Schematic of SV40 T-ag Assembly on Origin DNA

<div><p>The SV40 origin dsDNA is depicted as two ribbons. The SV40 T-ag N-terminal J domain is omitted for clarity. The SV40 T-ag domains are depicted as follows: the obd (yellow spheres), the helicase domain (yellow ellipsoids), and the flexible linker that connects them (green).</p> <p>(A) The T-ag obd binds its high-affinity GAGGC sites. The T-ag obd anchors the protein on the four GAGGC pentamers and thus orients the helicase domain for appropriate DNA strand selection in the subsequent steps. The obds on P2 and P4 are shown as transparent spheres to indicate that they are not crucial for single-hexamer formation but are required for unwinding. The helicase domains may interact as monomers with the DNA in a non–sequence-specific manner at this point.</p> <p>(B) Once the origin has been recognized by the obds, the two helicase domains each hexamerize around one strand of DNA. As a result, one strand goes through the central channel of the helicase domain, and the other traverses the surface of the helicase domain. This is consistent with the crystal structure of the E1 helicase domain with ssDNA [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050023#pbio-0050023-b020" target="_blank">20</a>], and this model is similar to that proposed for other hexameric helicases [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050023#pbio-0050023-b050" target="_blank">50</a>]. It is not known whether the DNA at site II is melted at this point or not. Twelve obds are now in close proximity and may now interact with one another despite relatively weak affinities.</p> <p>(C) Interaction between the two hexamers could occur through a series of obd–obd structural rearrangements wherein these domains transition from the site-specific complex with the A1 and B2 loops fully engaged with the DNA, to a structure where loop B3 makes contacts across a pair of obds (possibly as spirals). The A1 and B2 loops are now oriented away from the central channel (and are proximal to the helicase domains). The open ring spiral hexamer of the T-ag obd would allow access of ssDNA from the outside surface of the helicase domain to the center of the channel. This channel is positively charged and sufficiently wide (approximately 30 Å) to accommodate two ssDNA strands moving in opposite directions. It is likely that the obds are dynamic and fluctuate between differing states of interaction, including aclosed hexameric ring, depending on the requirement to interact with ssDNA, dsDNA, or other factors, such as the SSB hRPA. The double hexamer is now assembled and ready to recruit other host factors necessary for replication (for a review, see [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050023#pbio-0050023-b003" target="_blank">3</a>]).</p>