Harrigan Jeanine A, Wilson David M, Prasad Rajendra, Opresko Patricia L, Beck Gad, May Alfred, Wilson Samuel H, Bohr Vilhelm A
Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, MD, USA.
Nucleic Acids Res. 2006 Jan 30;34(2):745-54. doi: 10.1093/nar/gkj475. Print 2006.
Genome instability is a characteristic of cancer and aging, and is a hallmark of the premature aging disorder Werner syndrome (WS). Evidence suggests that the Werner syndrome protein (WRN) contributes to the maintenance of genome integrity through its involvement in DNA repair. In particular, biochemical evidence indicates a role for WRN in base excision repair (BER). We have previously reported that WRN helicase activity stimulates DNA polymerase beta (pol beta) strand displacement synthesis in vitro. In this report we demonstrate that WRN exonuclease activity can act cooperatively with pol beta, a polymerase lacking 3'-5' proofreading activity. Furthermore, using small interference RNA technology, we demonstrate that WRN knockdown cells are hypersensitive to the alkylating agent methyl methanesulfonate, which creates DNA damage that is primarily repaired by the BER pathway. In addition, repair assays using whole cell extracts from WRN knockdown cells indicate a defect in long patch (LP) BER. These findings demonstrate that WRN plays a direct role in the repair of methylation-induced DNA damage, and suggest a role for both WRN helicase and exonuclease activities together with pol beta during LP BER.
基因组不稳定是癌症和衰老的一个特征，也是早衰疾病沃纳综合征（WS）的一个标志。有证据表明，沃纳综合征蛋白（WRN）通过参与DNA修复，有助于维持基因组完整性。特别是，生化证据表明WRN在碱基切除修复（BER）中发挥作用。我们之前报道过，WRN解旋酶活性在体外可刺激DNA聚合酶β（polβ）链置换合成。在本报告中，我们证明WRN核酸外切酶活性可与polβ协同作用，polβ是一种缺乏3'-5'校对活性的聚合酶。此外，利用小干扰RNA技术，我们证明敲低WRN的细胞对烷化剂甲磺酸甲酯高度敏感，甲磺酸甲酯会造成主要由BER途径修复的DNA损伤。另外，使用来自敲低WRN细胞的全细胞提取物进行的修复分析表明，长片段（LP）BER存在缺陷。这些发现表明WRN在甲基化诱导的DNA损伤修复中起直接作用，并提示在LP BER过程中，WRN解旋酶和核酸外切酶活性与polβ共同发挥作用。
