Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide.
Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide.
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发现聚合酶诱导的氧化核苷酸的细胞毒性。
DOI：
10.1038/nature13886
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发表时间：
2015-01-29
期刊：
影响因子：
64.8
通讯作者：
Wilson, Samuel H.
中科院分区：
文献类型：
--
作者：
Freudenthal, Bret D.;Beard, William A.;Perera, Lalith;Shock, David D.;Kim, Taejin;Schlick, Tamar;Wilson, Samuel H.
氧化应激促进基因组不稳定性和人类疾病。常见的氧化核苷是8-氧代-7，8-二氢-2 '-脱氧鸟苷，既存在于DNA中（8-氧代-G），也存在于游离核苷酸中（8-氧代-dGTP）。核苷酸库特别容易受到氧化损伤。因此，细胞编码一种酶（MutT/MTH 1），可以去除游离的氧化核苷酸。这种净化功能是癌细胞存活和调节E.大肠杆菌抗生素敏感性的DNA聚合酶（pol）依赖的方式。聚合酶如何区分受损和未受损的核苷酸还不清楚。鉴于氧化核苷酸在诱变、癌症治疗和细菌抗生素中的作用，这种分析是必不可少的。即使具有细胞消毒活性，核苷酸池也含有足够的8-氧代-dGTP以促进诱变。这是由双编码潜能引起的，其中8-oxo-dGTP（anti）碱基对与胞嘧啶（Cy）和8-oxodGTP（syn）利用其Hoogsteen边缘与腺嘌呤（Ad）碱基配对。在这里，我们利用延时晶体学来跟踪与Ad或Cy相对的8-oxo-dGTP插入与人DNA pol β，以揭示插入分别适应于顺式或反式构象。对于8-氧代-dGTP（抗）插入，新型二价金属缓解加合的鸟嘌呤碱基与氧化的核苷酸的三磷酸之间的排斥相互作用。对于任一种模板碱基，随着酶在催化后重新打开，碱基之间的氢键相互作用丧失，导致细胞毒性切口DNA修复中间体。将结构快照与动力学和计算分析相结合，揭示了8-oxodGTP在插入过程中如何利用电荷调制，从而导致DNA修复中间体被阻断。
Oxidative stress promotes genomic instability and human diseases. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2’-deoxyguanosine found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP). Nucleotide pools are especially vulnerable to oxidative damage. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival and to modulate E. coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner. How polymerase discriminates between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics. Even with cellular sanitizing activities, nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine (Cy) and 8-oxodGTP(syn) utilizes its Hoogsteen edge to base pair with adenine (Ad). Here we utilized time-lapse crystallography to follow 8-oxo-dGTP insertion opposite Ad or Cy with human DNA pol β, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxodGTP utilizes charge modulation during insertion that can lead to a blocked DNA repair intermediate.
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DNA聚合酶β的结构和机制。
影响因子：
2.9
作者：
Beard WA;Wilson SH
通讯作者：
Wilson SH
影响因子：
3
作者：
CHIRLIAN, LE;FRANCL, MM
通讯作者：
FRANCL, MM
DOI：
10.1107/s0907444904019158
发表时间：
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影响因子：
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作者：
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通讯作者：
Cowtan, K
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影响因子：
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作者：
Beard, WA;Shock, DD;Wilson, SH
通讯作者：
Wilson, SH