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(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
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(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
reaction mechanism, overview
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(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
reaction mechanism, two different routes, overview
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(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
the enzyme catalyzes the spore photoproduct repair reaction and combines specific features of radical S-adenosyl-L-methionine and DNA repair enzymes to enable a complex radical-based repair reaction to take place
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
the enzyme catalyzes the spore photoproduct repair reaction via a radical mediated direct reverse mechanism. At the 1+ oxidation state, the [4Fe-4S] cluster provides an electron to the S-adenosyl-L-methionine, which binds to the cluster in a bidentate manner as the fourth and fifth ligands, to reductively cleave the C-S bond associated with the sulfonium ion in S-adenosyl-L-methionine, generating a reactive 5'-deoxyadenosyl radical. This 5'-dA radical abstracts the proR hydrogen atom from the C6 carbon of spore photoproduct to initiate the repair process. The resulting spore photoproduct radical subsequently fragments to generate a putative thymine methyl radical, which accepts a back-donated H atom to yield the repaired thymidylyl-(3'->5')-thymidylate. Cys141 is involved in the catalytic mechanism as the potential H atom donor to the thymine methyl radical, reaction mechanism, detailed overview. A a thiyl radical is subsequently generated on Cys141
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(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
the enzyme is a radical S-adenosyl-L-methionine enzyme, which uses a [4Fe-4S]1+ cluster to reduce the S-adenosyl-L-methionine, generating a catalytic 5'-deoxyadenosyl radical. This in turn abstracts an H atom from spore product, generating an spore product radical that undergoes beta scission to form a repaired 5'-thymine and a 3'-thymine allylic radical. A conserved cysteine donates an H atom to the thymine radical, resulting in a putative thiyl radical. Two conserved tyrosines are also critical in enzyme catalysis. One, Y99, is downstream of the cysteine, suggesting that the enzyme uses a hydrogen atom transfer pathway with a pair of cysteine-tyrosine residues to regenerate S-adenosyl-L-methionine. The other tyrosine, Y97, has a structural role to facilitate S-adenosyl-L-methionine binding. It may also contribute to the S-adenosyl-L-methionine regeneration process by interacting with the putative Y99 radical and/or 5-dA radical intermediates to lower the energy barrier for the second H-abstraction step. Irreversible first step and tightly coupled radical relay mechanism
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(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
SP repair is initiated by abstraction of a H atom from C6 of SP. The SP(C6) substrate radical is thought to promote a radical-mediated beta-scission of the C-C bond linking the two thymines; the resulting product radical then abstracts an H atom to generate repaired thymine. S-adenosyl-L-methionine as a substrate utilize a defined dinucleotide or dinucleoside SP, rather than SP in intact DNA, suggesting the possibility that stoichiometric SAM cleavage is favored with non-optimal substrates
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
SPL is a radical S-adenosylmethionine (SAM) enzyme, reaction mechanism, detailed overview. Radicals are quenched to close the catalytic cycle. The characteristic CXXXCXXC motif is involved in catalysis, although other tri-cysteine motifs may also facilitate this radical chemistry. Enzyme SPL directly directly reverts SP thymine photodimer. The conserved solvent-accessible cysteine 140 is the intrinsic hydrogen atom donor
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
SPL is a radical S-adenosylmethionine (SAM) enzyme, reaction mechanism, detailed overview. Radicals are quenched to close the catalytic cycle. The characteristic CXXXCXXC motif is involved in catalysis, although other tri-cysteine motifs may also facilitate this radical chemistry. Enzyme SPL directly reverts SP thymine photodimer. The conserved solvent-accessible cysteine 141 is the intrinsic hydrogen atom donor to the thymine radical
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
SPL is a radical S-adenosylmethionine (SAM) enzyme, via a 3'-thymine allylic radical intermediate, reaction mechanism, detailed overview
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
SPL is a radical S-adenosylmethionine (SAM) enzyme, via a 3'-thymine allylic radical intermediate, reaction mechanism, detailed overview
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
SPL is a radical S-adenosylmethionine (SAM) enzyme, via a 3'-thymine allylic radical intermediate, reaction mechanism, detailed overview
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
spore photoproduct lyase (SPL) repairs 5-thyminyl-5,6-dihydrothymine (i.e., the spore photoproduct (SP)) using a radical transfer pathway that includes at least a cysteine and a tyrosine in germinating endospores. The cysteine (at position 74) and tyrosine are located on the opposite sides of a substrate binding pocket that has to collapse to bring the two residues into proximity, enabling the C->Y radical passage
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
thymine dimers can be repaired via direct reversal mechanism
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
reaction mechanism, overview
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(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
the enzyme is a radical S-adenosyl-L-methionine enzyme, which uses a [4Fe-4S]1+ cluster to reduce the S-adenosyl-L-methionine, generating a catalytic 5'-deoxyadenosyl radical. This in turn abstracts an H atom from spore product, generating an spore product radical that undergoes beta scission to form a repaired 5'-thymine and a 3'-thymine allylic radical. A conserved cysteine donates an H atom to the thymine radical, resulting in a putative thiyl radical. Two conserved tyrosines are also critical in enzyme catalysis. One, Y99, is downstream of the cysteine, suggesting that the enzyme uses a hydrogen atom transfer pathway with a pair of cysteine-tyrosine residues to regenerate S-adenosyl-L-methionine. The other tyrosine, Y97, has a structural role to facilitate S-adenosyl-L-methionine binding. It may also contribute to the S-adenosyl-L-methionine regeneration process by interacting with the putative Y99 radical and/or 5-dA radical intermediates to lower the energy barrier for the second H-abstraction step. Irreversible first step and tightly coupled radical relay mechanism
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(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
the enzyme catalyzes the spore photoproduct repair reaction via a radical mediated direct reverse mechanism. At the 1+ oxidation state, the [4Fe-4S] cluster provides an electron to the S-adenosyl-L-methionine, which binds to the cluster in a bidentate manner as the fourth and fifth ligands, to reductively cleave the C-S bond associated with the sulfonium ion in S-adenosyl-L-methionine, generating a reactive 5'-deoxyadenosyl radical. This 5'-dA radical abstracts the proR hydrogen atom from the C6 carbon of spore photoproduct to initiate the repair process. The resulting spore photoproduct radical subsequently fragments to generate a putative thymine methyl radical, which accepts a back-donated H atom to yield the repaired thymidylyl-(3'->5')-thymidylate. Cys141 is involved in the catalytic mechanism as the potential H atom donor to the thymine methyl radical, reaction mechanism, detailed overview. A a thiyl radical is subsequently generated on Cys141
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(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
thymine dimers can be repaired via direct reversal mechanism
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(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
SPL is a radical S-adenosylmethionine (SAM) enzyme, reaction mechanism, detailed overview. Radicals are quenched to close the catalytic cycle. The characteristic CXXXCXXC motif is involved in catalysis, although other tri-cysteine motifs may also facilitate this radical chemistry. Enzyme SPL directly reverts SP thymine photodimer. The conserved solvent-accessible cysteine 141 is the intrinsic hydrogen atom donor to the thymine radical
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(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
SPL is a radical S-adenosylmethionine (SAM) enzyme, via a 3'-thymine allylic radical intermediate, reaction mechanism, detailed overview
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(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
SP repair is initiated by abstraction of a H atom from C6 of SP. The SP(C6) substrate radical is thought to promote a radical-mediated beta-scission of the C-C bond linking the two thymines; the resulting product radical then abstracts an H atom to generate repaired thymine. S-adenosyl-L-methionine as a substrate utilize a defined dinucleotide or dinucleoside SP, rather than SP in intact DNA, suggesting the possibility that stoichiometric SAM cleavage is favored with non-optimal substrates
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(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
spore photoproduct lyase (SPL) repairs 5-thyminyl-5,6-dihydrothymine (i.e., the spore photoproduct (SP)) using a radical transfer pathway that includes at least a cysteine and a tyrosine in germinating endospores. The cysteine (at position 74) and tyrosine are located on the opposite sides of a substrate binding pocket that has to collapse to bring the two residues into proximity, enabling the C->Y radical passage
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(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
SPL is a radical S-adenosylmethionine (SAM) enzyme, via a 3'-thymine allylic radical intermediate, reaction mechanism, detailed overview
