Biochemical Characterization of an Exonuclease from Arabidopsis thaliana Reveals Similarities to the DNA Exonuclease of the Human Werner Syndrome Protein*-猫眼课题宝

拟南芥核酸外切酶的生化表征揭示了与人类维尔纳综合征蛋白 DNA 核酸外切酶的相似性*

DOI：

发表时间：

2003

期刊：

Journal of Biological Chemistry

影响因子：

4.8

通讯作者：

中科院分区：

生物学2区

文献类型：

作者：

H. Plchova;F. Hartung;H. Puchta

人Werner综合征蛋白（hWRN-p）具有DNA解旋酶和核酸外切酶活性，对基因组的稳定性至关重要。植物中没有这种双功能蛋白的同源物，但令人惊讶的是，拟南芥基因组含有一个小的开放阅读框（ORF），该ORF在大肠杆菌中的表达揭示了AtWRN-exo-p的外切核酸酶活性与hWRN-p的外切核酸酶活性具有相似性，但也存在一些显著差异。p.该蛋白质在3′ → 5′方向上对DNA双链体的凹陷链起作用，但几乎不对单链DNA或平端双链体起作用。与Werner核酸外切酶相比，AtWRNexo-p也能够消化3′-突出链。具有凹陷的3′-PO 4和3′-OH末端的DNA以类似的程度降解。AtWRNexo-p水解含有错配碱基的双链体的3′-凹陷链末端。AtWRNexo-p需要二价阳离子Mg 2+来获得活性，其可以被Mn 2+取代。脱嘌呤位点、胆固醇加合物和氧化性DNA损伤（如8-氧代腺嘌呤和8-氧代鸟嘌呤）抑制或阻断该酶。其他DNA修饰，包括尿嘧啶，次黄嘌呤和乙烯腺嘌呤，没有抑制AtWRNexo-p。一个保守的残基内的核酸外切酶结构域（E135 A）的突变完全废除了外切核酸活性。我们的研究结果表明，一种WRN样核酸外切酶活性似乎是动物和植物DNA代谢的共同特征。

The human Werner syndrome protein (hWRN-p) possessing DNA helicase and exonuclease activities is essential for genome stability. Plants have no homologue of this bifunctional protein, but surprisingly the Arabidopsis genome contains a small open reading frame (ORF) (AtWRNexo) with homology to the exonuclease domain of hWRN-p. Expression of this ORF in Escherichia coli revealed an exonuclease activity for AtWRN-exo-p with similarities but also some significant differences to hWRN-p. The protein digests recessed strands of DNA duplexes in the 3′ → 5′ direction but hardly single-stranded DNA or blunt-ended duplexes. In contrast to the Werner exonuclease, AtWRNexo-p is also able to digest 3′-protruding strands. DNA with recessed 3′-PO4 and 3′-OH termini is degraded to a similar extent. AtWRNexo-p hydrolyzes the 3′-recessed strand termini of duplexes containing mismatched bases. AtWRNexo-p needs the divalent cation Mg2+ for activity, which can be replaced by Mn2+. Apurinic sites, cholesterol adducts, and oxidative DNA damage (such as 8-oxoadenine and 8-oxoguanine) inhibit or block the enzyme. Other DNA modifications, including uracil, hypoxanthine and ethenoadenine, did not inhibit AtWRNexo-p. A mutation of a conserved residue within the exonuclease domain (E135A) completely abolished the exonucleolytic activity. Our results indicate that a type of WRN-like exonuclease activity seems to be a common feature of the DNA metabolism of animals and plants.