Carmichael E P, Roome J M, Wahl A F
Department of Cellular and Molecular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut 06492.
Mol Cell Biol. 1993 Jan;13(1):408-20. doi: 10.1128/mcb.13.1.408-420.1993.
The inverted repeat domain (IR domain) within the simian virus 40 origin of replication is the site of initial DNA melting prior to the onset of DNA synthesis. The domain had previously been shown to be bound by a cellular factor in response to DNA damage. We demonstrate that two distinct cellular components bind opposite strands of the IR domain. Replication protein A (RPA), previously identified as a single-stranded DNA binding protein required for origin-specific DNA replication in vitro, is shown to have a preference for the pyrimidine-rich strand. A newly described component, IR factor B (IRF-B), specifically recognizes the opposite strand. IRF-B binding activity in nuclear extract varies significantly with cell proliferation and the cell cycle, so that binding of IRF-B to the IR domain is negatively correlated with the onset of DNA synthesis. Loss of IRF-B binding from the nucleus also occurs in response to cellular DNA damage. UV cross-linking indicates that the core binding component of IRF-B is a protein of ca. 34 kDa. We propose that RPA and IRF-B bind opposite strands of the IR domain and together may function in the regulation of origin activation.
猿猴病毒40复制起点内的反向重复结构域（IR结构域）是DNA合成开始前初始DNA解链的位点。此前已表明该结构域会因DNA损伤而被一种细胞因子结合。我们证明了两种不同的细胞成分结合在IR结构域的相反链上。复制蛋白A（RPA），先前被鉴定为体外特异性起点DNA复制所需的单链DNA结合蛋白，显示出对富含嘧啶链的偏好。一种新描述的成分，IR因子B（IRF-B），特异性识别相反链。核提取物中的IRF-B结合活性随细胞增殖和细胞周期而显著变化，因此IRF-B与IR结构域的结合与DNA合成的开始呈负相关。细胞核中IRF-B结合的丧失也会因细胞DNA损伤而发生。紫外线交联表明IRF-B的核心结合成分是一种约34 kDa的蛋白质。我们提出RPA和IRF-B结合在IR结构域的相反链上，并且可能共同参与起点激活的调控。
