Direct H atom abstraction from spore photoproduct C-6 initiates DNA repair in the reaction catalyzed by spore photoproduct lyase: Evidence for a reversibly generated adenosyl radical intermediate
Direct H atom abstraction from spore photoproduct C-6 initiates DNA repair in the reaction catalyzed by spore photoproduct lyase: Evidence for a reversibly generated adenosyl radical intermediate
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DOI：
10.1021/ja017784g
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发表时间：
2002-03-27
影响因子：
15
通讯作者：
Broderick, JB
中科院分区：
文献类型：
--
作者：
Cheek, J;Broderick, JB
孢子光产物（SP）裂解酶是目前发现的唯一一种非光活化的嘧啶二聚体裂解酶，它催化SP（5-胸腺嘧啶-5，6-二氢胸腺嘧啶）直接转化为胸腺嘧啶单体。与DNA光裂合酶不同，SP裂合酶不含黄素辅因子，并且不需要光来激活。相反，初步研究指出SP裂解酶中存在铁硫簇，并且需要S-腺苷甲硫氨酸（S-adenosylmethionine，S-Met）来获得催化活性，这表明SP裂解酶属于铁硫簇和S-腺苷甲硫氨酸依赖性自由基酶的增长组。在这里，我们提供的证据，作为一个可逆的脱氧腺苷自由基发生器的作用，它启动修复从SP的C-6的氢原子的提取。6- 3 H-SP，而不是甲基-3 H-SP，与SP裂解酶和3 H-Met的反应，导致3 H转移到3 H-Met，而没有观察到氚标记的5 '-脱氧腺苷。当5 '-氚标记的胸腺嘧啶蛋氨酸用于与未标记的SP反应时，观察到3 H转移到修复的胸腺嘧啶单体中。这些结果表明，5 '-脱氧腺苷自由基中间体的可逆产生，它直接与DNA损伤反应，启动自由基介导的β-断裂。我们还表明，蛋氨酸是一种催化辅因子，在营业额不消耗。总之，这些结果支持了一种新的基于自由基的紫外线诱导的DNA损伤修复机制。
Spore photoproduct (SP) lyase, which catalyzes the direct reversal of SP (5-thyminyl-5,6-dihydrothymine) to thymine monomers, is the only identified nonphotoactivatable pyrimidine dimer lyase. Unlike DNA photolyase, SP lyase does not contain a flavin cofactor and does not require light for activation. Instead, preliminary studies point to the presence of an iron−sulfur cluster in SP lyase and the requirement forS-adenosylmethionine (AdoMet) for catalytic activity, suggesting that SP lyase belongs to the growing group of iron−sulfur cluster and AdoMet-dependent radical enzymes. Here we provide evidence for the role of AdoMet as a reversible deoxyadenosyl radical generator, which initiates repair by hydrogen atom abstraction from C-6 of SP. Reaction of 6-3H-SP, but not methyl-3H-SP, with SP lyase and AdoMet results in transfer of3H to AdoMet, while no tritiated 5‘-deoxyadenosine is observed. When 5‘-tritiated AdoMet is used in the reaction with unlabeled SP, transfer of3H into the repaired thymine monomers is observed. These results point to thereversiblegeneration of a 5‘-deoxyadenosyl radical intermediate, which reacts directly with the DNA lesion to initiate a radical-mediated β-scission. We also demonstrate that AdoMet is a catalytic cofactor that is not consumed during turnover. Together, these results support a novel radical-based mechanism for the repair of UV-induced DNA damage.