Understanding the structural basis of replication initiation in AAV

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的核酸酶无关，并提供了HUH超家族成员的第一个观点。Rep与SV 40 T抗原的起始结合结构域同源（Luo等人，1996）和牛乳头瘤病毒的复制起始蛋白E1（Enemark等，2000年）。结合催化所需的金属离子的HUH残基与带有两个活性位点酪氨酸残基的螺旋会聚以产生酶活性位点裂缝。随后Rep核酸酶结构域与代表AAV基因组的两个特定区域的寡核苷酸的共晶体结构显示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，其构成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复合物的模型，该复合物准备切割病毒DNA并开始解旋，作为复制的前奏。位点特异性整合似乎也始于人类19号染色体相关序列的切口。 尽管据信Rep组装为六聚体解旋酶，但以前未观察到这样的组装，并且Rep多聚化的模式仍然是有争议的。我们已经确定了在腺相关病毒DNA序列上组装全长Rep的六聚体形式所需的条件，并使用结合试验和DNA足迹方法来确定腺相关病毒病毒DNA上Rep结合位点的限制。我们可以在具有3'单链延伸的单链和dsDNA底物上产生Rep的六聚体复合物，并且结晶试验正在进行中。 此外，我们已经定义了删除分析的限制的多聚化基序的共和党。六聚体组装的形成是不依赖于特定的病毒DNA序列或核苷酸的存在。我们已经将与双链DNA复合的最小多聚化结构域结晶化，并且正在对这些进行晶体学分析。 Enemark，E.J.，Chen，G.，Vaughn，D.E.，Stenlund，A.，和Joshua-Tor，L.（2000）Mol. 6号牢房149-158 Flotte，T.R.等（2005）Pediatric Res. 58，1143-1147。 Hickman，A. B.，Ronning，D.R.，Kotin，R.M.，和Dyda，F.等人（2002）Mol. 10号牢房327-337 我，D.S.和Muzyczka，N.等（1990）Cell 61，447-457. 勒贝克角和Douar，A.M.（2006）Gene Ther. 13，805-813。 Lee，H.C.，Kim，S.J.，Kim，K.S.，Shin，H.C.，和Yoon，J.W.等人（2000）Nature 408，483-488. Luo，X.，桑福德，D.G.，布洛克，私人助理，和Bachovchin，W. W.等（1996）Nat.Struct.Biol.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.