Enzymatic and DNA binding properties of purified WRN protein: high affinity binding to single-stranded DNA but not to DNA damage induced by 4NQO
Enzymatic and DNA binding properties of purified WRN protein: high affinity binding to single-stranded DNA but not to DNA damage induced by 4NQO
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DOI：
10.1093/nar/27.17.3557
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发表时间：
1999-09-01
影响因子：
14.9
通讯作者：
Bohr, VA
中科院分区：
文献类型：
--
作者：
Orren, DK;Brosh, RM;Bohr, VA
WRN基因突变导致Werner综合征，这是一种常染色体隐性遗传疾病，其中许多衰老特征加速。WRN基因产物的一级序列表明其可能在DNA代谢的某些方面起作用。该纯化方案去除了在单个Ni 2+亲和层析步骤后持续存在的核酸酶和拓扑异构酶污染物，并允许明确解释DNA底物上的WRNp酶活性。纯化的WRNp具有DNA依赖性ATP酶和解旋酶活性，这与其与RecQ亚家族蛋白质的同源性一致。与双链DNA相比，WRNp与单链DNA的结合亲和力也更高。然而，WRNp对各种类型的DNA损伤（包括4NQO处理期间形成的加合物）的亲和力并不比对未损伤DNA的亲和力高。我们的研究结果证实，WRNp在DNA代谢中发挥作用，尽管这种作用似乎不是DNA损伤的特异性识别。
Mutations in the WRN gene result in Werner syndrome, an autosomal recessive disease in which many characteristics of aging are accelerated, A probable role in some aspect of DNA metabolism is suggested by the primary sequence of the WRN gene product, A recombinant His-tagged WRN protein (WRNp) was overproduced in insect cells using the baculovirus system and purified to near homogeneity by several chromatographic steps. This purification scheme removes both nuclease and topoisomerase contaminants that persist following a single Ni2+ affinity chromatography step and allows for unambiguous interpretation of WRNp enzymatic activities on DNA substrates. Purified WRNp has DNA-dependent ATPase and helicase activities consistent with its homology to the RecQ subfamily of proteins. The protein also binds with higher affinity to single-stranded DNA than to double-stranded DNA, However, WRNp has no higher affinity for various types of DNA damage, including adducts formed during 4NQO treatment, than for undamaged DNA. Our results confirm that WRNp has a role in DNA metabolism, although this role does not appear to be the specific recognition of damage in DNA.