Biochemical and biological characterization of wild-type and ATPase-deficient Cockayne syndrome B repair protein
Biochemical and biological characterization of wild-type and ATPase-deficient Cockayne syndrome B repair protein
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DOI：
10.1074/jbc.273.19.11844
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发表时间：
1998-05-08
影响因子：
4.8
通讯作者：
Vermeulen, W
中科院分区：
文献类型：
--
作者：
Citterio, E;Rademakers, S;Vermeulen, W
Cockayne综合征（CS）是一种核苷酸切除修复障碍，其特征是阳光（UV）敏感性和严重的发育问题。有两个基因参与其中：CSA和CSB。这两种蛋白质在优先修复来自活性基因的转录阻断损伤中起着至关重要的作用。在这项研究中，我们报告了杆状病毒产生的HA-His（6）-标签的CSB蛋白（dtCSB）的纯化和表征，使用高效的三步纯化方案。在CS-B成纤维细胞中显微注射dtCSB蛋白表明其在体内具有生物学功能。dtCSB表现出DNA依赖性ATP酶活性，受到裸DNA以及核小体DNA的刺激。使用结构定义的DNA寡核苷酸，我们表明，双链DNA和双链DNA的部分单链字符，但不是真正的单链DNA作为有效的辅助因子CSB ATP酶活性。使用各种底物，没有明显的DNA解旋的dtCSB可以检测到，发现与其他SNF 2/SWI 2家族蛋白。通过定点突变，CSB的NTP结合基序中不变的赖氨酸残基被一个物理化学相关的精氨酸取代，正如预期的那样，该突变消除了ATP酶活性。令人惊讶的是，突变体蛋白仍然能够部分挽救在CS-B成纤维细胞中显微注射UV后恢复RNA合成的缺陷。这些结果表明，保守的核苷酸结合结构域的完整性对于CSB的体内功能是重要的，但也独立于ATP水解的其他性质可能有助于CSB的生物学功能。
Cockayne syndrome (CS) is a nucleotide excision repair disorder characterized by sun (UV) sensitivity and severe developmental problems. Two genes have been shown to be involved: CSA and CSB. Both proteins play an essential role in preferential repair of transcription-blocking lesions from active genes. In this study we report the purification and characterization of baculovirus-produced HA-His(6)-tagged CSB protein (dtCSB), using a highly efficient three-step purification protocol. Microinjection of dtCSB protein in CS-B fibroblasts shows that it is biologically functional in vivo. dtCSB exhibits DNA-dependent ATPase activity, stimulated by naked as well as nucleosomal DNA. Using structurally defined DNA oligonucleotides, we show that double-stranded DNA and double-stranded DNA with partial single-stranded character but not true single-stranded DNA act as efficient cofactors for CSB ATPase activity. Using a variety of substrates, no overt DNA unwinding by dtCSB could be detected, as found with other SNF2/SWI2 family proteins. By site-directed mutagenesis the invariant lysine residue in the NTP-binding motif of CSB was substituted with a physicochemically related arginine, As expected, this mutation abolished ATPase activity. Surprisingly, the mutant protein was nevertheless able to partially rescue the defect in recovery of RNA synthesis after UV upon microinjection in CS-B fibroblasts, These results indicate that integrity of the conserved nucleotide-binding domain is important for the in vivo function of CSB but that also other properties independent from ATP hydrolysis may contribute to CSB biological functions.