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(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
SPL is a radical S-adenosylmethionine (SAM) enzyme, reaction mechanism, detailed overview. Radicals are quenched to close the catalytic cycle. The characteristic CXXXCXXC motif is involved in catalysis, although other tri-cysteine motifs may also facilitate this radical chemistry. Enzyme SPL directly directly reverts SP thymine photodimer. The conserved solvent-accessible cysteine 140 is the intrinsic hydrogen atom donor
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(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
SPL is a radical S-adenosylmethionine (SAM) enzyme, via a 3'-thymine allylic radical intermediate, reaction mechanism, detailed overview
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(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
the enzyme catalyzes the spore photoproduct repair reaction and combines specific features of radical S-adenosyl-L-methionine and DNA repair enzymes to enable a complex radical-based repair reaction to take place
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(5''R)-alpha-5''(6''H)-bithymine + S-adenosyl-L-methionine
thymidylyl-(3'-5')-thymidylate + 5'-deoxyadenosine + L-methionine
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'-5')-thymidylate
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the enzyme establishes a complex radical transfer cascade and creates a cysteine and a tyrosyl radical dyade to establish repair. This allows the enzyme to solve topological and energetic problems associated with the radical based repair reaction
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(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double helical DNA) + S-adenosyl-L-methionine + 2 H+
thymidylyl-(3'-5')-thymidylate (in DNA) + 5'-deoxyadenosine + L-methionine
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine + S-adenosyl-L-methionine
thymidylyl-(3'->5')-thymidylate + 5'-deoxyadenosine + L-methionine
(5R)-CH2-spore photoproduct + S-adenosyl-L-methionine
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a dinucleotide spore photodroduct isostere (5R)-CH2SP is prepared, which contains a neutral CH2 moiety between the two thymine residues instead of a phosphate. ROESY spectroscopic, DFT computational, and enzymatic studies of this (5R)-CH2SP compound prove that it possesses similar properties with the (5R) spore photoproduct species
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(5S)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double helical DNA)
thymidylyl-(3'-5')-thymidylate (in DNA)
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assay with a synthetic dinucleotide SP lesion substrate and with smallDNAsingle strands, which contain one SP lesion at a defined site
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5,6-dihydro-5-(thymidin-7-yl)thymidine (in double helical DNA)
thymidylyl-(3'-5')-thymidylate (in DNA)
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5-(alpha-thyminyl)-5,6-dihydrothymidine
thymidylyl-(3'-5')-thymidylate
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the enzyme repairs 5-(alpha-thyminyl)-5,6-dihydrothymidine in DNA
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5-thyminyl-5,6 dihydrothymine + S-adenosyl-L-methionine
thymidylyl-(3'-5')-thymidylate + 5'-deoxyadenosine + L-methionine
additional information
?
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(5''R)-alpha-5''(6''H)-bithymine + S-adenosyl-L-methionine
thymidylyl-(3'-5')-thymidylate + 5'-deoxyadenosine + L-methionine
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SPL repairs specifically the 5R isomer. (5''R)-alpha-5''(6''H)-bithymine is the diastereomer produced upon UV irradiation of a TpT dinucleotide
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?
(5''R)-alpha-5''(6''H)-bithymine + S-adenosyl-L-methionine
thymidylyl-(3'-5')-thymidylate + 5'-deoxyadenosine + L-methionine
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SPL repairs specifically the 5R isomer
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?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
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?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
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i.e. spore photoproduct, an in situ monomerization
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?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
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the reaction is highly stereo-selective
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?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
in double-helical DNA
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?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
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in double-helical DNA
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?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
in double-helical DNA
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?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
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in double-helical DNA
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?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
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?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
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the reaction is highly stereo-selective
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?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
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?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
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i.e. spore photoproduct, an in situ monomerization
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?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
in double-helical DNA
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?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
in double-helical DNA
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-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
-
in double-helical DNA
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?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
in double-helical DNA
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?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
in double-helical DNA
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?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
in double-helical DNA
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?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
in double-helical DNA
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?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double helical DNA) + S-adenosyl-L-methionine + 2 H+
thymidylyl-(3'-5')-thymidylate (in DNA) + 5'-deoxyadenosine + L-methionine
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?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double helical DNA) + S-adenosyl-L-methionine + 2 H+
thymidylyl-(3'-5')-thymidylate (in DNA) + 5'-deoxyadenosine + L-methionine
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?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double helical DNA) + S-adenosyl-L-methionine + 2 H+
thymidylyl-(3'-5')-thymidylate (in DNA) + 5'-deoxyadenosine + L-methionine
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the reduced enzyme rapidly and completely repairs the 5R-diastereomer of a synthetic dinucleotide SP, whereas no repair occurs with the 5S-diastereomer
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(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double helical DNA) + S-adenosyl-L-methionine + 2 H+
thymidylyl-(3'-5')-thymidylate (in DNA) + 5'-deoxyadenosine + L-methionine
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?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double helical DNA) + S-adenosyl-L-methionine + 2 H+
thymidylyl-(3'-5')-thymidylate (in DNA) + 5'-deoxyadenosine + L-methionine
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the reduced enzyme rapidly and completely repairs the 5R-diastereomer of a synthetic dinucleotide SP, whereas no repair occurs with the 5S-diastereomer
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?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double helical DNA) + S-adenosyl-L-methionine + 2 H+
thymidylyl-(3'-5')-thymidylate (in DNA) + 5'-deoxyadenosine + L-methionine
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assay with a synthetic dinucleotide SP lesion substrate and with smallDNAsingle strands, which contain one SP lesion at a defined site
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?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine + S-adenosyl-L-methionine
thymidylyl-(3'->5')-thymidylate + 5'-deoxyadenosine + L-methionine
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no repair is observed for the (5S) diasteroisomer
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?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine + S-adenosyl-L-methionine
thymidylyl-(3'->5')-thymidylate + 5'-deoxyadenosine + L-methionine
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no repair is observed for the (5S) diasteroisomer
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?
5-thyminyl-5,6 dihydrothymine + S-adenosyl-L-methionine
thymidylyl-(3'-5')-thymidylate + 5'-deoxyadenosine + L-methionine
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?
5-thyminyl-5,6 dihydrothymine + S-adenosyl-L-methionine
thymidylyl-(3'-5')-thymidylate + 5'-deoxyadenosine + L-methionine
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via organic synthesis and DNA photochemistry, the two H-atoms at the C6 carbon (6-HproS or 6-HproR position) are selectively labeled with a deuterium in a dinucleotide spore photoproduct TpT substrate. Monitoring the deuterium migration in enzyme catalysis reveals that it is the 6-HproR atom of spore photoproduct that is abstracted by the 5'-dA radical. The abstracted deuterium is not returned to the resulting TpT after enzymatic catalysis, an H-atom from the aqueous buffer is incorporated into TpT instead
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additional information
?
