Major oxidative products of cytosine are substrates for the nucleotide incision repair pathway
Major oxidative products of cytosine are substrates for the nucleotide incision repair pathway
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DOI：
10.1016/j.dnarep.2006.08.001
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发表时间：
2007-01-04
期刊：
影响因子：
3.8
通讯作者：
Ishchenko, Alexander A.
中科院分区：
文献类型：
--
作者：
Daviet, Stephane;Couve-Privat, Sophie;Ishchenko, Alexander A.
最常见的自发或由电离辐射引起的点突变是C->；T转变，涉及胞嘧啶作为靶标。氧化胞嘧啶类化合物是氧化应激引起的最丰富和最具突变性的DNA损伤。DNA糖基酶启动的碱基切除修复(BER)途径被认为是去除这些损伤的主要途径。然而，在另一种核苷酸切割修复(NIR)途径中，脱嘌呤/脱嘧啶(AP)内切酶以不依赖于DNA糖基酶的方式将DNA双链5‘切割到氧化损伤的碱基上。在这里，我们表征了人主要AP内切酶APE1对5-羟基-2‘-脱氧胞苷(5ohC)和α-异构体2’-脱氧胞苷(UDC)残基的底物特异性。反应的表观动力学参数表明，APE1和DNA糖基酶/AP裂解酶hNth1和hNeil1修复5ohC的效率很低。然而，由于APE1的细胞浓度极高，在无细胞提取物中，近红外是对5ohC的主要活性。为了解决近红外功能的生理作用，我们描述了自然发生的APE1变体，包括氨基酸替换(E126A、E126D和D148E)和N-末端截断形式(N Delta 31、N Delta 35和N Delta 61)。正如预期的那样，所有APE1突变体都具有熟练的AP内切酶活性，但截短形式的突变体显示NIR和3‘-gt；5’外切酶活性降低，这表明这两个功能是遗传连锁的，并受相同的氨基酸残基控制。此外，APE1催化的NIR和3‘->；5’外切酶活性都在双链DNA中受损碱基的5‘侧产生单链缺口，而在AP位点没有。我们假设，核苷酸切割和核酸外切酶降解的生物化学耦合可能有助于消除聚集的DNA损伤。我们的数据表明，近红外是BER途径的备份系统，以消除体内胞嘧啶的氧化损伤。(C)2006爱思唯尔B.V.保留所有权利。
Most common point mutations occurring spontaneously or induced by ionizing radiation are C -> T transitions implicating cytosine as the target. Oxidative cytosine derivatives are the most abundant and mutagenic DNA damage induced by oxidative stress. Base excision repair (BER) pathway initiated by DNA glycosylases is thought to be the major pathway for the removal of these lesions. However, in alternative nucleotide incision repair (NIR) pathway the apurinic/apyrimidinic (AP) endonucleases incise DNA duplex 5' to an oxidatively damaged base in a DNA glycosylase-independent manner. Here, we characterized the substrate specificity of human major AP endonuclease, Ape1, towards 5-hydroxy-2'deoxycytidine (5ohC) and alpha-anomeric 2'-deoxycytidine (udC) residues. The apparent kinetic parameters of the reactions suggest that Ape1 and the DNA glycosylases/AP lyases, hNth1 and hNeil1 repair 5ohC with a low efficiency. Nevertheless, due to the extremely high cellular concentration of Ape1, NIR was the major activity towards 5ohC in cell-free extracts. To address the physiological role of NIR function, we have characterized naturally occurring Ape1 variants including amino acids substitutions (E126A, E126D and D148E) and N-terminal truncated forms (N Delta 31, N Delta 35 and N Delta 61). As expected, all Ape1 mutants had proficient AP endonuclease activity, however, truncated forms showed reduced NIR and 3' -> 5' exonuclease activities indicating that these two functions are genetically linked and governed by the same amino acid residues. Furthermore, both Ape1-catalyzed NIR and 3' -> 5' exonuclease activities generate a single-strand gap at the 5' side of a damaged base but not at an AP site in duplex DNA. We hypothesized that biochemical coupling of the nucleotide incision and exonuclease degradation may serve to remove clustered DNA damage. Our data suggest that NIR is a backup system for the BER pathway to remove oxidative damage to cytosines in vivo. (c) 2006 Elsevier B.V. All rights reserved.