SAMHD1 is a single-stranded nucleic acid binding protein with no active site-associated nuclease activity.
SAMHD1 is a single-stranded nucleic acid binding protein with no active site-associated nuclease activity.
复制标题
DOI：
10.1093/nar/gkv633
复制
发表时间：
2015-07-27
影响因子：
14.9
通讯作者：
Stivers JT
中科院分区：
文献类型：
--
作者：
Seamon KJ;Sun Z;Shlyakhtenko LS;Lyubchenko YL;Stivers JT
HIV-1限制性因子SAMHD 1是一种由具有dNTP三磷酸水解酶活性（dNTR）的鸟嘌呤核苷酸激活的四聚体酶。除了这种确定的活性外，关于这种酶是否也具有单链DNA和/或RNA 3′-5′核酸外切酶活性，还有一系列相互矛盾的报道。SAMHD 1使用三个层析步骤纯化，在这三个层析步骤中，DNA酶活性与dNTR酶活性在很大程度上分离，但RNA酶活性持续存在。令人惊讶的是，我们发现，催化和核苷酸激活位点突变体的SAMHD 1没有dNTR酶活性保留核酸外切酶的活动。因此，外切核酸酶活性不能与任何已知的dNTP结合位点相关联。单体SAMHD 1被发现优先结合到单链RNA，而所需的dNTR作用的四聚体形式结合弱。ssRNA结合，而不是ssDNA，诱导高阶寡聚体状态，这与结合dNTP的四聚体形式不同。我们的结论是，在SAMHD 1制剂中检测到的痕量核酸外切酶活性来自与SAMHD 1共纯化的持久性污染物，而不是来自HD活性位点。一个体内模型的建议，其中SAMHD 1之间的相互排斥的功能的ssRNA结合和dNTP水解取决于dNTP池水平和病毒ssRNA的存在交替。
The HIV-1 restriction factor SAMHD1 is a tetrameric enzyme activated by guanine nucleotides with dNTP triphosphate hydrolase activity (dNTPase). In addition to this established activity, there have been a series of conflicting reports as to whether the enzyme also possesses single-stranded DNA and/or RNA 3′-5′ exonuclease activity. SAMHD1 was purified using three chromatography steps, over which the DNase activity was largely separated from the dNTPase activity, but the RNase activity persisted. Surprisingly, we found that catalytic and nucleotide activator site mutants of SAMHD1 with no dNTPase activity retained the exonuclease activities. Thus, the exonuclease activity cannot be associated with any known dNTP binding site. Monomeric SAMHD1 was found to bind preferentially to single-stranded RNA, while the tetrameric form required for dNTPase action bound weakly. ssRNA binding, but not ssDNA, induces higher-order oligomeric states that are distinct from the tetrameric form that binds dNTPs. We conclude that the trace exonuclease activities detected in SAMHD1 preparations arise from persistent contaminants that co-purify with SAMHD1 and not from the HD active site. An in vivo model is suggested where SAMHD1 alternates between the mutually exclusive functions of ssRNA binding and dNTP hydrolysis depending on dNTP pool levels and the presence of viral ssRNA.