Cited by 14 publications

References 26 publications

Crystal Structures and Repair Studies Reveal the Identity and the Base‐Pairing Properties of the UV‐Induced Spore Photoproduct DNA Lesion

Crystal Structures and Repair Studies Reveal the Identity and the Base‐Pairing Properties of the UV‐Induced Spore Photoproduct DNA Lesion

Spore Photoproduct Lyase Catalyzes Specific Repair of the 5R but Not the 5S Spore Photoproduct

Nucleotides and Nucleic Acids; Oligo- and Polynucleotides

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