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。我们已经确定了在AAV DNA序列上组装全长Rep的必要条件，并使用结合分析和DNA足迹方法确定了AAV病毒DNA上Rep结合位点的界限。我们可以在具有3‘单链延伸的单链和dsDNA底物上生成Rep的六聚体络合物，结晶试验正在进行中。 此外，我们通过缺失分析定义了REP的多聚化基序的界限。六聚体组装的形成不依赖于特定的病毒DNA序列或核苷酸的存在。我们已经结晶了与双链DNA络合的最小多聚化结构域，并对其进行了结晶学分析。 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 determined the conditions necessary to assemble a hexameric form of full-length Rep on AAV DNA sequences, and have used binding assays and DNA footprinting methods to determine the limits of the Rep binding site on AAV viral DNA. We can generate hexameric complexes of Rep on both single-stranded and dsDNA substrates which have a 3' single-strand extension, and crystallization trials are underway. Furthermore, we have defined by deletion analysis the limits of the multimerization motif of Rep. The formation of the hexameric assembly is not dependent on specific viral DNA sequences or on the presence of nucleotides. We have crystallized the minimum multimerization domain complexed with double-stranded DNA and crystallographic analysis of these 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.