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内切核酸内切酶结构域的结构（Hickman等，2002）表明，它与所有其他结构表征的核酸酶无关，并提供了Huh超家族成员的第一视图。 REP与SV40 T抗原的原点结合结构域（Luo等，1996）和牛乳头瘤病毒的复制起始蛋白E1（Enemark等，2000）同源。结合催化所需的金属离子的HuH残基与螺旋轴承均匀，并带有两个活性位点酪氨酸残基，以形成酶的活性位点裂解。随后用代表AAV基因组的两个特定区域的寡核苷酸的REP核酸酶结构域的共结晶结构表明，核酸酶结构域使用两个不同的蛋白质表面来识别其DNA靶标。一个表面结合了病毒基因组尖端的发夹，另一个表面识别出接近构成REP结合位点的基因组末端的重复的四核苷酸序列。这些结构使我们能够提出一个模型，用于组装六聚体rep-DNA复合物，该模型有望划分病毒DNA，并开始将其放松为复制的前奏。似乎特定地点的集成也可能始于人类染色体19中相关序列的划痕。 尽管据认为REP作为六聚体解旋酶组装，但以前尚未观察到此类组件，并且REP多聚合的模式仍然存在争议。最近，我们一直在研究rep的N末端截断版本，其中核酸内切酶域缺失。 Rep的这一部分作为单链和DSDNA底物的六聚体组装，并且不依赖于特定的病毒DNA序列或核苷酸的存在。 我们确定了负责DNA多聚化的蛋白质的一部分，并且我们能够使用电子显微镜直接可视化这些六聚体配合物。结晶试验正在进行中。 E.J. Enemark，Chen，G.，Vaughn，D.E。，Stenlund，A。和Joshua-Tor，L。（2000）Mol。单元6，149-158。 Flotte，T.R。 （2005年）小儿res。 58，1143-1147。 Hickman，A.B.，Ronning，D.R.，Kotin，R.M。和Dyda，F。（2002）Mol。单元10，327-337。 IM，D.S。和Muzyczka，N。（1990）Cell 61，447-457。 Le Bec，C。和Douar，A.M。 （2006）基因。 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W.W. Bachovchin （1996）Nat。结构。生物。 3，1034-1039。