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the (5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine lesion does not absolutely need to be contained within a single or double-stranded DNA for recognition and repaired by the enzyme
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additional information
?
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the enzyme recognizes and repairs a range of spore photoproduct containing DNA substrates, ranging from dinucleotide and dinucleoside spore photoproduct to spore photoproduct containing single- and double-stranded DNA. The fastest reaction rate employs a single-stranded spore photoproduct containing GGSPGG 6-mer as the substrate, while the double-stranded spore photoproduct-containing plasmid DNA also supports a fast repair reaction
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additional information
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DNA conformation during in vivo repair and dinucleotide SP TpT flipping process, overview
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additional information
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SP-containing (prepared via UV irradiation) pUC 18 plasmid or Escherichia coli genomic DNA is used as the substrate, or synthesized SP TpT or SP TT-containing 10-mer, 13-mer and 20-mer ss/ds oligonucleotides are used. Stable isotope dilution mass spectrometry (SID-MRM-MS) substrate and product analysis, enzyme substrate specificity and activity analysis, overview. Spore photoproduct within DNA is a surprisingly poor substrate for its designated repair enzyme, the spore photoproduct lyase. Minor DNA conformational changes induced by SP TpT may explain low SPL activity in vitro. Slow SP TT repair may also be due to small conformational changes
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additional information
?
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the enzyme recognizes and repairs a range of spore photoproduct containing DNA substrates, ranging from dinucleotide and dinucleoside spore photoproduct to spore photoproduct containing single- and double-stranded DNA. The fastest reaction rate employs a single-stranded spore photoproduct containing GGSPGG 6-mer as the substrate, while the double-stranded spore photoproduct-containing plasmid DNA also supports a fast repair reaction
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?
additional information
?
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SP-containing (prepared via UV irradiation) pUC 18 plasmid or Escherichia coli genomic DNA is used as the substrate, or synthesized SP TpT or SP TT-containing 10-mer, 13-mer and 20-mer ss/ds oligonucleotides are used. Stable isotope dilution mass spectrometry (SID-MRM-MS) substrate and product analysis, enzyme substrate specificity and activity analysis, overview. Spore photoproduct within DNA is a surprisingly poor substrate for its designated repair enzyme, the spore photoproduct lyase. Minor DNA conformational changes induced by SP TpT may explain low SPL activity in vitro. Slow SP TT repair may also be due to small conformational changes
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additional information
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DNA conformation during in vivo repair and dinucleotide SP TpT flipping process, overview
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additional information
?
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solution phase dynamics of the DNA repair enzyme spore photoproduct lyase as probed by H/D exchange with HDX as a probe to explore the solution phase conformational dynamics in SPL upon binding to undamaged DNA and dinucleotide 5R-SPTpT and dinucleoside 5R-SP substrates. A 6-mer oligonucleotide, 5'-GCAAGT-3', is used as substrate, which is cleaved to and complement 5'-ACT and TGC-3', overview. Mass spectrometric substrate and product analyses. Enzyme SPL has low affinity for substrate or DNA in the absence of S-adenosyl-L-methionine
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additional information
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substrate and product analysis by mass spectrometry
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additional information
?
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substrate and product analysis by mass spectrometry
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?
additional information
?
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solution phase dynamics of the DNA repair enzyme spore photoproduct lyase as probed by H/D exchange with HDX as a probe to explore the solution phase conformational dynamics in SPL upon binding to undamaged DNA and dinucleotide 5R-SPTpT and dinucleoside 5R-SP substrates. A 6-mer oligonucleotide, 5'-GCAAGT-3', is used as substrate, which is cleaved to and complement 5'-ACT and TGC-3', overview. Mass spectrometric substrate and product analyses. Enzyme SPL has low affinity for substrate or DNA in the absence of S-adenosyl-L-methionine
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additional information
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substrate and product analysis by mass spectrometry
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?
additional information
?
-
synthesis and DNA incorporation of a DNA SP lesion analogue lacking the phosphodiester backbone is reported. Repair studies show that the 5'-3' (5R) analogue, incorporated in oligonucleotides, is efficiently repaired
-
-
?
additional information
?
-
DNA conformation during in vivo repair and dinucleotide SP TpT flipping process, overview
-
-
?
additional information
?
-
activity of the reconstituted wild-type enzyme and the enzyme mutants is measured using a 13-mer oligonucleotide containing the dinucleoside SP (5'-CAGCGGT-TGCAGG-3') as substrate, mass spectrometric poduct analysis, overview. The repair of the SP containing DNA leads to two oligonucleotides: the 7 mer (5'-CAGCGGT-3') and the 6 mer (5'-TGCAGG-3')
-
-
?
additional information
?
-
activity of the reconstituted wild-type enzyme and the enzyme mutants is measured using a 13-mer oligonucleotide containing the dinucleoside SP (5'-CAGCGGT-TGCAGG-3') as substrate, mass spectrometric poduct analysis, overview. The repair of the SP containing DNA leads to two oligonucleotides: the 7 mer (5'-CAGCGGT-3') and the 6 mer (5'-TGCAGG-3')
-
-
?
additional information
?
-
DNA conformation during in vivo repair and dinucleotide SP TpT flipping process, overview
-
-
?
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(5''R)-alpha-5''(6''H)-bithymine + S-adenosyl-L-methionine
thymidylyl-(3'-5')-thymidylate + 5'-deoxyadenosine + L-methionine
-
SPL repairs specifically the 5R isomer. (5''R)-alpha-5''(6''H)-bithymine is the diastereomer produced upon UV irradiation of a TpT dinucleotide
-
-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'-5')-thymidylate
-
the enzyme establishes a complex radical transfer cascade and creates a cysteine and a tyrosyl radical dyade to establish repair. This allows the enzyme to solve topological and energetic problems associated with the radical based repair reaction
-
-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double helical DNA) + S-adenosyl-L-methionine + 2 H+
thymidylyl-(3'-5')-thymidylate (in DNA) + 5'-deoxyadenosine + L-methionine
5,6-dihydro-5-(thymidin-7-yl)thymidine (in double helical DNA)
thymidylyl-(3'-5')-thymidylate (in DNA)
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-
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-
?
additional information
?
-
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
-
-
-
-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
in double-helical DNA
-
-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
-
in double-helical DNA
-
-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
in double-helical DNA
-
-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
-
in double-helical DNA
-
-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
-
-
-
-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
in double-helical DNA
-
-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
in double-helical DNA
-
-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
-
in double-helical DNA
-
-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
in double-helical DNA
-
-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
in double-helical DNA
-
-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
in double-helical DNA
-
-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
in double-helical DNA
-
-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double helical DNA) + S-adenosyl-L-methionine + 2 H+
thymidylyl-(3'-5')-thymidylate (in DNA) + 5'-deoxyadenosine + L-methionine
-
-
-
-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double helical DNA) + S-adenosyl-L-methionine + 2 H+
thymidylyl-(3'-5')-thymidylate (in DNA) + 5'-deoxyadenosine + L-methionine
-
-
-
-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double helical DNA) + S-adenosyl-L-methionine + 2 H+
thymidylyl-(3'-5')-thymidylate (in DNA) + 5'-deoxyadenosine + L-methionine
-
-
-
-
?
additional information
?
-
-
the (5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine lesion does not absolutely need to be contained within a single or double-stranded DNA for recognition and repaired by the enzyme
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-
?
additional information
?
-
DNA conformation during in vivo repair and dinucleotide SP TpT flipping process, overview
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-
?
additional information
?
-
DNA conformation during in vivo repair and dinucleotide SP TpT flipping process, overview
-
-
?
additional information
?
