Coupling of the nucleotide incision and 3′ → 5′ exonuclease activities in Escherichia coli endonuclease IV: Structural and genetic evidences
Coupling of the nucleotide incision and 3′ → 5′ exonuclease activities in Escherichia coli endonuclease IV: Structural and genetic evidences
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DOI：
10.1016/j.mrfmmm.2009.08.017
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发表时间：
2010-03-01
影响因子：
2.3
通讯作者：
Saparbaev, Murat K.
中科院分区：
文献类型：
--
作者：
Golan, Gali;Ishchenko, Alexander A.;Saparbaev, Murat K.
有氧呼吸产生的活性氧(ROS)是细胞代谢的副产物，可损伤DNA。氧化性DNA损伤的复杂性需要几个修复途径的作用。氧化的DNA碱基是两条重叠通路的底物：碱基切除修复(BER)和核苷酸切开修复(NIR)。在BER途径中，DNA糖基酶裂解异常碱基和脱氧核糖之间的N-糖基键，留下基本位置或单链DNA断裂。或者，在近红外途径中，脱嘌呤/脱嘧啶(AP)内切酶在氧化损伤的核苷酸旁边切割双链DNA 5‘。多功能大肠杆菌内切酶IV(NFO)参与了BER和NIR信号转导途径。NFO切割受损残基的双链DNA 5‘，但也具有内在的3’-gt；5‘外切酶活性。在这里。我们证明了NFO催化的近红外和核酸外切酶活性可以在5，6-二氢尿嘧啶残基的5‘侧产生单链缺口。此外，我们还发现携带氨基酸替换的NFO突变体H69A和G149D同时缺乏近红外和核酸外切酶活性，这表明这两种功能是由相同的氨基酸残基遗传连锁的。NFO-H69A突变体的晶体结构显示活性中心锌原子(锌原子)的丢失和催化中心的重排，但酶的整体构象没有明显变化。我们假设这些微小的变化强烈地影响了NFO的DNA结合。亲和力降低可能导致DNA螺旋的不同扭折角，这反过来会阻碍NFO突变体的核苷酸切割和核酸外切酶活性，但对其AP内切酶功能的影响较小。基于生化和遗传数据，我们提出了一个模型，在修复双链簇状DNA损伤时，核苷酸切割与3‘-gt；5’外切酶活性相结合，防止形成致命的双链断裂。(C)2009爱思唯尔B.V.保留所有权利。
Aerobic respiration generates reactive oxygen species (ROS) as a by-product of cellular metabolism which can damage DNA. The complex nature of oxidative DNA damage requires actions of several repair pathways. Oxidized DNA bases are substrates for two overlapping pathways: base excision repair (BER) and nucleotide incision repair (NIR). In the BER pathway a DNA glycosylase cleaves the N-glycosylic bond between the abnormal base and deoxyribose, leaving either an abasic site or single-stranded DNA break. Alternatively, in the NIR pathway, an apurinic/apyrimidinic (AP) endonuclease incises duplex DNA 5 ' next to oxidatively damaged nucleotide. The multifunctional Escherichia coli endonuclease IV (Nfo) is involved in both BER and NIR pathways. Nfo incises duplex DNA 5' of a damaged residue but also possesses an intrinsic 3' -> 5' exonuclease activity. Herein. we demonstrate that Nfo-catalyzed NIR and exonuclease activities can generate a single-strand gap at the 5' side of 5,6-dihydrouracil residue. Furthermore, we show that Nfo mutants carrying amino acid substitutions H69A and G149D are deficient in both NIR and exonuclease activities, suggesting that these two functions are genetically linked and governed by the same amino acid residues. The crystal structure of Nfo-H69A mutant reveals the loss of one of the active site zinc atoms (Zn1) and rearrangements of the catalytic site, but no gross changes in the overall enzyme conformation. We hypothesize that these minor changes strongly affect the DNA binding of Nfo. Decreased affinity may lead to a different kinking angle of the DNA helix and this in turn thwart nucleotide incision and exonuclease activities of Nfo mutants but to lesser extent of their AP endonuclease function. Based on the biochemical and genetic data we propose a model where nucleotide incision coupled to 3' -> 5' exonuclease activity prevents formation of lethal double-strand breaks when repairing bi-stranded clustered DNA damage. (C) 2009 Elsevier B.V. All rights reserved.