Direct fluorescence monitoring of DNA base excision repair.
Direct fluorescence monitoring of DNA base excision repair.
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DOI：
10.1002/anie.201108135
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发表时间：
2012-02-13
影响因子：
16.6
通讯作者：
Kool, Eric T.
中科院分区：
文献类型：
--
作者：
Ono, Toshikazu;Wang, Shenliang;Koo, Chi-Kin;Engstrom, Lisa;David, Sheila S.;Kool, Eric T.
尿嘧啶是DNA中不需要的成分，因为它是由胞嘧啶自发脱氨产生的这种水解反应促进突变，因为在DNA复制过程中产生的U-G对可能被误读。因此，多种细胞酶已经进化到可以检测DNA中的尿嘧啶，并在复制之前将其移除在大肠杆菌中，尿嘧啶DNA糖基化酶（UDG）酶起保护细菌基因组的作用。在人类中，存在类似的酶活性，包括蛋白质UNG1/2， SMUG和TDG.[3]这些酶将尿嘧啶从DNA螺旋中翻转出来，并将其从脱氧核糖中切割出来，在原来的位置上留下一个基本位点UDG酶的化学传感器可以在多个方面发挥作用。这种探针有助于酶的机理研究此外，如果这种探针可以在细胞中发挥作用，它们可以用于筛选酶突变体的活性，并探索DNA修复途径的生物学调节。最后，DNA修复酶抑制剂目前在抗癌治疗方法中受到强烈关注；因此，这些酶的简单点亮报告基因也可以用于筛选候选药物。先前开发尿嘧啶糖基酶荧光传感器的努力受到尺寸大、间接读数和性能差的限制。设计通常依赖于28-39个核苷酸的大型双链DNA结构在尿嘧啶被移除和DNA链最终被切割后，构象的改变（双工结构的丧失）产生了信号。据报道，信号增强4至8倍；这些信号仅间接报告尿嘧啶去糖基化，因为它们需要随后的DNA降解和/或解绕步骤才能看到信号。尽管这样的DNA结构能够在体外发挥作用，但这种结构在生物介质（如细胞提取物）中的功能可能会因细胞内DNA解绕活动、核酸酶降解和其他DNA结合蛋白（如单链结合蛋白）产生的错误信号而进一步复杂化，这些错误信号都可能导致双链完整性的丧失。尽管先前的一份报告描述了在哺乳动物细胞中产生的具有39个碱基DNA结构的信号，但[7a]没有进行排除这些可能的背景信号来源的对照实验。最后，这种结构的大尺寸增加了成本，复杂性和降低了细胞内摄取的可能性。人们早就知道，UDG酶在单链dna上也显示出活性一份较早的报告描述了大肠杆菌UDG的体外动力学试验，该试验利用含有荧光染料2-氨基嘌呤（2AP）的双链和单链DNA，该染料在DNA中猝灭；据报道，UDG使荧光增加3- 8倍。ap脱氧核苷在DNA中是一种方便的探针，因为它像DNA碱基一样堆叠；然而，它的荧光效率低，在紫外区发光我们的目标是为UDG活动构建比以前的例子更简单、更小、更有效的传感器。作为利用含有荧光DNA碱基替换物的小合成DNA低聚物构建酶传感器项目的一部分，我们观察到荧光团芘特别容易被DNA碱基胸腺嘧啶猝灭，这是一种由光诱导电荷转移（PICT）机制产生的现象芘作为荧光团特别有用，因为它具有高量子产率，强大的亮度，并且相对于2AP向红色偏移约40 nm，允许在可见区域进行检测。芘脱氧核苷(Y)，一种非天然的DNA核苷，已被用于…
Uracil is an undesired component of DNA, as it arises from spontaneous deamination of cytosine.[1] This hydrolysis reaction promotes mutations, since the resulting U–G pair can be misread during DNA replication. As a result, multiple cellular enzymes have evolved to detect uracil in DNA and remove it prior to replication.[2] In E. coli uracil DNA glycosylase (UDG) enzyme functions to guard the bacterial genome. In humans, similar enzyme activities exist, including the proteins UNG1/2, SMUG, and TDG.[3] These enzymes flip uracil out of the DNA helix and cleave it from its deoxyribose sugar, leaving an abasic site in its place.[4] Chemical sensors of UDG enzymes could be useful in multiple respects. Mechanistic studies of the enzymes can benefit from such probes.[5] In addition, if such probes could function in cells, they could be used to screen activity of enzyme mutants, and to probe biological regulation of DNA repair pathways. Finally, inhibitors of DNA repair enzymes are currently receiving intense interest in anticancer therapeutic approaches;[6] thus simple light-up reporters of such enzymes could be also useful in screening drug candidates. Previous efforts to develop fluorescent sensors for uracilglycosylase enzymes have been limited by large size, by their indirect readout and by poor performance. Designs have generally relied on large duplex DNA constructs of 28–39 nucleotides.[7] Signals were generated by changes in conformation (loss of duplex structure) which results after uracil is removed and the DNA strand is ultimately cleaved. Signal enhancements of 4-to 8-fold were reported; these signals report only indirectly on the uracil deglycosylation because they require subsequent steps of DNA degradation and/or unwinding before the signal is seen. Although such DNA constructs were able to function in vitro, function of such structures in biological media such as cell extracts can be further complicated by false signals from DNA-unwinding activities in the cell, from nuclease degradation, and from other DNA-binding proteins (such as single-strand-binding proteins) that can all lead to loss of duplex integrity. Although one previous report describes signals generated in mammalian cells with a 39-base DNA construct,[7a] control experiments to rule out these likely sources of background signal were not performed. Finally, the large size of such constructs adds cost, complexity and lowers the likelihood of intracellular uptake.It has been known for some time that UDG enzymes also show activity on single-stranded DNAs.[8] One earlier report described an in vitro kinetics assay for E. coli UDG making use of double-and single-stranded DNAs containing the fluorescent dye 2-aminopurine (2AP), which is quenched in DNA; increases in fluorescence of 3-to 8-fold were reported with UDG.[9] 2AP deoxynucleoside is a convenient probe in DNA as it stacks like a DNA base; however, it has low fluorescence efficiency and emits in the UV region.[10] Our aim was to construct sensors for UDG activity that are simpler, smaller and more effective than previous examples. As part of a program to build enzyme sensors from small synthetic DNA oligomers containing fluorescent DNA base replacements,[11] we observed that the fluorophore pyrene is especially well quenched by the DNA base thymine, a phenomenon that occurs by the photoinduced charge transfer (PICT) mechanism.[12] Pyrene is especially useful as a fluorophore because it has a high quantum yield, robust brightness, and is shifted to the red by ca. 40 nm relative to 2AP, allowing for detection in the visible region. Pyrene deoxyriboside (Y), an nonnatural DNA nucleoside, has been used …
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影响因子：
64.8
作者：
Matray, TJ;Kool, ET
通讯作者：
Kool, ET
影响因子：
15
作者：
Guckian, KM;Schweitzer, BA;Kool, ET
通讯作者：
Kool, ET
DOI：
10.1073/pnas.71.9.3649
发表时间：
1974-01-01
影响因子：
11.1
作者：
LINDAHL, T
通讯作者：
LINDAHL, T
影响因子：
4.9
作者：
Babu, BR;Wengel, J
通讯作者：
Wengel, J
影响因子：
2.9
作者：
Jiang, YL;Stivers, JT;Song, FH
通讯作者：
Song, FH