Freudenthal Bret D, Beard William A, Perera Lalith, Shock David D, Kim Taejin, Schlick Tamar, Wilson Samuel H
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, PO Box 12233, Research Triangle Park, North Carolina 27709-2233, USA.
1] Department of Chemistry, New York University, and NYU-ECNU Center for Computational Chemistry at NYU Shanghai, 10th Floor Silver Center, 100 Washington Square East, New York, New York 10003, USA [2] Courant Institute of Mathematical Sciences, New York University, 251 Mercer Street, New York, New York 10012, USA.
Nature. 2015 Jan 29;517(7536):635-9. doi: 10.1038/nature13886. Epub 2014 Nov 17.
Oxidative stress promotes genomic instability and human diseases. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2'-deoxyguanosine, which is 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 Escherichia coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner. How polymerases discriminate 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 and 8-oxo-dGTP(syn) uses its Hoogsteen edge to base pair with adenine. Here we use time-lapse crystallography to follow 8-oxo-dGTP insertion opposite adenine or cytosine with human 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-oxo-dGTP uses charge modulation during insertion that can lead to a blocked DNA repair intermediate.
氧化应激会促进基因组不稳定和引发人类疾病。一种常见的氧化核苷是8-氧代-7,8-二氢-2'-脱氧鸟苷，它既存在于DNA中（8-氧代-G），也以游离核苷酸的形式存在（8-氧代-dGTP）。核苷酸池特别容易受到氧化损伤。因此，细胞编码了一种酶（MutT/MTH1）来清除游离的氧化核苷酸。这种清除功能对于癌细胞的存活以及以DNA聚合酶（pol）依赖的方式调节大肠杆菌对抗生素的敏感性是必需的。目前人们对聚合酶如何区分受损和未受损核苷酸的了解还不够深入。鉴于氧化核苷酸在诱变、癌症治疗和细菌抗生素方面的作用，这种分析至关重要。即使有细胞的净化活动，核苷酸池中仍含有足够的8-氧代-dGTP来促进诱变。这源于其双重编码潜力，即8-氧代-dGTP（反式）与胞嘧啶碱基配对，而8-氧代-dGTP（顺式）利用其Hoogsteen边缘与腺嘌呤碱基配对。在这里，我们使用延时晶体学技术追踪人pol β在腺嘌呤或胞嘧啶对面插入8-氧代-dGTP的过程，结果表明插入分别以顺式或反式构象进行。对于8-氧代-dGTP（反式）的插入，一种新型二价金属减轻了加合鸟嘌呤碱基与氧化核苷酸三磷酸之间的排斥相互作用。无论模板碱基是什么，在催化后酶重新打开时，碱基之间的氢键相互作用都会丧失，从而导致细胞毒性的带切口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-氧代-dGTP在插入过程中如何利用电荷调节导致DNA修复中间体受阻。
