A Molecular Switch in a Replication Machine Defined by an Internal Competition for Protein Rings  Vytautas Naktinis, Jennifer Turner, Mike O'Donnell  Cell  Volume 84, Issue 1, Pages 137-145 (January 1996) DOI: 10.1016/S0092-8674(00)81000-4

Figure 1 Core and γ Complex Interact with β in an Order Dictated by the DNA Structure (A) Clamp loading. (B) Elongation. (C) Clamp unloading. The γ complex and core are connected through the τ subunit. This study shows that both core and γ complex compete for the same surface of β. Further, the DNA structure determines the outcome. The γ complex binds β preferentially in the absence of DNA. But once β is on DNA, core develops a higher affinity for β, and the γ complex is excluded. Upon replication to a nick, core looses affinity to β, and γ complex recycles it from DNA. Cell 1996 84, 137-145DOI: (10.1016/S0092-8674(00)81000-4)

Figure 2 Core and γ Complex Interact near the C-Terminus of β (A) β2 crystal structure. A protein kinase motif was engineered onto the C-termini of the β dimer that extrude from one face of the ring. The βPK was incubated with the indicated subunit, protein kinase was added, and timepoints were analyzed on a polyacrylamide gel. The subunits were δ (the β-interacting subunit of the γ complex), α (DNA polymerase of core), δ′ (a subunit of γ complex), and τ (the subunit that organizes two cores and one γ complex into a particle). (B) Kinase protection assays. On the left are autoradiograms of the polyacrylamide gels. On the right is the quantitation of the autoradiograms: no addition is shown by open circles, δ by closed circles, δ′ by closed triangles, τ by open squares, and α by open diamonds. Cell 1996 84, 137-145DOI: (10.1016/S0092-8674(00)81000-4)

Figure 3 Protein Footprinting of β Interaction with γ Complex and Core The first four lanes in (A) and (B) are cleavage markers in which [35S]βPK was cleaved with specific proteases (Asp or Gln; lanes 1 and 2, respectively) and chemicals (Asn–Gly and Met; lanes 3 and 4, respectively). (A) Lanes 5–9 are treatment of [32S]βPK with pronase E and V8 protease. Lane 5 is β alone. Lane 6 is in the presence of excess δ. Lane 7 is in the presence of excess δ, as well as addition of unlabeled β equivalent to δ. Lanes 8 and 9 were as in lanes 6 and 7, except γ complex was used in place of δ. (B) Lanes 5–9 are as in (A), but using α or core. The arrows in (A) and (B) point to the cleavage fragment that is protected by δ, γ complex, α, and core. Cell 1996 84, 137-145DOI: (10.1016/S0092-8674(00)81000-4)

Figure 4 Site Mutants of β Show the C-Terminus Is Essential for Activity and Interaction with γ Complex and Core (A) Activity assays. (B) Binding assays. The last four amino acids of β were mutated to Ala (see top diagram). In (A), β mutants were titrated into replication assays: β with a wild-type C-terminus is shown by closed circles, L366A by open squares, R365A by closed squares, M364A by closed triangles, and P363A by open circles. In (B), interaction of β mutants with δ (top) and core (bottom) was evaluated by SPR. Cell 1996 84, 137-145DOI: (10.1016/S0092-8674(00)81000-4)

Figure 5 γ Complex and Core Compete for β Gel filtration analysis of a mixture of γ complex and core with substoichiometric β shows that γ complex binds β in exclusion of core. (A)–(D) are SDS–polyacrylamide gel analysis of column fractions eluted during analysis of β and a 4-fold molar excess of γ complex (A); β and a 4-fold molar excess of core (B); core and γ complex each in 4-fold molar excess over β (C); β alone (D). Cell 1996 84, 137-145DOI: (10.1016/S0092-8674(00)81000-4)

Figure 6 Core Binds β Tighter than γ Complex in the Presence of Primed DNA The β ring was assembled onto primed M13mp18 ssDNA and gel filtered to separate protein bound to DNA from protein in free solution. [35S]βPK, [3H]γ complex, and [3H]core were used to follow proteins in column fractions. (A) and (B) were the same except that in (A) [3H]γ complex and unlabeled core were used and in (B) [3H]core and unlabeled γ complex were used. Open circles are [35S]βPK, and closed circles are [3H]γ complex (A) or [3H]core (B). Cell 1996 84, 137-145DOI: (10.1016/S0092-8674(00)81000-4)

Figure 7 The γ Complex Removes Clamps from DNA Equivalent samples of [3H]β clamp–DNA complexes were incubated at 37°C in the presence (open circles) or absence (closed circles) of γ complex. At 1, 10, 30, and 60 min, a sample was removed from the reaction and assayed by gel filtration. Cell 1996 84, 137-145DOI: (10.1016/S0092-8674(00)81000-4)

Figure 8 Core Polymerase Protects Clamps from Being Removed by γ Complex, but Not after Completion of the Template Core (with τ) was bound to β on a gapped plasmid and then treated with γ complex either under conditions of idling with two dNTPs to prevent filling the gap (left) or in the presence of all four dNTPs to fill the gap (right). The reactions were then gel filtered to resolve [3H]β clamps that had been removed from DNA from those that bound to DNA. Cell 1996 84, 137-145DOI: (10.1016/S0092-8674(00)81000-4)

Figure 9 Model Illustrating How Core and γ Complex Coordinate Their Actions with β Rings in a Working Holoenzyme Particle Two DNA polymerase cores act on the two strands of a chromosome for simultaneous replication of both strands (diagram A). Each polymerase is shown with its own β sliding clamp for high processivity. Owing to the antiparallel orientation of the strands of duplex DNA, the lagging strand proceeds in the direction opposite fork movement, resulting in a loop. During extension of a lagging strand fragment, the γ complex loads a new clamp on an upstream RNA primer (diagrams A–B). Upon completing a fragment, the lagging polymerase disengages from its clamp, leaving the β ring on the DNA (diagrams B–C). The upstream β clamp falls into position with the lagging strand core for the next Okazaki fragment (diagrams C–D). In going from diagram D to diagram E, γ complex removes the used β clamp from the completed Okazaki fragment. An important feature in this model is that γ complex binds β tighter than core when β is off DNA (as in diagram A), but after assembly onto a primed site, the β ring associates preferentially with core (as in diagram C). The γ complex regains access to β on DNA only when orphaned by core upon completing a fragment. Cell 1996 84, 137-145DOI: (10.1016/S0092-8674(00)81000-4)