Tsurumi T, Kishore J, Yokoyama N, Fujita M, Daikoku T, Yamada H, Yamashita Y, Nishiyama Y
Laboratory of Viral Oncology, Aichi Cancer Center Research Institute, Nagoya, Japan.
J Gen Virol. 1998 May;79 ( Pt 5):1257-64. doi: 10.1099/0022-1317-79-5-1257.
The Epstein-Barr virus (EBV) ssDNA-binding protein (SSB) encoded by the BALF2 gene is one of the essential replication proteins in the lytic phase of EBV DNA replication. In order to obtain the amount of EBV SSB required for characterization, a recombinant baculovirus containing the complete sequence of the BALF2 open reading frame under the control of the baculovirus polyhedrin promoter was constructed. Insect cells infected with the recombinant virus produced a protein of 130 kDa, recognized by anti-BALF2 protein-specific polyclonal antibody. The overexpressed EBV SSB was purified homogeneously from the cytosolic fraction of the recombinant virus-infected cells. The purified protein displaced short DNA strands from their complementary sequences in the single-stranded form of M13. The helix-destabilizing activity was neutralized by the anti-BALF2 protein-specific antibody. Maximum unwinding occurred at EBV SSB concentrations exceeding saturation level of the DNA substrate. The DNA unwinding reaction mediated by the EBV SSB was highly cooperative and extremely rapid. The reaction displayed no directionality and required neither ATP nor MgCl2, two essential cofactors for DNA helicase activity. The helix-destabilizing property of the EBV SSB may function to melt out secondary structures on the ssDNA template, thereby facilitating the movement of the EBV DNA polymerase.
由BALF2基因编码的爱泼斯坦-巴尔病毒（EBV）单链DNA结合蛋白（SSB）是EBV DNA复制裂解期的必需复制蛋白之一。为了获得用于特性鉴定所需的EBV SSB量，构建了一种重组杆状病毒，其在杆状病毒多角体蛋白启动子的控制下含有BALF2开放阅读框的完整序列。用重组病毒感染昆虫细胞产生了一种130 kDa的蛋白质，可被抗BALF2蛋白特异性多克隆抗体识别。从重组病毒感染细胞的胞质部分中均匀纯化出过表达的EBV SSB。纯化的蛋白质从M13单链形式的互补序列中置换出短DNA链。螺旋解链活性被抗BALF2蛋白特异性抗体中和。在EBV SSB浓度超过DNA底物饱和水平时发生最大解链。由EBV SSB介导的DNA解链反应具有高度协同性且极其迅速。该反应没有方向性，既不需要ATP也不需要MgCl2，而这两者是DNA解旋酶活性的两个必需辅助因子。EBV SSB的螺旋解链特性可能起到融化单链DNA模板上二级结构的作用，从而促进EBV DNA聚合酶的移动。
