酶促切割介导的延伸停滞使 DNA 中位点特异性尿嘧啶修饰的准确识别和定量成为可能,Analytical Chemistry

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酶促切割介导的延伸停滞使 DNA 中位点特异性尿嘧啶修饰的准确识别和定量成为可能
Analytical Chemistry ( IF 6.7 ) Pub Date : 2023-05-16 , DOI: 10.1021/acs.analchem.3c01410
Tong-Tong Ji ₁ , Neng-Bin Xie _{1,

2} , Jiang-Hui Ding ₁ , Min Wang ₁ , Xia Guo ₁ , Ying-Ying Chen ₁ , Si-Yu Yu ₂ , Yu-Qi Feng _{1,

2} , Bi-Feng Yuan _{1,

2,

3}

Affiliation

DNA 中的化学修饰对 DNA 的结构和功能具有深远的影响。尿嘧啶是一种天然存在的 DNA 修饰，可源于胞嘧啶的脱氨作用，或源于 dUTP 在 DNA 复制过程中错误掺入 DNA。DNA 中的尿嘧啶会危及基因组稳定性，因为它们有可能产生有害突变。深入了解尿嘧啶修饰的功能需要准确确定其位点以及在基因组中的含量。在此，我们表征了尿嘧啶-DNA 糖基化酶 (UDG) 家族酶 (UdgX-H109S) 的新成员可以选择性地切割含尿嘧啶的单链 DNA (ssDNA) 和双链 DNA (dsDNA)。基于UdgX-H109S的这一独特属性，我们开发了一种酶解介导的延伸停滞 (ECES) 方法，用于基因组 DNA 中尿嘧啶的位点特异性检测和定量。在 ECES 方法中，UdgX-H109S 特异性识别并切割来自 dsDNA 的尿嘧啶的N -糖苷键并生成脱嘌呤/脱嘧啶 (AP) 位点，该位点可被 APE1 打断以形成单核苷酸缺口。然后通过 qPCR 评估和量化 UdgX-H109S 的特异性切割。通过开发的 ECES 方法，我们证明了乳腺癌组织基因组 DNA 中 Chr4:50566961 位置的尿嘧啶水平显着降低。总的来说，ECES 方法已被证明在生物和临床样本的基因组 DNA 中尿嘧啶位点特异性定量中是准确且可重复的。

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Enzymatic Cleavage-Mediated Extension Stalling Enables Accurate Recognition and Quantification of Locus-Specific Uracil Modification in DNA

Chemical modifications in DNA have profound influences on the structures and functions of DNA. Uracil, a naturally occurring DNA modification, can originate from the deamination of cytosine or arise from misincorporation of dUTP into DNA during DNA replication. Uracil in DNA will imperil genomic stability due to their potential in producing detrimental mutations. An in-depth understanding of the functions of uracil modification requires the accurate determination of its site as well as content in genomes. Herein, we characterized that a new member of the uracil-DNA glycosylase (UDG) family enzyme (UdgX-H109S) could selectively cleave both uracil-containing single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Based on this unique property of UdgX-H109S, we developed an enzymatic cleavage-mediated extension stalling (ECES) method for the locus-specific detection and quantification of uracil in genomic DNA. In the ECES method, UdgX-H109S specifically recognizes and cleaves the N-glycosidic bond of uracil from dsDNA and generates an apurinic/apyrimidinic (AP) site, which could be broken by APE1 to form a one-nucleotide gap. The specific cleavage by UdgX-H109S is then evaluated and quantified by qPCR. With the developed ECES approach, we demonstrated that the level of uracil at position Chr4:50566961 in genomic DNA of breast cancer tissues was significantly decreased. Collectively, the ECES method has been proved to be accurate and reproducible in the locus-specific quantification of uracil in genomic DNA from biological and clinical samples.

更新日期：2023-05-16

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