Understanding the structural basis of replication initiation in AAV

Rep内切酶结构域的结构(Hickman等人，2002年)表明它与所有其他结构特征的核酸酶无关，并提供了HH超家族成员的第一个视角。REP与SV40T抗原(Luo等人，1996)和牛乳头瘤病毒复制起始蛋白E1(Enemark等人，2000)的起始结合区同源。HH残基与催化所需的金属离子结合，与带有两个活性部位酪氨酸残基的螺旋汇合，形成酶活性部位裂解。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个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末端的发夹，另一个表面识别靠近基因组末端的重复的四核苷酸序列，构成Rep结合位点。这些结构使我们能够提出一个组装六聚体Rep-DNA复合体的模型，该复合体准备切割病毒DNA并开始解开它，作为复制的前奏。似乎定点整合也是从人类19号染色体相关序列的缺口开始的。 尽管人们认为Rep以六聚体解旋酶的形式组装，但这种组装以前没有被观察到，Rep多聚体的模式仍然存在争议。我们最近一直在研究N端截短的Rep，其中缺少核酸内切酶结构域。Rep的这一部分在单链和dsDNA底物上以六聚体的形式组装，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上多聚体的蛋白质部分，并能够用电子显微镜直接显示这些六聚体复合体。结晶试验正在进行中。 书名/作者/作者：En-Ej.牢房6,149-158。 Flotte，T.R.(2005)儿科研究报告58,1143-1147。 Hickman，A.B.，Ronning，D.R.，Kotin，R.M.和Dyda，F.(2002)Mol.牢房10,327-337。 Im，D.S.和Muzyczka，N.(1990)细胞61,447-457。 Le Bec，C.和Douar，A.M.(2006)吉恩·瑟尔。13,805-813 李华章，金圣杰，金建生，申建新，陈华清，尹俊伟(2000)自然，408,483-488. 罗翔，桑福德，D.G.，布洛克，P.A.和巴霍夫钦，W.W.(1996)NAT。结构。比奥尔。3,1034-1039。

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.