Brosh Robert M, Opresko Patricia L, Bohr Vilhelm A
Laboratory of Molecular Gerontology, National Institute of Aging-IRP, National Institutes of Health, Baltimore, Maryland, USA.
Methods Enzymol. 2006;409:52-85. doi: 10.1016/S0076-6879(05)09004-X.
Werner syndrome (WS) is a premature aging disorder characterized by genomic instability and increased cancer risk (Martin, 1978). The WRN gene product defective in WS belongs to the RecQ family of DNA helicases (Yu et al., 1996). Mutations in RecQ family members BLM and RecQ4 result in two other disorders associated with elevated chromosomal instability and cancer, Bloom syndrome and Rothmund-Thomson syndrome, respectively (for review see Opresko et al., 2004a). RecQ helicase mutants display defects in DNA replication, recombination, and repair, suggesting a role for RecQ helicases in maintaining genomic integrity. The WRN gene encodes a 1,432 amino acid protein that has several catalytic activities (Brosh and Bohr, 2002) (Fig. 1). WRN is a DNA-dependent ATPase and utilizes the energy from ATP hydrolysis to unwind double-stranded DNA. WRN is also a 3' to 5' exonuclease, consistent with the presence of three conserved exonuclease motifs homologous to the exonuclease domain of Escherichia coli DNA polymerase I and RNase D. Most recently, WRN (Machwe et al., 2005) and other human RecQ helicases (Garcia et al., 2004; Machwe et al., 2005; Sharma et al., 2005) have been reported to possess an intrinsic single-strand annealing activity. In addition to its catalytic activities, WRN interacts with a number of proteins involved in various aspects of DNA metabolism. To understand the role of WRN in the maintenance of genome stability, a number of laboratories have undertaken a thorough characterization of its molecular and cellular functions. Here, we describe methods and approaches used for the functional and mechanistic analysis of WRN helicase or exonuclease activity. Protocols for measuring ATP hydrolysis, DNA binding, and catalytic unwinding or exonuclease activity of WRN protein are provided. Application of these procedures should enable the researcher to address fundamental questions regarding the biochemical properties of WRN or related helicases or nucleases, which would serve as a platform for further investigation of its molecular and cellular functions.
沃纳综合征（WS）是一种早衰性疾病，其特征为基因组不稳定和癌症风险增加（Martin，1978年）。在WS中存在缺陷的WRN基因产物属于DNA解旋酶的RecQ家族（Yu等人，1996年）。RecQ家族成员BLM和RecQ4中的突变分别导致另外两种与染色体不稳定性升高和癌症相关的疾病，即布卢姆综合征和罗思蒙德 - 汤姆森综合征（有关综述见Opresko等人，2004a）。RecQ解旋酶突变体在DNA复制、重组和修复方面表现出缺陷，这表明RecQ解旋酶在维持基因组完整性中发挥作用。WRN基因编码一种具有多种催化活性的1432个氨基酸的蛋白质（Brosh和Bohr，2002年）（图1）。WRN是一种依赖DNA的ATP酶，并利用ATP水解产生的能量解开双链DNA。WRN也是一种3'至5'外切核酸酶，这与存在三个与大肠杆菌DNA聚合酶I和RNase D的外切核酸酶结构域同源的保守外切核酸酶基序一致。最近，有报道称WRN（Machwe等人，2005年）和其他人类RecQ解旋酶（Garcia等人，2004年；Machwe等人，2005年；Sharma等人，2005年）具有内在的单链退火活性。除了其催化活性外，WRN还与许多参与DNA代谢各个方面的蛋白质相互作用。为了了解WRN在维持基因组稳定性中的作用，许多实验室对其分子和细胞功能进行了全面表征。在这里，我们描述了用于WRN解旋酶或外切核酸酶活性的功能和机制分析的方法和途径。提供了测量WRN蛋白的ATP水解、DNA结合以及催化解旋或外切核酸酶活性的方案。应用这些程序应能使研究人员解决有关WRN或相关解旋酶或核酸酶生化特性的基本问题，这将为进一步研究其分子和细胞功能提供一个平台。
