Department of Medical, Surgical, Neurological, Metabolic Sciences, and Aging, 2nd Division of Neurology, Center for Rare Diseases and InterUniversity Center for Research in Neurosciences, University of Campania "Luigi Vanvitelli", Naples, Italy.
Research Institute on Terrestrial Ecosystems, National Research Council, Naples, Italy.
J Mol Med (Berl). 2019 Jul;97(7):991-1001. doi: 10.1007/s00109-019-01788-8. Epub 2019 May 1.
Base excision repair (BER) is a frontline repair mechanism that operates through the G phase of the cell cycle, which ensures the genome integrity by repairing thousands of DNA lesions due to endogenous and exogenous agents. Its correct functioning is fundamental for cell viability and the health of the organism. Uracil is one of the most prevalent lesions that appears in DNA arising by spontaneous or enzymatic deamination of cytosine or misincorporation of the deoxyuridine 5'-triphosphate nucleotide (dUTP) in place of deoxythymidine 5'-triphosphate (dTTP) during DNA replication. In the first pathway, the uracil will preferentially pair with adenine, leading to C:G → T:A transition. When uracil in DNA arises from misincorporation of dUTP instead of dTTP, this process will result in A:U pairs. Organisms counteract the mutagenic effects of uracil in DNA using the BER repair system, which is mediated by a member of the uracil-DNA glycosylase (UDG) superfamily. Several assays evaluating the in vitro BER enzyme activity have been described so far. Some of these measure the BER activity by an oligonucleotide incision assay using radiolabeled duplex oligo. Others use circular double-stranded DNA substrates containing a defined lesion. The novelty of our method resides in its rapidity and safety (radioactive free detection) as well as in the possibility of having a reliable quantitative determination of UDG activity in both cell and tissue extracts. We also demonstrated the effectiveness of our method in assessing UDG activity in cell lines with a reduced DNA repair capacity and in different kinds of tissues. KEY MESSAGES: • Base excision repair is a fundamental repair mechanism ensuring the genome integrity. • Uracil is one of the most prevalent lesions that appears in DNA. • The mutagenic effects of uracil in DNA are mitigated by the uracil-DNA glycosylase. • Several assays evaluating the in vitro BER activity have been described so far. • A safe and quantitative assay evaluating the in vitro UDG activity is required.
碱基切除修复（BER）是一种前沿的修复机制，通过细胞周期的 G 期运作，通过修复由于内源性和外源性试剂引起的数千个 DNA 损伤来确保基因组的完整性。其正确的功能对于细胞活力和生物体的健康至关重要。尿嘧啶是 DNA 中最常见的一种损伤，它是由胞嘧啶自发或酶促脱氨或脱氧尿苷 5′-三磷酸核苷酸（dUTP）错误掺入取代脱氧胸苷 5′-三磷酸（dTTP）而在 DNA 复制过程中产生的。在第一个途径中，尿嘧啶将优先与腺嘌呤配对，导致 C：G→T：A 转换。当 DNA 中的尿嘧啶是由 dUTP 而不是 dTTP 错误掺入引起时，这一过程将导致 A：U 配对。生物体使用 BER 修复系统来对抗 DNA 中尿嘧啶的诱变作用，该系统由尿嘧啶-DNA 糖基化酶（UDG）超家族的成员介导。到目前为止，已经描述了几种评估体外 BER 酶活性的测定方法。其中一些通过使用放射性标记的双链寡核苷酸进行寡核苷酸切口测定来测量 BER 活性。其他方法使用含有定义损伤的环状双链 DNA 底物。我们的方法的新颖之处在于其快速性和安全性（无放射性检测），以及在细胞和组织提取物中可靠地定量测定 UDG 活性的可能性。我们还证明了我们的方法在评估具有降低 DNA 修复能力的细胞系和不同类型组织中的 UDG 活性方面的有效性。
碱基切除修复是一种基本的修复机制，可确保基因组的完整性。
尿嘧啶是 DNA 中最常见的一种损伤。
尿嘧啶-DNA 糖基化酶减轻了 DNA 中尿嘧啶的诱变作用。
到目前为止，已经描述了几种评估体外 BER 活性的测定方法。
需要一种安全且定量的测定方法来评估体外 UDG 活性。
