Assembly and activation of the SV40 pre-replication complex in vitro.

<p>(<b>A</b>) Domain architecture of SV40 Tag. Three structured domains (yellow) (DnaJ chaperone domain, origin DNA binding domain [OBD], and helicase domain), composed of the zinc (Zn) and AAA+ ATPase sub-domains, are connected by flexible regions (white) (P, cluster of phosphorylated residues that regulates origin activation; HR, host range function). (<b>B</b>) Diagram of ADP-associated SV40 Tag double hexamer bound to the duplex SV40 core origin of DNA replication (EP, central palindrome, AT), with non-origin DNA protruding from the complex (adapted from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002994#ppat.1002994-Cuesta1" target="_blank">[6]</a>). (<b>C</b>) 3D cryo-electron microscopy reveals two conformations (parallel, displaced) of ADP-associated hypo-phosphorylated SV40 Tag double hexamer on SV40 origin DNA as in (B) (adapted from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002994#ppat.1002994-Cuesta1" target="_blank">[6]</a>). A hypothetical conformation for the activated double hexamer is shown at the right. Dashed lines suggest potential paths of the DNA strands through each protein conformation. (<b>D</b>) Stages of SV40 replication. I, Tag dodecamer assembled on duplex SV40 DNA as in (B); II, hypo-phosphorylated Tag dodecamer activated as in (C) unwinds DNA bidirectionally <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002994#ppat.1002994-Wessel1" target="_blank">[13]</a> and may assemble host proteins (not shown here) into two sister replisomes that interact physically through the central lobe of the Tag dodecamer; III, hyper-phosphorylation of Tag disrupts interactions between the hexamers <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002994#ppat.1002994-Fanning2" target="_blank">[7]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002994#ppat.1002994-Weisshart1" target="_blank">[8]</a>, releasing the replisomes to progress independently along the template chromatin; IV, replication forks converge slowly, accompanied by DNA decatenation, to complete replication, which may involve additional host proteins <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002994#ppat.1002994-Bullock1" target="_blank">[3]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002994#ppat.1002994-Shi1" target="_blank">[14]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002994#ppat.1002994-Zhao1" target="_blank">[18]</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002994#ppat.1002994-Rohaly1" target="_blank">[21]</a>.