Understanding the structural basis of replication initiation in AAV

Rep内切酶结构域的结构揭示了它与所有其他结构表征的核酸酶无关，并提供了HUH超家族成员的第一个视图。Rep与SV40 T抗原的起始结合域（Luo et al., 1996）和牛乳头瘤病毒的复制起始蛋白E1 （Enemark et al., 2000）同源。HUH残基结合催化所需的金属离子，与带有两个活性位点酪氨酸残基的螺旋聚集在一起，形成酶活性位点的间隙。在相关的病毒起源结合蛋白中，该区域被额外的残基遮挡。Rep核酸酶结构域与代表AAV基因组两个区域的寡核苷酸的后续共晶结构表明，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。一个表面与病毒基因组最尖端的发夹结合，另一个表面识别靠近基因组末端的重复四核苷酸序列，构成Rep结合位点。这些结构使我们提出了一个六聚体Rep-DNA复合体组装的模型，该复合体准备切断病毒DNA并开始将其解绕，作为复制的前奏。位点特异性整合似乎也可能始于人类第19号染色体相关序列上的一个缺口。

The structure of the Rep endonuclease domain 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. In the related viral origin binding proteins, this region is occluded by extra residues. Subsequent co-crystal structures of the Rep nuclease domain with oligonucleotides representing two 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 been attempting to determine 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 are able to generate hexameric complexes of Rep on dsDNA substrates which have a 3' single-strand extension, and 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. 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.