-
DNA conformation during in vivo repair and dinucleotide SP TpT flipping process, overview
-
-
?
additional information
?
-
DNA conformation during in vivo repair and dinucleotide SP TpT flipping process, overview
-
-
?
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evolution
-
the enzyme is a member of the radical SAM superfamily
evolution
-
the enzyme is a member of the so-called radical SAM superfamily, which is defined by the characteristic CXXXCXXC motif. The three cysteine residues serve as ligands respectively for three irons in the [4Fe-4S] cluster, with the fourth iron being coordinated by the S-adenosylmethionine in a bi-dentate manner, with its amino and carboxylate moieties serving as the fourth and fifth ligands to the cluster. SPL and the DNA photolyase, EC 4.1.99.3, show amino acid sequence homolgy and might have descended from a common ancestral protein
evolution
-
the enzyme is the first member of the radical SAM superfamily (comprising more than 44000 members) to bear a catalytically operating hydrogen atom transfer chain that is essential for S-adenosyl-L-methionine regeneration after the catalytic cycle
evolution
unlike DNA photolyases, EC 4.1.99.3, SP lyase belongs to the emerging superfamily of radical S-adenosyl-L-methionine (SAM) enzymes
evolution
enzyme SPL belongs to the radical SAM (S-adenosylmethionine) enzyme superfamily. The superfamily is defined by the characteristic tri-cysteinyl motif: CX3CX2C that binds to a [4Fe-4S] cluster
evolution
spore photoproduct lyase (SPL) is a member of the radical S-adenosyl-L-methionine (SAM) superfamily, the family members utilize S-adenosyl-Lmethionine (SAM) and a redox active [4Fe-4S] cluster to carry out diverse radical reactions including rearrangements, sulfur insertions and oxidations. Electron-nuclear double resonance and X-ray crystallography of several members of the superfamily have shown that the unique iron is coordinated by the amino and carboxylate moieties of S-adenosyl-L-methionine. An innersphere electron transfer from a reduced [4Fe-4S]+ cluster to the sulfonium of SAM leads to homolytic S-C(5') bond cleavage to generate a 5'-deoxyadenosyl radical (dAdo) intermediate, which abstracts a hydrogen atom from substrate to initiate a radical transformation
evolution
the enzyme is a member of the radical SAM superfamily, but SP lyase is the only known radical SAM enzyme to date catalyzing DNA repair. Clostridia SP lyases possess a conserved cysteine residue upstream the radical SAM motif (Cys74), which is substituted by a serine residue in Bacilli SP lyases
evolution
the enzyme is a member of the radical SAM superfamily, but SP lyase is the only known radical SAM enzyme to date catalyzing DNA repair. Clostridia SP lyases possess a conserved cysteine residue upstream the radical SAM motif (Cys74), which is substituted by a serine residue in Bacilli SP lyases. Cys74 in Ca SP lyase occupies a critical location similar to Cys140 in Gt SP lyase
evolution
the enzyme is a member of the radical SAM superfamily, but SP lyase is the only known radical SAM enzyme to date catalyzing DNA repair. Cys74 in Ca SP lyase occupies a critical location similar to Cys140 in Gt SP lyase
evolution
the enzyme is a member of the radical SAM superfamily, which is defined by the characteristic CXXXCXXC motif
evolution
the enzyme is a member of the radical SAM superfamily, which is defined by the characteristic CXXXCXXC motif
evolution
-
the enzyme is a member of the radical SAM superfamily
-
evolution
-
the enzyme is the first member of the radical SAM superfamily (comprising more than 44000 members) to bear a catalytically operating hydrogen atom transfer chain that is essential for S-adenosyl-L-methionine regeneration after the catalytic cycle
-
evolution
-
the enzyme is a member of the so-called radical SAM superfamily, which is defined by the characteristic CXXXCXXC motif. The three cysteine residues serve as ligands respectively for three irons in the [4Fe-4S] cluster, with the fourth iron being coordinated by the S-adenosylmethionine in a bi-dentate manner, with its amino and carboxylate moieties serving as the fourth and fifth ligands to the cluster. SPL and the DNA photolyase, EC 4.1.99.3, show amino acid sequence homolgy and might have descended from a common ancestral protein
-
evolution
-
the enzyme is a member of the radical SAM superfamily, which is defined by the characteristic CXXXCXXC motif
-
evolution
-
the enzyme is a member of the radical SAM superfamily, but SP lyase is the only known radical SAM enzyme to date catalyzing DNA repair. Clostridia SP lyases possess a conserved cysteine residue upstream the radical SAM motif (Cys74), which is substituted by a serine residue in Bacilli SP lyases
-
evolution
-
spore photoproduct lyase (SPL) is a member of the radical S-adenosyl-L-methionine (SAM) superfamily, the family members utilize S-adenosyl-Lmethionine (SAM) and a redox active [4Fe-4S] cluster to carry out diverse radical reactions including rearrangements, sulfur insertions and oxidations. Electron-nuclear double resonance and X-ray crystallography of several members of the superfamily have shown that the unique iron is coordinated by the amino and carboxylate moieties of S-adenosyl-L-methionine. An innersphere electron transfer from a reduced [4Fe-4S]+ cluster to the sulfonium of SAM leads to homolytic S-C(5') bond cleavage to generate a 5'-deoxyadenosyl radical (dAdo) intermediate, which abstracts a hydrogen atom from substrate to initiate a radical transformation
-
evolution
-
enzyme SPL belongs to the radical SAM (S-adenosylmethionine) enzyme superfamily. The superfamily is defined by the characteristic tri-cysteinyl motif: CX3CX2C that binds to a [4Fe-4S] cluster
-
evolution
-
the enzyme is a member of the radical SAM superfamily, but SP lyase is the only known radical SAM enzyme to date catalyzing DNA repair. Clostridia SP lyases possess a conserved cysteine residue upstream the radical SAM motif (Cys74), which is substituted by a serine residue in Bacilli SP lyases. Cys74 in Ca SP lyase occupies a critical location similar to Cys140 in Gt SP lyase
-
evolution
-
the enzyme is a member of the radical SAM superfamily, which is defined by the characteristic CXXXCXXC motif
-
evolution
-
the enzyme is a member of the radical SAM superfamily, but SP lyase is the only known radical SAM enzyme to date catalyzing DNA repair. Cys74 in Ca SP lyase occupies a critical location similar to Cys140 in Gt SP lyase
-
evolution
-
unlike DNA photolyases, EC 4.1.99.3, SP lyase belongs to the emerging superfamily of radical S-adenosyl-L-methionine (SAM) enzymes
-
malfunction
-
the enzyme C141A mutant produces thymidylyl-(3'->5')-thymidylate likely via an unprecedented thymine radical cation reduction (proton coupled electron transfer) mechanism
malfunction
contrary to the C140A mutant, the double mutant repaires the DNA lesion without generating DNA adducts. Furthermore, in the crystal structure, the mutant S76C is shown to be at 4.1 A from the methylene bridge of SP which is a shorter distance than the one reported for the wild-type enzyme
malfunction
mutation at the remote glycine 168 residue alters the enzyme 3D structure, subsequently reducing the SPL activity by changing the positions of the essential amino acids involved in the radical transfer process
malfunction
-
the enzyme C141A mutant produces thymidylyl-(3'->5')-thymidylate likely via an unprecedented thymine radical cation reduction (proton coupled electron transfer) mechanism
-
malfunction
-
mutation at the remote glycine 168 residue alters the enzyme 3D structure, subsequently reducing the SPL activity by changing the positions of the essential amino acids involved in the radical transfer process
-
malfunction
-
contrary to the C140A mutant, the double mutant repaires the DNA lesion without generating DNA adducts. Furthermore, in the crystal structure, the mutant S76C is shown to be at 4.1 A from the methylene bridge of SP which is a shorter distance than the one reported for the wild-type enzyme
