Understanding the structural basis of replication initiation in AAV

了解 AAV 复制起始的结构基础

• 批准号: 8741430
• 负责人: Frederick Dyda
• 金额: $ 40.52万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8741430
• 关键词: Active Sites; Amyotrophic Lateral Sclerosis; Binding; Binding Sites; Bovine papillomavirus E1 protein; Cells; Characteristics; Childhood; Chromosomes, Human, Pair 19; Cleaved cell; Complex; Crystallization; Cystic Fibrosis; DNA; DNA Integration; DNA Sequence; DNA biosynthesis; DNA-dependent ATPase-endonuclease; Dependovirus; Diabetes Mellitus; Disease; Disease remission; Electron Microscopy; Enzymes; Exhibits; Foundations; Gene Transduction Agent; Genes; Genome; Goals; Hemophilia A; Human; Human Chromosomes; Insulin; Ions; Lead; Light; Location; Mediating; Medical; Membrane Proteins; Metals; Modeling; Molecular; Mus; Nature; Nucleotides; Oligonucleotides; Parvovirus; Process; Property; Proteins; Rattus; Replication Initiation; Replication-Associated Process; Reporting; Research; Resolution; SV40 T Antigens; Shorthand; Sickle Cell Anemia; Single-Stranded DNA; Site; Specificity; Structure; Surface; System; Tetranucleotide Repeat; Time; Tyrosine; Viral; Viral Genome; Viral Proteins; Virus; analog; base; design; endonuclease; gene correction; gene therapy; helicase; human DNA; human disease; improved; interest; member; nuclease; prevent; programs; site-specific integration; structural biology; success; vector; viral DNA

The structure of the Rep endonuclease domain (Hickman et al., 2002) revealed that it is unrelated to all other structurally characterized nucleases and provided the first view of an HUH superfamily member. Rep is homologous to the origin binding domains of the SV40 T antigen (Luo et al., 1996) and replication initiation protein E1 of bovine papillomavirus (Enemark et al., 2000). The HUH residues, which bind the catalytically required metal ion, converge with a helix bearing the two active site tyrosine residues to create the enzyme active site cleft. Subsequent co-crystal structures of the Rep nuclease domain with oligonucleotides representing two specific regions of the AAV genome showed that the nuclease domain uses two different protein surfaces to recognize its DNA target. One surface binds a hairpin that is at the very tip of the viral genome and the other recognizes a repeated tetranucleotide sequence close to the genome ends that constitutes the Rep binding site. These structures allowed us to propose a model for the assembly of a hexameric Rep-DNA complex that is poised to nick the viral DNA and begin unwinding it as a prelude to replication. It seems likely that site-specific integration also begins with a nick at a related sequence in human chromosome 19. Although it is believed that Rep assembles as a hexameric helicase, such assemblies had not previously been observed and the mode of Rep multimerization remains controversal. We have recently been studying an N-terminally truncated version of Rep in which the endonuclease domain is missing. This portion of Rep assembles as a hexamer on both single-stranded and dsDNA substrates, and is not dependent on specific viral DNA sequences or on the presence of nucleotides. We identified the portion of the protein responsible for multimerization on DNA, and we have been able to directly visualize these hexameric complexes using electron microscopy. Crystallization trials are underway. Enemark, E.J., Chen, G., Vaughn, D.E., Stenlund, A., and Joshua-Tor, L. (2000) Mol. Cell 6, 149-158. Flotte, T.R. (2005) Pediatric Res. 58, 1143-1147. Hickman, A.B., Ronning, D.R., Kotin, R.M., and Dyda, F. (2002) Mol. Cell 10, 327-337. Im, D.S. and Muzyczka, N. (1990) Cell 61, 447-457. Le Bec, C. and Douar, A.M. (2006) Gene Ther. 13, 805-813. Lee, H.C., Kim, S.J., Kim, K.S., Shin, H.CV., and Yoon, J. W. (2000) Nature 408, 483-488. Luo, X., Sanford, D.G., Bullock, P.A., and Bachovchin, W.W. (1996) Nat. Struct. Biol. 3, 1034-1039.

Rep 核酸内切酶结构域的结构（Hickman 等人，2002）揭示了它与所有其他结构特征核酸酶无关，并提供了 HUH 超家族成员的第一个视图。 Rep 与 SV40 T 抗原的起始结合域（Luo 等，1996）和牛乳头瘤病毒的复制起始蛋白 E1（Enemark 等，2000）同源。结合催化所需金属离子的 HUH 残基与带有两个活性位点酪氨酸残基的螺旋会聚，形成酶活性位点裂缝。随后 Rep 核酸酶结构域与代表 AAV 基因组两个特定区域的寡核苷酸的共晶结构表明，核酸酶结构域使用两种不同的蛋白质表面来识别其 DNA 靶标。一个表面结合位于病毒基因组最顶端的发夹，另一个表面识别靠近基因组末端的重复四核苷酸序列，该序列构成Rep结合位点。这些结构使我们能够提出一种六聚 Rep-DNA 复合物的组装模型，该复合物准备在病毒 DNA 上产生切口并开始将其解开，作为复制的前奏。位点特异性整合似乎也始于人类 19 号染色体相关序列上的切口。 尽管人们认为 Rep 组装为六聚体解旋酶，但以前从未观察到此类组装，并且 Rep 多聚化模式仍然存在争议。我们最近一直在研究 Rep 的 N 末端截短版本，其中核酸内切酶结构域缺失。 Rep 的这一部分在单链和 dsDNA 底物上组装为六聚体，并且不依赖于特定的病毒 DNA 序列或核苷酸的存在。 我们鉴定了负责 DNA 多聚化的蛋白质部分，并且我们已经能够使用电子显微镜直接可视化这些六聚体复合物。结晶试验正在进行中。 Enemark, E.J.、Chen, G.、Vaughn, D.E.、Stenlund, A. 和 Joshua-Tor, L. (2000) Mol。 6 号牢房，149-158。 弗洛特，T.R. (2005)儿科研究。 58、1143-1147。 Hickman, A.B.、Ronning, D.R.、Kotin, R.M. 和 Dyda, F. (2002) Mol。 10 号牢房，327-337。 Im, D.S. 和 Muzyczka, N. (1990) Cell 61, 447-457。 Le Bec, C. 和 Douar, A.M. （2006）吉恩·瑟尔。 13、805-813。 Lee, H.C.、Kim, S.J.、Kim, K.S.、Shin, H.CV. 和 Yoon, J. W. (2000) Nature 408, 483-488。 Luo, X.、Sanford, D.G.、Bullock, P.A. 和 Bachovchin, W.W. (1996) 国家。结构。生物。 3、1034-1039。