1. The Crystal Structure of the Bifunctional Primase-Helicase of Bacteriophage T7 
Eric A. Toth, Ying Li, Michael R. Sawaya, Yifan Cheng, Tom Ellenberger 
Molecular Cell 
Volume 12, Issue 5, Pages (November 2003)
DOI: /S (03)

2. Figure 1 Crystallographic Structure of the T7 Primase-Helicase
(A) A ribbon diagram of T7 primase-helicase shows that the helicase domains (red) and associated DNA binding loops (yellow) are arranged in a ring with approximate 7-fold rotational symmetry. This view shows the C-terminal side of the ring.
(B) A view of the N-terminal side of the ring shows seven primase domains (blue) are loosely arrayed in different orientations on top of the helicase ring (red). The active sites of the primases (gold balls) roughly face the circumference of the ring and point toward the neighboring subunit. One of the primase domains, designated with an orange ball, tilts toward the center of the ring where its active site is positioned for interaction with DNA passing through the central channel.
(C) A schematic diagram, oriented as in (B), shows the relative orientations of the primase domains (blue balls with N-terminal “hats”) and their active sites (light blue dots). The inward facing primase domain has an orange hat. The underlying helicase domains (red) are shown with yellow arrows that point toward the helicase active site. Ribbon diagrams were created with the program RIBBONS (Carson, 1997) and rendered with POV-Ray (Amundsen et al., 2000).
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2 Summary of Electron Microscopic Images
(A) Where applicable, particles are listed as number picked/number classifiable. 63 kD-WT is a mutant (M64G) designed to prevent simultaneous expression of both the 56 kD and 63 kD isoforms.
(B) The top two panels are representative particles from electron micrographs of the heptameric (left) and hexameric (right) forms of 63 kD-WT. The bottom two panels are class averages of these two forms.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3 Crown-Shaped Appearance of the T7 Primase-Helicase
A side view of the surface of the primase-helicase shows that the primase domains (top of figure) are swapped onto neighboring helicase domains (bottom), which pack together in a closed ring. An extended “clasp” between the primase and helicase domains packs against the adjacent subunit and contributes most of the contacts between subunits (cf. Figure 4). The primase domains make few contacts with one another and they are oriented differently in all seven subunits (cf. Figure 1). This loose-packed arrangement would allow a DNA template to bind to the primase active site in the gap between adjacent primase domains. The surface rendering was generated with the program MOLMOL (Koradi et al., 1996) and rendered with POV-Ray (Amundsen et al., 2000).
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4 Subunit Packing in T7 Primase-Helicase
(A) A view along the circumference of the ring. The helicase (red) and primase (blue) domains are joined by a segment termed the “clasp” (yellow) that packs against a neighboring subunit shown as a surface representation (gray). The clasp consists of helix A from the helicase domain (Sawaya et al., 1999) and surrounding residues, including two flexible joints, termed “swivels.” The experimentally phased electron density (black mesh) clearly shows the path of the clasp, which positions the primase domain over the helicase domain of the neighboring subunit in a right-handed, domain-swapping arrangement. One swivel allows the primase domain to roll around on the surface of the helicase, as evidenced by the different orientations seen in the crystal (cf. Figures 1B and 1C). Note the severe tilt of the primase domain of the gray surface toward the center of the ring. The other swivel allows movement of the helicase domain, as previously proposed in the mechanism of DNA unwinding (Singleton et al., 2000).
(B) The subunit packing interface as viewed from inside the ring reveals the positions of the loops containing residues that are important for DNA binding activity (yellow) and the active site of the helicase, denoted by the P loop (cyan). At the C terminus of the helicase domain is an acidic segment that participates in interactions with T7 DNA polymerase (Notarnicola et al., 1997). The primase domain (blue, in [A] colored gray) is markedly tilted inward toward the center of the ring (cf. Figures 1B and 1C).
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5 Proposed Role of the Heptameric T7 Helicase
The expanded ring formed by the heptameric primase-helicase accommodates dsDNA in the central channel, whereas the smaller hexameric helicase (Singleton et al., 2000) appears to only accommodate a single strand of DNA. A DNA remodeling activity of the T7 primase-helicase was recently described (Kaplan and O'Donnell, 2002) with translocation on dsDNA that is consistent with the larger size of the heptameric ring.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6
A Conserved Glycine Correctly Positions the P Loop of the Helicase
The crystallized primase-helicase contains two inactivating mutations (G317V, K318M) in the Walker A (P loop) motif of the helicase. The superimposed structures of the mutant (green) and wild-type (white) proteins are shown in stereo along with the nucleotide (transparent orange) bound to the wild-type helicase (Sawaya et al., 1999). The main chain dihedral angles of G317 in the wild-type protein are forbidden to valine. Therefore, the G317V mutation causes a twist in the backbone that brings the neighboring residue M316 into the nucleotide binding pocket and prevents nucleotide binding.
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7 Model for Coupled DNA Unwinding and Primer Synthesis
The helicase encircles the lagging strand of the replication fork, catalyzing the unwinding of DNA in a 5′→3′ direction. The negative charge of the C-terminal surface of the helicase (Sawaya et al., 1999) could assist in displacing the 3′ tail of the DNA. The primase is initially disengaged during DNA unwinding by the helicase, and then it moves into position to synthesize a primer. The zinc binding domain of the primase (yellow ball) engages the priming site on the DNA along with the RNA polymerase domain (peach oval) of the primase (Kato et al., 2003). Following primer synthesis, the primase opens up and the zinc binding domain alone delivers the primed DNA template to DNA polymerase to initiate synthesis of an Okazaki fragment (not pictured here). The articulated motions seen in the crystal structure of T7 primase-helicase reconcile the continual unwinding of DNA by the helicase with the periodic RNA synthesis by the primase that initiates each Okazaki fragment.
Molecular Cell  , DOI: ( /S (03) )