-
physiological function
-
the overwhelming majority of DNA photoproducts in UV-irradiated spores is a unique thymine dimer called spore photoproduct, SP, or 5-thymine-5,6-dihydrothymine. This lesion is repaired by the spore photoproduct lyase enzyme that directly reverts 5-thymine-5,6-dihydrothymine to two unmodified thymines. The SP lyase enzyme demonstrates an aspect of the diversity of DNA repair mechanisms in living organisms
physiological function
-
spore photoproduct lyase repairs a covalent UV-induced thymine dimer, spore photoproduct, in germinating endospores and is responsible for endospores' strong UV resistance. The spore photoproduct is rapidly repaired by the metalloenzyme spore photoproduct lyase when spores start germinating
physiological function
-
spore photoproduct lyase repairs a covalent UV-induced thymine dimer, spore photoproduct, in germinating endospores and is responsible for strong UV resistance of endospores
physiological function
-
spore photoproduct lyase repairs a special thymine dimer 5-thyminyl-5,6-dihydrothymine, which is commonly called spore photoproduct at the bacterial early germination phase. Spore photoproduct is the exclusive DNA photo-damage product in bacterial endospores. Its generation and swift repair by the enzyme are responsible for the spores' extremely high UV resistance
physiological function
on exposure of spores to UV radiation, an unique methylene bridged thymine dimer, 5-thyminyl-5,6-dihydrothymine (spore photoproduct or SP) accumulates as the main photoproduct. This accumulated photo-damage is rapidly repaired upon spore germination. Spore photoproduct lyase (SPL) catalyzes the repair of the UV lesion spore photoproduct (SP) in a reaction dependent on S-adenosyl-L-methionine (SAM)
physiological function
spore photoproduct lyase (SPL) catalyzes the direct reversal of a specific DNA photoproduct, 5-thyminyl-5,6-dihydrothymine (spore photoproduct or SP), back to two thymines. The methylene-bridged thymine dimer SP is the primary photoproduct when bacterial spores are subjected to UV radiation, and is rapidly repaired upon spore germination
physiological function
spore photoproduct lyase (SPL) catalyzes the direct reversal of a thymine dimer 5-thyminyl-5,6 dihydrothymine, i.e., the spore photoproduct (SP) to two thymine residues in germinating endospores
physiological function
spore photoproduct lyase (SPL) repairs 5-thyminyl-5,6-dihydrothymine, a thymine dimer that is also called the spore photoproduct (SP), in germinating endospores. SPL is a radical S-adenosylmethionine (SAM) enzyme, utilizing the 5'-deoxyadenosyl radical generated by SAM reductive cleavage reaction to revert SP to two thymine residues
physiological function
spore photoproduct lyase (SPL) repairs 5-thyminyl-5,6-dihydrothymine, a thymine dimer that is also called the spore photoproduct (SP), in germinating endospores. SPL is a radical S-adenosylmethionine (SAM) enzyme, utilizing the 5'-deoxyadenosyl radical generated by SAM reductive cleavage reaction to revert SP to two thymine residues
physiological function
the radical SAM enzyme, spore photoproduct lyase, requires an H-atom transfer (HAT) pathway to catalyze DNA repair
physiological function
UV radiation triggers the formation of 5-thyminyl-5,6-dihydrothymine, i.e., the spore photoproduct (SP), in the genomic DNA of bacterial endospores. These SPs, if not repaired in time, may lead to genome instability and cell death. SP is mainly repaired by spore photoproduct lyase (SPL) during spore outgrowth via an unprecedented protein-harbored radical transfer pathway that is composed of at least a cysteine and two tyrosine residues. 5-thyminyl-5,6-dihydrothymine, i.e. the spore photoproduct (SP), is the dominant DNA photolesion found in bacterial endospores
physiological function
-
spore photoproduct lyase repairs a covalent UV-induced thymine dimer, spore photoproduct, in germinating endospores and is responsible for endospores' strong UV resistance. The spore photoproduct is rapidly repaired by the metalloenzyme spore photoproduct lyase when spores start germinating
-
physiological function
-
spore photoproduct lyase repairs a covalent UV-induced thymine dimer, spore photoproduct, in germinating endospores and is responsible for strong UV resistance of endospores
-
physiological function
-
spore photoproduct lyase repairs a special thymine dimer 5-thyminyl-5,6-dihydrothymine, which is commonly called spore photoproduct at the bacterial early germination phase. Spore photoproduct is the exclusive DNA photo-damage product in bacterial endospores. Its generation and swift repair by the enzyme are responsible for the spores' extremely high UV resistance
-
physiological function
-
UV radiation triggers the formation of 5-thyminyl-5,6-dihydrothymine, i.e., the spore photoproduct (SP), in the genomic DNA of bacterial endospores. These SPs, if not repaired in time, may lead to genome instability and cell death. SP is mainly repaired by spore photoproduct lyase (SPL) during spore outgrowth via an unprecedented protein-harbored radical transfer pathway that is composed of at least a cysteine and two tyrosine residues. 5-thyminyl-5,6-dihydrothymine, i.e. the spore photoproduct (SP), is the dominant DNA photolesion found in bacterial endospores
-
physiological function
-
spore photoproduct lyase (SPL) repairs 5-thyminyl-5,6-dihydrothymine, a thymine dimer that is also called the spore photoproduct (SP), in germinating endospores. SPL is a radical S-adenosylmethionine (SAM) enzyme, utilizing the 5'-deoxyadenosyl radical generated by SAM reductive cleavage reaction to revert SP to two thymine residues
-
physiological function
-
on exposure of spores to UV radiation, an unique methylene bridged thymine dimer, 5-thyminyl-5,6-dihydrothymine (spore photoproduct or SP) accumulates as the main photoproduct. This accumulated photo-damage is rapidly repaired upon spore germination. Spore photoproduct lyase (SPL) catalyzes the repair of the UV lesion spore photoproduct (SP) in a reaction dependent on S-adenosyl-L-methionine (SAM)
-
physiological function
-
spore photoproduct lyase (SPL) catalyzes the direct reversal of a specific DNA photoproduct, 5-thyminyl-5,6-dihydrothymine (spore photoproduct or SP), back to two thymines. The methylene-bridged thymine dimer SP is the primary photoproduct when bacterial spores are subjected to UV radiation, and is rapidly repaired upon spore germination
-
physiological function
-
spore photoproduct lyase (SPL) catalyzes the direct reversal of a thymine dimer 5-thyminyl-5,6 dihydrothymine, i.e., the spore photoproduct (SP) to two thymine residues in germinating endospores
-
physiological function
-
the radical SAM enzyme, spore photoproduct lyase, requires an H-atom transfer (HAT) pathway to catalyze DNA repair
-
physiological function
-
spore photoproduct lyase (SPL) repairs 5-thyminyl-5,6-dihydrothymine, a thymine dimer that is also called the spore photoproduct (SP), in germinating endospores. SPL is a radical S-adenosylmethionine (SAM) enzyme, utilizing the 5'-deoxyadenosyl radical generated by SAM reductive cleavage reaction to revert SP to two thymine residues
-
additional information
-
spectral analysis of the purified recombinant enzyme, overview
additional information
active site structure, DNA lesion recognition, and substrate binding which involve a beta-hairpin structure, overview. S-adenosyl-L-methionine and a conserved cysteine residue are perfectly positioned in the active site for hydrogen atom abstraction from the dihydrothymine residue of the lesion and donation to the alpha-thyminyl radical moiety, respectively. Structure comparison of wild-type and C140 mutant enzymes, overview
additional information
-
residue C141 is solvent exposable and no other protein residue locates between the thymidylyl-(3'->5')-thymidylate radical and the C141 in the wild--type enzyme reaction pathway
additional information
-
