A rat RuvB-like protein, TIP49, is a germ cell-enriched novel DNA helicase.

我们从大鼠肝脏中分离到一种新的核蛋白，其分子质量为49 kDa(TIP49)。TIP49在结构上与几种细菌RuvB相似，并显示Walker A和B基序。重要的是，TIP49的生化特性产生了以下重要的发现。1)TIP49具有受单链DNA和DNA解旋酶活性刺激的ATPase活性。2)TIP49与三磷酸腺苷特异性结合。3)TIP49在睾丸中大量表达，在脾、胸腺和肺中中等水平表达。在小鼠的生精小管中，从粗线期晚期精母细胞到圆形精子细胞的生殖系中，该蛋白被限制性地观察到。结果表明，TIP49是一种新的DNA解旋酶，可能参与重组和转录等核过程。抗TIP49抗体的免疫荧光分析显示，TIP49具有典型的点状核染色模式，表明TIP49属于大分子结构I…更多的在原子核中。此外，甘油梯度分析表明，TIP49在核提取液中以大分子复合体的形式存在。有趣的是，我们在多发性肌炎和自身免疫性肝炎等自身免疫性疾病患者的血清中检测到高水平的TIP49自身抗体。抗TIP49抗体可作为临床诊断和核内结构研究的有用探针。我们还证明了TIP49相关基因(S)在从人类到古生物的各种生物中都存在。广泛的同源性搜索发现了真核生物中另一类与TIP49相关的基因，命名为TIP49b。在以往的报道中，TIP49a仅被简称为TIP49。系统发育研究表明，古生菌的TIP49基因属于TIP49b的祖先，而不是TIP49a，TIP49a是从TIP49b进化而来的，与TIP49a一样，TIP49b在睾丸和胸腺中也有大量表达。酶分析表明，TIP49b是一种单链DNA激活的ATPase和依赖于ATP的DNA解旋酶。TIP49a和TIP49b相互结合，并在一个细胞内包含在同一个约700 kDa的复合体中。我们发现TIP49b和TIP49a基因是酿酒酵母生长所必需的基因，提示TIP49b不能补充TIP49a的功能，反之亦然。从这些观察中，我们认为TIP49b在参与dna代谢的细胞过程中扮演的角色较少。

We have isolated a novel nuclear protein with a molecular mass of 49 kDa (TIP49) from rat liver. TIP49 showed structural resemblance to several bacterial RuvBs and also displayed Walker A and B motifs. Importantly, biochemical characterization of TIP49 yielded the following significant findings. 1) TIP49 possessed ATPase activity that was stimulated by single-stranded DNA and DNA helicase activity. 2) TIP49 specifically interacted with ATP. 3) TIP49 was abundantly expressed in testes and moderately in spleen, thymus, and lung. In mouse seminiferous tubules, the protein was restrictively observed in germ lineages from late pachytene spermatocytes to round spermatids. From these observations, we propose that TIP49 is a novel DNA helicase and may play a role in nuclear processes such as recombination and transcription.Immunofluorescence analysis using anti-TIP49 antibody showed a typical dot-shaped nuclear staining pattern, suggesting that TIP49 is included in a macromolecular structure i … More n the nucleus. Moreover, glycerol gradient analysis demonstrated that TIP49 is present in a macromolecular complex in nuclear extract. Interestingly, we detected a high level of autoantibodies against TIP49 in sera of patients with autoimmune diseases such as polymyositis and autoimmune hepatitis. The anti-TIP49 antibodies can be useful probes for clinical diagnosis and for investigation of intranuclear structure. We also demonstrated that TIP49-related gene(s) in variety organisms from human to archaea. An extensive homology search revealed another class of TIP49-related gene in the eukaryotes, designated as TIP49b. TIP49a is just called as simply TIP49 in previous reports. A phylogenetical study suggested that archaeal TIP49 genes belong to the TIP49b ancestor but not the TIP49a one and that TIP49a evolved from TIP49b in accordance with divergence of archaea and eukarya.Like TIP49a, TIP49b was abundantly expressed in the testis and thymus. Enzyme assays revealed that TIP49b was an single-stranded DNA stimulated ATPase and ATP-dependent DNA helicase. TIP49a and TIP49b bound to each other and were included in the same complex of 〜700 kDa in a cell. We found that TIP49b was an essential gene for the growth of S. cerevisiae, as is the TIP49a gene, suggesting that TIP49b does not complement the TIP49a function and vice versa. From these observation, we suggest that TIP49b plays an essential role in the cellular processes involved in DNA metabolism Less