Mason Penelope A, Boubriak Ivan, Robbins Timothy, Lasala Ralph, Saunders Robert, Cox Lynne S
Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
Age (Dordr). 2013 Jun;35(3):793-806. doi: 10.1007/s11357-012-9411-0. Epub 2012 May 5.
Werner syndrome (WS) is a rare late-onset premature ageing disease showing many of the phenotypes associated with normal ageing, and provides one of the best models for investigating cellular pathways that lead to normal ageing. WS is caused by mutation of WRN, which encodes a multifunctional DNA replication and repair helicase/exonuclease. To investigate the role of WRN protein's unique exonuclease domain, we have recently identified DmWRNexo, the fly orthologue of the exonuclease domain of human WRN. Here, we fully characterise DmWRNexo exonuclease activity in vitro, confirming 3'-5' polarity, demonstrating a requirement for Mg(2+), inhibition by ATP, and an ability to degrade both single-stranded DNA and duplex DNA substrates with 3' or 5' overhangs, or bubble structures, but with no activity on blunt ended DNA duplexes. We report a novel active site mutation that ablates enzyme activity. Lesional substrates containing uracil are partially cleaved by DmWRNexo, but the enzyme pauses on such substrates and is inhibited by abasic sites. These strong biochemical similarities to human WRN suggest that Drosophila can provide a valuable experimental system for analysing the importance of WRN exonuclease in cell and organismal ageing.
沃纳综合征（WS）是一种罕见的迟发性早衰疾病，表现出许多与正常衰老相关的表型，为研究导致正常衰老的细胞途径提供了最佳模型之一。WS由WRN基因突变引起，WRN基因编码一种多功能DNA复制和修复解旋酶/核酸外切酶。为了研究WRN蛋白独特的核酸外切酶结构域的作用，我们最近鉴定出了DmWRNexo，它是人类WRN核酸外切酶结构域在果蝇中的同源物。在这里，我们全面表征了DmWRNexo在体外的核酸外切酶活性，证实了其3'-5'极性，证明了对Mg(2+)的需求，ATP对其有抑制作用，并且它能够降解具有3'或5'突出端或泡状结构的单链DNA和双链DNA底物，但对平端DNA双链体无活性。我们报道了一种消除酶活性的新型活性位点突变。含有尿嘧啶的损伤底物被DmWRNexo部分切割，但该酶在此类底物上会停顿，并被无碱基位点抑制。这些与人类WRN的强烈生化相似性表明，果蝇可以为分析WRN核酸外切酶在细胞和机体衰老中的重要性提供一个有价值的实验系统。