residues Cys140 and Tyr98 are important for establishing catalytic turnover. While the allyl radical is situated and reduced at the 3'-side, transfer of the radical center back to the 5'-dAdoH requires moving the radical back to the 50-part in the active side over a distance of roughly 10 A. This creates next to a topological problem also an energetic obstacle, because regeneration of the adenosyl radical by the thiyl radical would be endothermic. The enzyme uses a further tyrosyl radical intermediate to solve the energetic and topological problem
additional information
-
the enzyme protects at least 9 nucleotides in the spore photoproduct containing DNA strand with 5 nucleotides 3' to and 2 nucleotides 5' to the spore photoproduct damage, suggesting that the phosphates included in this region may be involved in the binding interaction with the enzyme
additional information
a cysteine and two tyrosine residues are located in proximity and able to participate in the radical transfer process during the enzyme catalysis
additional information
-
a cysteine and two tyrosine residues are located in proximity and able to participate in the radical transfer process during the enzyme catalysis
additional information
active site structure of Gt SPL in complex with substrate and S-adenosyl-L-methionine, Cys140, Tyr96, and Tyr98 are active site residues, overview
additional information
combined Mössbauer, multi-edge X-ray absorption spectroscopic, and density functional theoretical study of theradical SAM enzyme spore photoproduct lyase, detailed overview. SPL requires S-adenosyl-L-methionine (SAM) and a redox active [4Fe-4S] cluster for catalysis
additional information
conformational changes associated with cofactor and substrate binding may serve to provide a solvent inaccessible and protected active site to safely catalyze radical reactions
additional information
Cys141, Tyr97, and Tyr99 are active site residues
additional information
the catalytic the cysteine residue is localized in a loop within the active site and in a very close proximity to the substrate at a distance of 4.5 A from the methylene bridge of the SP dimer
additional information
the catalytic the cysteine residue is localized in a loop within the active site and in a very close proximity to the substrate at a distance of 4.5 A from the methylene bridge of the SP dimer
additional information
-
the catalytic the cysteine residue is localized in a loop within the active site and in a very close proximity to the substrate at a distance of 4.5 A from the methylene bridge of the SP dimer
additional information
the repair of dinucleotide SP TpT by SPL(Ca) is 8-10-fold slower than that by SPL from Bacillus subtilis. The process also generates a large portion of the aborted product TpTSO2-. SPL(Ca) exhibits apparent (DV) kinetic isotope effects (KIEs) of about 6 and abnormally large competitive (DV/K) KIEs (about 20), both of which are much larger than the KIEs observed in from Bacillus subtilis. Clostridium acetobutilicum SPL(Ca) possesses a flexible active site and readily undergoes conformational changes during catalysis. Apparent (DV) kinetics isotope effect (KIE) determination, competitive (DV/K) KIE determination, and HDX-MS analysis of enzyme reaction and structure, overview
additional information
-
the repair of dinucleotide SP TpT by SPL(Ca) is 8-10-fold slower than that by SPL from Bacillus subtilis. The process also generates a large portion of the aborted product TpTSO2-. SPL(Ca) exhibits apparent (DV) kinetic isotope effects (KIEs) of about 6 and abnormally large competitive (DV/K) KIEs (about 20), both of which are much larger than the KIEs observed in from Bacillus subtilis. Clostridium acetobutilicum SPL(Ca) possesses a flexible active site and readily undergoes conformational changes during catalysis. Apparent (DV) kinetics isotope effect (KIE) determination, competitive (DV/K) KIE determination, and HDX-MS analysis of enzyme reaction and structure, overview
additional information
-
residue C141 is solvent exposable and no other protein residue locates between the thymidylyl-(3'->5')-thymidylate radical and the C141 in the wild--type enzyme reaction pathway
-
additional information
-
the enzyme protects at least 9 nucleotides in the spore photoproduct containing DNA strand with 5 nucleotides 3' to and 2 nucleotides 5' to the spore photoproduct damage, suggesting that the phosphates included in this region may be involved in the binding interaction with the enzyme
-
additional information
-
a cysteine and two tyrosine residues are located in proximity and able to participate in the radical transfer process during the enzyme catalysis
-
additional information
-
Cys141, Tyr97, and Tyr99 are active site residues
-
additional information
-
the catalytic the cysteine residue is localized in a loop within the active site and in a very close proximity to the substrate at a distance of 4.5 A from the methylene bridge of the SP dimer
-
additional information
-
spectral analysis of the purified recombinant enzyme, overview
-
additional information
-
conformational changes associated with cofactor and substrate binding may serve to provide a solvent inaccessible and protected active site to safely catalyze radical reactions
-
additional information
-
combined Mössbauer, multi-edge X-ray absorption spectroscopic, and density functional theoretical study of theradical SAM enzyme spore photoproduct lyase, detailed overview. SPL requires S-adenosyl-L-methionine (SAM) and a redox active [4Fe-4S] cluster for catalysis
-
additional information
-
the repair of dinucleotide SP TpT by SPL(Ca) is 8-10-fold slower than that by SPL from Bacillus subtilis. The process also generates a large portion of the aborted product TpTSO2-. SPL(Ca) exhibits apparent (DV) kinetic isotope effects (KIEs) of about 6 and abnormally large competitive (DV/K) KIEs (about 20), both of which are much larger than the KIEs observed in from Bacillus subtilis. Clostridium acetobutilicum SPL(Ca) possesses a flexible active site and readily undergoes conformational changes during catalysis. Apparent (DV) kinetics isotope effect (KIE) determination, competitive (DV/K) KIE determination, and HDX-MS analysis of enzyme reaction and structure, overview
-
additional information
-
active site structure of Gt SPL in complex with substrate and S-adenosyl-L-methionine, Cys140, Tyr96, and Tyr98 are active site residues, overview
-
additional information
-
the catalytic the cysteine residue is localized in a loop within the active site and in a very close proximity to the substrate at a distance of 4.5 A from the methylene bridge of the SP dimer
-
additional information
-
active site structure, DNA lesion recognition, and substrate binding which involve a beta-hairpin structure, overview. S-adenosyl-L-methionine and a conserved cysteine residue are perfectly positioned in the active site for hydrogen atom abstraction from the dihydrothymine residue of the lesion and donation to the alpha-thyminyl radical moiety, respectively. Structure comparison of wild-type and C140 mutant enzymes, overview
-
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G168A
site-directed mutagenesis, the mutant exhibits 3-4fold reduced enzyme activity compared to the wild-type enzyme, the mutant exhibits a smaller apparent kinetic isotope effect (KIE) but a bigger competitive KIE than the wild-type SPL
G168R
site-directed mutagenesis, the mutant exhibits 80fold reduced enzyme activity compared to the wild-type enzyme, the mutant exhibits a smaller apparent kinetic isotope effect (KIE) but a bigger competitive KIE than the wild-type SPL. G168R mutant also produces a large portion of the abortive repair product TpT-SO2-, the formation of which indicates that cysteine 141 is no longer well positioned as the H-donor to the thymine allylic radical intermediate
Y97A
-
site-directed mutagenesis, the mutation disrupts the interaction between the phenol ring of the Y97 and the adenine ring of S-adenosyl-L-methionine, subsequently affecting S-adenosyl-L-methionine binding and/or reductive cleavage, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme
