Latency-associated nuclear antigen (LANA) cooperatively binds to two sites within the terminal repeat, and both sites contribute to the ability of LANA to suppress transcription and to facilitate DNA replication
Latency-associated nuclear antigen (LANA) cooperatively binds to two sites within the terminal repeat, and both sites contribute to the ability of LANA to suppress transcription and to facilitate DNA replication
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DOI：
10.1074/jbc.m203489200
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发表时间：
2002-07-26
影响因子：
4.8
通讯作者：
Renne, R
中科院分区：
文献类型：
--
作者：
Garber, AC;Hu, JH;Renne, R
卡波西肉瘤相关疱疹病毒的潜伏相关核抗原 (LANA) 是一种多功能蛋白，在转录调节和附加型维持中发挥重要作用。 LANA 也是一种 DNA 结合蛋白，已被证明可以特异性结合末端重复区域内的区域。在这里，我们对 LANA 的 DNA 结合活性进行了详细分析，结果表明它结合了两个相距 22 bp 的位点。我们使用电泳迁移率变动分析对结合位点进行定量分析，确定高亲和力位点的 K-d 为 1.51 +/- 0.16 nM。对位点末端附近核苷酸贡献的检查表明，核心结合位点由 16 bp 组成，其中 13 个在两个位点之间是保守的。对每个位点单独和串联的亲和力分析表明，与第二个位点的结合主要是由于与第一个位点的协同作用。通过删除和点突变，我们发现这两个位点都有助于 LANA 抑制转录和促进 DNA 复制的能力。此外，我们还表明 LANA 执行这些功能的能力与其对该区域中位点的亲和力成正比。两个位点之间的亲和力、间距和协同结合与 Epstein-Barr 病毒二分体对称元件 oriP 相似，表明这些相关 DNA 肿瘤病毒的潜在复制需要这样的元件。
The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus is a multifunctional protein with important roles in both transcriptional regulation and episomal maintenance. LANA is also a DNA-binding protein and has been shown to specifically bind to a region within the terminal repeat. Here, we have performed a detailed analysis of the DNA-binding activity of LANA and show that it binds two sites separated by 22 bp. We used electrophoretic mobility shift assay to quantitatively analyze the binding sites and determined that the K-d of the high affinity site is 1.51 +/- 0.16 nM. Examination of the contribution of nucleotides near the ends of the site showed that the core binding site consists of 16 bp, 13 of which are conserved between both sites. Analysis of the affinity of each site alone and in tandem revealed that the binding to the second site is primarily due to cooperativity with the first site. Using deletion and point mutations, we show that both sites contribute to the ability of LANA to suppress transcription and to facilitate DNA replication. In addition, we show that the ability of LANA to carry out these functions is directly proportional to its affinity for the sites in this region. The affinities, spacing, and cooperative binding between the two sites is similar to that of the Epstein-Barr virus dyad symmetry element oriP, suggesting a requirement for such an element in latent replication of these related DNA tumor viruses.