DNA Structure Modulates the Oligomerization Properties of the AAV Initiator Protein Rep68
DNA Structure Modulates the Oligomerization Properties of the AAV Initiator Protein Rep68
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DOI：
10.1371/journal.ppat.1000513
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发表时间：
2009-07-01
期刊：
影响因子：
6.7
通讯作者：
Linden, R. Michael
中科院分区：
文献类型：
--
作者：
Mansilla-Soto, Jorge;Yoon-Robarts, Miran;Linden, R. Michael
Rep 68是腺相关病毒（AAV）的多功能蛋白，腺相关病毒是一种细小病毒，主要以其作为基因治疗载体的前景而闻名。除了其作为病毒DNA复制中的起始剂的作用之外，Rep 68对于AAV基因组位点特异性整合到人染色体19中是必需的。Rep 68是超家族3（SF 3）解旋酶的成员，沿着有充分研究的起始蛋白猿猴病毒40大T抗原（SV 40-LTag）和牛乳头瘤病毒（BPV）E1。在结构上，SF 3解旋酶共有两个结构域，DNA起始相互作用结构域（DNA origin interaction domain，AAA）和AAA（+）马达结构域。AAA（+）马达结构域也是细胞引发剂的结构特征，并且其充当引发剂寡聚化的平台。在这里，我们研究了Rep 68寡聚化在体外不同的DNA底物的存在下，使用各种生物物理技术和冷冻EM。我们发现AAV起源的dsDNA区域促进含有5个Rep 68亚基的复合物的形成。有趣的是，非特异性ssDNA促进了双环Rep 68的形成，这是一种由LTag和E1起始蛋白形成的已知结构。Rep 68环对称性为8重，因此不同于由其它SF 3解旋酶形成的六聚体环。然而，与LTag和E1类似，Rep 68环是头对头定向的，这表明复合物的DNA解旋是双向进行的。这种新的Rep 68四级结构需要DNA结合和AAA（+）结构域，表明在体外寡聚化过程中这些区域之间的协同性。我们的研究清楚地表明Rep 68可以通过两种不同的寡聚化途径寡聚化，这取决于Rep 68结构域的DNA结构和协同性。这些发现提供了深入了解Rep 68的动力学和寡聚体适应性，并作为理解这种多功能蛋白在AAV DNA复制和位点特异性整合过程中的作用的一步。
Rep68 is a multifunctional protein of the adeno-associated virus (AAV), a parvovirus that is mostly known for its promise as a gene therapy vector. In addition to its role as initiator in viral DNA replication, Rep68 is essential for site-specific integration of the AAV genome into human chromosome 19. Rep68 is a member of the superfamily 3 (SF3) helicases, along with the well-studied initiator proteins simian virus 40 large T antigen (SV40-LTag) and bovine papillomavirus (BPV) E1. Structurally, SF3 helicases share two domains, a DNA origin interaction domain (OID) and an AAA(+) motor domain. The AAA(+) motor domain is also a structural feature of cellular initiators and it functions as a platform for initiator oligomerization. Here, we studied Rep68 oligomerization in vitro in the presence of different DNA substrates using a variety of biophysical techniques and cryo-EM. We found that a dsDNA region of the AAV origin promotes the formation of a complex containing five Rep68 subunits. Interestingly, non-specific ssDNA promotes the formation of a double-ring Rep68, a known structure formed by the LTag and E1 initiator proteins. The Rep68 ring symmetry is 8-fold, thus differing from the hexameric rings formed by the other SF3 helicases. However, similiar to LTag and E1, Rep68 rings are oriented head-to-head, suggesting that DNA unwinding by the complex proceeds bidirectionally. This novel Rep68 quaternary structure requires both the DNA binding and AAA(+) domains, indicating cooperativity between these regions during oligomerization in vitro. Our study clearly demonstrates that Rep68 can oligomerize through two distinct oligomerization pathways, which depend on both the DNA structure and cooperativity of Rep68 domains. These findings provide insight into the dynamics and oligomeric adaptability of Rep68 and serve as a step towards understanding the role of this multifunctional protein during AAV DNA replication and site-specific integration.