Y97A/Y99A
-
site-directed mutagenesis, inactive mutant, the Y97 mutation disrupts the interaction between the phenol ring of the Y97 and the adenine ring of S-adenosyl-L-methionine, subsequently affecting S-adenosyl-L-methionine binding and/or reductive cleavage
Y97F
-
site-directed mutagenesis, the mutation disrupts the interaction between the phenol ring of the Y97 and the adenine ring of S-adenosyl-L-methionine, subsequently affecting S-adenosyl-L-methionine binding and/or reductive cleavage, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme
Y98A
-
site-directed mutagenesis, the mutant's active site architecture, activity, and kinetics are similar to the wild-type enzyme
Y98F
-
site-directed mutagenesis, the mutant's active site architecture, activity, and kinetics are similar to the wild-type enzyme
Y99A
-
site-directed mutagenesis, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme
Y99F
-
site-directed mutagenesis, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme
G168A
-
site-directed mutagenesis, the mutant exhibits 3-4fold reduced enzyme activity compared to the wild-type enzyme, the mutant exhibits a smaller apparent kinetic isotope effect (KIE) but a bigger competitive KIE than the wild-type SPL
-
G168R
-
site-directed mutagenesis, the mutant exhibits 80fold reduced enzyme activity compared to the wild-type enzyme, the mutant exhibits a smaller apparent kinetic isotope effect (KIE) but a bigger competitive KIE than the wild-type SPL. G168R mutant also produces a large portion of the abortive repair product TpT-SO2-, the formation of which indicates that cysteine 141 is no longer well positioned as the H-donor to the thymine allylic radical intermediate
-
Y97F
-
site-directed mutagenesis, the mutation disrupts the interaction between the phenol ring of the Y97 and the adenine ring of S-adenosyl-L-methionine, subsequently affecting S-adenosyl-L-methionine binding and/or reductive cleavage, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme
-
Y98A
-
site-directed mutagenesis, the mutant's active site architecture, activity, and kinetics are similar to the wild-type enzyme
-
Y99F
-
site-directed mutagenesis, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme
-
C140G
-
site-directed mutagenesis, the mutant still shows catalytic turnover, but reduced activity, due to a replacement of the thiyl radical by a thermodynamically comparable glycyl radical16 in the catalytic cycle
Y98F
-
the mutant shows in addition a reduced substrate binding affinity, which indicates that the phenolic hydroxyl group is important to organize the substrate in the active site
C141A
-
site-directed mutagenesis, the mutant repairs spore photoproduct at a rate which is about 3fold slower than the wild-type enzyme, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme. S-adenosyl-L-methionine plays a non-catalytical role in the mutant in contrast to the wild-type enzyme
C141A
-
site-directed mutagenesis, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme
C141A
site-directed mutagenesis, mutant enzyme activity analysis using an SP dinucleoside and an SP-containing oligonucleotide
C141A
site-directed mutagenesis, spores carrying the C141A mutation are sensitive to UV irradiation
C141A
-
site-directed mutagenesis, the mutant repairs spore photoproduct at a rate which is about 3fold slower than the wild-type enzyme, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme. S-adenosyl-L-methionine plays a non-catalytical role in the mutant in contrast to the wild-type enzyme
-
C141A
-
site-directed mutagenesis, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme
-
C141A
-
site-directed mutagenesis, spores carrying the C141A mutation are sensitive to UV irradiation
-
C141A
-
site-directed mutagenesis, mutant enzyme activity analysis using an SP dinucleoside and an SP-containing oligonucleotide
-
C140A
site-directed mutagenesis
C140A
site-directed mutagenesis, the mutant shows a similar protein and substrate binding structure compared to the wild-type enzyme, but 2.5fold reduced repair activity
C140A
-
site-directed mutagenesis, the mutant still shows catalytic turnover, but reduced activity, due to a replacement of the thiyl radical by a thermodynamically comparable glycyl radical16 in the catalytic cycle
C140A
site-directed mutagenesis, mutant enzyme activity analysis using an SP dinucleoside and an SP-containing oligonucleotide
C140A/S76C
site-directed mutagenesis
C140A/S76C
site-directed mutagenesis, contrary to the C140A mutant, the double mutant repaires the DNA lesion without generating DNA adducts. Furthermore, in the crystal structure, the mutant S76C is shown to be at 4.1 A from the methylene bridge of SP which is a shorter distance than the one reported for the wild-type enzyme
C140S
site-directed mutagenesis, the mutant shows a similar protein and substrate binding structure compared to the wild-type enzyme, but reduced repair activity
C140S
site-directed mutagenesis, mutant enzyme activity analysis using an SP dinucleoside and an SP-containing oligonucleotide
C140A
-
site-directed mutagenesis
-
C140A
-
site-directed mutagenesis, mutant enzyme activity analysis using an SP dinucleoside and an SP-containing oligonucleotide
-
C140A
-
site-directed mutagenesis, the mutant shows a similar protein and substrate binding structure compared to the wild-type enzyme, but 2.5fold reduced repair activity
-
C140A/S76C
-
site-directed mutagenesis
-
C140A/S76C
-
site-directed mutagenesis, contrary to the C140A mutant, the double mutant repaires the DNA lesion without generating DNA adducts. Furthermore, in the crystal structure, the mutant S76C is shown to be at 4.1 A from the methylene bridge of SP which is a shorter distance than the one reported for the wild-type enzyme
-
C140S
-
site-directed mutagenesis, mutant enzyme activity analysis using an SP dinucleoside and an SP-containing oligonucleotide
-
C140S
-
site-directed mutagenesis, the mutant shows a similar protein and substrate binding structure compared to the wild-type enzyme, but reduced repair activity
-
additional information
-
compared to tyrosine, phenylalanine retains the aromatic ring, but does not support the radical propagation reaction due to the loss of the OH moiety. [4Fe-4S] clusters remains intact in the Y->F mutants
additional information
wild-type SPL, and mutant SPLs G168A and G168R exhibit indistinguishable circular dichroism spectra indicating that the mutation of glycine to alanine and arginine at the position 168 does not markedly alter the protein secondary structures
additional information
-
wild-type SPL, and mutant SPLs G168A and G168R exhibit indistinguishable circular dichroism spectra indicating that the mutation of glycine to alanine and arginine at the position 168 does not markedly alter the protein secondary structures
additional information
-
compared to tyrosine, phenylalanine retains the aromatic ring, but does not support the radical propagation reaction due to the loss of the OH moiety. [4Fe-4S] clusters remains intact in the Y->F mutants
-
additional information
-
wild-type SPL, and mutant SPLs G168A and G168R exhibit indistinguishable circular dichroism spectra indicating that the mutation of glycine to alanine and arginine at the position 168 does not markedly alter the protein secondary structures
-
additional information
by rational engineering, the enzyme's HAT pathway is rewired. Possible development of improved catalysts based on the radical SAM enzyme scaffold
additional information
-
by rational engineering, the enzyme's HAT pathway is rewired. Possible development of improved catalysts based on the radical SAM enzyme scaffold
-
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Sun, Y.; Palasingam, K.; Nicholson, W.L.
High-pressure liquid chromatography assay for quantitatively monitoring spore photoproduct repair mediated by spore photoproduct lyase during germination of uv-irradiated Bacillus subtilis spores
Anal. Biochem.
221
61-65
1994
Bacillus subtilis
brenda
Chandor, A.; Berteau, O.; Douki, T.; Gasparutto, D.; Sanakis, Y.; Ollagnier-de-Choudens, S.; Atta, M.; Fontecave, M.
Dinucleotide spore photoproduct, a minimal substrate of the DNA repair spore photoproduct lyase enzyme from Bacillus subtilis
J. Biol. Chem.
281
26922-26931
2006
Bacillus subtilis
brenda
Pieck, J.C.; Hennecke, U.; Pierik, A.J.; Friedel, M.G.; Carell, T.
Characterization of a new thermophilic spore photoproduct lyase from Geobacillus stearothermophilus (SplG) with defined lesion containing DNA substrates
J. Biol. Chem.
281
36317-36326
2006
Geobacillus stearothermophilus
brenda
Chandra, T.; Silver, S.C.; Zilinskas, E.; Shepard, E.M.; Broderick, W.E.; Broderick, J.B.
Spore photoproduct lyase catalyzes specific repair of the 5R but not the 5S spore photoproduct
J. Am. Chem. Soc.
131
2420-2421
2009
Clostridium acetobutylicum
brenda
Heil, K.; Kneuttinger, A.C.; Schneider, S.; Lischke, U.; Carell, T.
Crystal structures and repair studies reveal the identity and the base-pairing properties of the UV-induced spore photoproduct DNA lesion
Chemistry
17
9651-9657
2011
Geobacillus stearothermophilus (A0A0E0TDM3)
brenda
Lin, G.; Chen, C.H.; Pink, M.; Pu, J.; Li, L.
Chemical synthesis, crystal structure and enzymatic evaluation of a dinucleotide spore photoproduct analogue containing a formacetal linker
Chemistry
17
9658-9668
2011
Bacillus subtilis
brenda
Yang, L.; Lin, G.; Liu, D.; Dria, K.J.; Telser, J.; Li, L.
Probing the reaction mechanism of spore photoproduct lyase (SPL) via diastereoselectively labeled dinucleotide SP TpT substrates
J. Am. Chem. Soc.
133
10434-10447
2011
Bacillus subtilis
brenda
Silver, S.C.; Chandra, T.; Zilinskas, E.; Ghose, S.; Broderick, W.E.; Broderick, J.B.
Complete stereospecific repair of a synthetic dinucleotide spore photoproduct by spore photoproduct lyase
J. Biol. Inorg. Chem.
15
943-955
2010
Clostridium acetobutylicum, Clostridium acetobutylicum ATCC 824D
brenda
Yang, L.; Lin, G.; Nelson, R.S.; Jian, Y.; Telser, J.; Li, L.
Mechanistic studies of the spore photoproduct lyase via a single cysteine mutation
Biochemistry
51
7173-7188
2012
Bacillus subtilis, Bacillus subtilis 168
brenda
Yang, L.; Nelson, R.S.; Benjdia, A.; Lin, G.; Telser, J.; Stoll, S.; Schlichting, I.; Li, L.
A radical transfer pathway in spore photoproduct lyase
Biochemistry
52
3041-3050
2013
Bacillus subtilis, Bacillus subtilis 168
brenda
Li, L.
Mechanistic studies of the radical SAM enzyme spore photoproduct lyase (SPL)
Biochim. Biophys. Acta
1824
1264-1277
2012
Bacillus subtilis, Bacillus subtilis 168
brenda
Kneuttinger, A.C.; Heil, K.; Kashiwazaki, G.; Carell, T.
The radical SAM enzyme spore photoproduct lyase employs a tyrosyl radical for DNA repair
Chem. Commun. (Camb. )
49
722-724
2013
Geobacillus thermodenitrificans
brenda
Benjdia, A.; Heil, K.; Barends, T.R.; Carell, T.; Schlichting, I.
Structural insights into recognition and repair of UV-DNA damage by spore photoproduct lyase, a radical SAM enzyme
Nucleic Acids Res.
40
9308-9318
2012
Geobacillus thermodenitrificans (A4IQU1), Geobacillus thermodenitrificans NG80-2 (A4IQU1)
brenda
Benjdia, A.; Heil, K.; Winkler, A.; Carell, T.; Schlichting, I.
Rescuing DNA repair activity by rewiring the H-atom transfer pathway in the radical SAM enzyme, spore photoproduct lyase
Chem. Commun. (Camb.)
50
14201-14204
2014
Geobacillus thermodenitrificans (A4IQU1), Geobacillus thermodenitrificans NG80-2 (A4IQU1)
brenda
Yang, L.; Jian, Y.; Setlow, P.; Li, L.
Spore photoproduct within DNA is a surprisingly poor substrate for its designated repair enzyme-The spore photoproduct lyase
DNA Repair
53
31-42
2017
Bacillus subtilis (P37956), Bacillus subtilis 168 (P37956)
brenda
Ghose, S.; Hilmer, J.K.; Bothner, B.; Broderick, J.B.
Solution phase dynamics of the DNA repair enzyme spore photoproduct lyase as probed by H/D exchange
FEBS Lett.
588
3023-3029
2014
Clostridium acetobutylicum (Q97L63), Clostridium acetobutylicum ATCC 824 / DSM 792 / JCM 1419 / LMG 5710 / VKM B-1787 (Q97L63)
brenda
Yang, L.; Li, L.
Insights into the activity change of spore photoproduct lyase induced by mutations at a peripheral glycine residue
Front. Chem.
5
14
2017
Bacillus subtilis (P37956), Bacillus subtilis, Bacillus subtilis 168 (P37956)
brenda
Yang, L.; Li, L.
Spore photoproduct lyase the known, the controversial, and the unknown
J. Biol. Chem.
290
4003-4009
2015
Geobacillus thermodenitrificans (A4IQU1), Bacillus subtilis (P37956), Bacillus subtilis 168 (P37956), Geobacillus thermodenitrificans NG80-2 (A4IQU1)
brenda
Silver, S.C.; Gardenghi, D.J.; Naik, S.G.; Shepard, E.M.; Huynh, B.H.; Szilagyi, R.K.; Broderick, J.B.
Combined Moessbauer spectroscopic, multi-edge X-ray absorption spectroscopic, and density functional theoretical study of the radical SAM enzyme spore photoproduct lyase
J. Biol. Inorg. Chem.
19
465-483
2014
Clostridium acetobutylicum (Q97L63), Clostridium acetobutylicum ATCC 824 / DSM 792 / JCM 1419 / LMG 5710 / VKM B-1787 (Q97L63)
brenda
Yang, L.; Adhikari, J.; Gross, M.L.; Li, L.
Kinetic isotope effects and hydrogen/deuterium exchange reveal large conformational changes during the catalysis of the Clostridium acetobutylicum spore photoproduct lyase
Photochem. Photobiol.
93
331-342
2017
Clostridium acetobutylicum (Q97L63), Clostridium acetobutylicum, Clostridium acetobutylicum ATCC 824 / DSM 792 / JCM 1419 / LMG 5710 / VKM B-1787 (Q97L63)
brenda
Berteau, O.; Benjdia, A.
DNA repair by the radical SAM enzyme spore photoproduct lyase from biochemistry to structural investigations
Photochem. Photobiol.
93
67-77
2017
Geobacillus thermodenitrificans (A4IQU1), Bacillus subtilis (P37956), Bacillus subtilis, Clostridium acetobutylicum (Q97L63), Bacillus subtilis 168 (P37956), Clostridium acetobutylicum ATCC 824 / DSM 792 / JCM 1419 / LMG 5710 / VKM B-1787 (Q97L63), Geobacillus thermodenitrificans NG80-2 (A4IQU1)
brenda