1. Volume 19, Issue 6, Pages 805-815 (September 2005)
A Sliding-Clamp Toolbelt Binds High- and Low-Fidelity DNA Polymerases Simultaneously 
Chiara Indiani, Peter McInerney, Roxana Georgescu, Myron F. Goodman, Mike O’Donnell 
Molecular Cell 
Volume 19, Issue 6, Pages (September 2005)
DOI: /j.molcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1 Pol IV Gains Control of β and DNA from a Stalled Pol III*
(A) Dissociation time of stalled Pol III* from β. Upon dissociation of the stalled Pol III* from DNA, it is captured by the excess M13Gori challenge DNA. At timed intervals, dATP and [α32P]dTTP are added to initiate a 20 s burst of synthesis, sufficient time for Pol III*-β to replicate either substrate to the complete RFII duplex circle.
(B) Time course of primer extension on a M13mp18 ssDNA substrate by either Pol III* (plus β) or Pol IV (plus β and γ complex).
(C) Different types of DNA polymerases are titrated into a reaction containing stalled Pol III*-β on primed M13mp18 ssDNA. After 10 s, a 20 s burst of synthesis is initiated. RFII products were analyzed in native agarose gels.
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3. Figure 2 The β Clamp Mediates p/t Control between Pol IV and Pol III*
(A) Pol IV is titrated into a stalled S. aureus (Sa) Pol C–β on primed M13mp18 ssDNA. After 10 s, synthesis is initiated upon adding remaining dNTPs, then quenched after a further 1 min.
(B) Pol IV is titrated into stalled E. coli (Ec) Pol III core or S. aureus Pol C bound to E. coli β. After 10 s, synthesis is initiated upon adding remaining dNTPs, then quenched after a further 1 min. Results using Sa Pol C are shown in the top gel. The control reaction with Ec Pol III core is shown below.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Pol III* and Pol IV Bind the β Clamp at the Same Time
The formation of a ternary complex between Pol IVOG (donor), Pol III αAR (acceptor), and E. coli β was monitored by FRET.
(A) Model of E. coli Pol IV attached to β encircling DNA. The Pol IV model is based on the structures of E. coli C-terminal domain of Pol IV (PDB code: 1UNN) (Bunting et al., 2003) and two related Pol IV/Din B type-Y class polymerases, Sulfolobus solfataricus (PDB code: 1JXL) (Ling et al., 2001), and human DNA Pol κ (PDB code: 1T94) (Uljon et al., 2004), using the Modeller 8.0 program (Marti-Renom et al., 2000). Pol IV was positioned on β by superimposition of the E. coli Pol IV model on the structure of E. coli β bound to a C-terminal domain of E. coli Pol IV (Bunting et al., 2003). The DNA is a B form duplex placed through the center of the β ring and perpendicular to the plane of the ring.
(B) Reaction mixtures contained 75 nM β2, 150 nM Pol IVOG, and the following amounts of Pol III αAR: 0 nM, blue triangles; 150 nM, red squares; 300 nM, yellow triangles; 600 nM, red triangles. Fluorescence resulting from nonsensitized Pol III αAR excitation was subtracted as described in Experimental Procedures.
(C) Control to assess whether the FRET signal requires β. Fluorescence emission spectra of 150 nM Pol IVOG alone (blue squares) or spectra corrected as described in Experimental Procedures of: 150 nM Pol IVOG plus either 150 nM Pol III αAR (blue diamonds), 300 nM Pol III αAR (red squares), or 600 nM Pol III αAR (yellow triangles).
(D) Control using β monomer mutant to determine whether the dimer is needed for FRET. Experimental conditions were the same as described in (B).
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Pol III* Rapidly Regains the p/t from a Moving Pol IV
(A) Pol IV (0.5 pmol) was assembled with β on primed DNA (using γ complex), then synthesis was initiated upon adding remaining dNTPs. After 10 s, 1.2 pmol Pol III* was added and reactions were quenched after 10, 20, 40, and 60 s. The plot shows the quantitation of DNA synthesis.
(B) The indicated amount of Pol IV is added to a stalled Pol III*-β 10 s before initiating DNA synthesis. Reactions were quenched after 10, 20, 30, 45, and 60 s.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 A Moving Pol III* Is Refractory to Pol IV
(A) Time course of primer extension on M13mp18 ssDNA by Pol III*-β using the nucleotide analog dAMPPNP.
(B) The indicated amount of Pol IV was added to a stalled Pol III* either 10 s before or 10 s after initiating synthesis with dAMPPNP. The total time of DNA synthesis was 90 s. Results of adding Pol IV to the moving Pol III* are shown in the top gel. The bottom gel shows results of adding Pol IV to the stalled Pol III*-β before initiating synthesis.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Pol III and Pol IV Remain Bound to β During Both Switches
(A) A β monomer mutant was used as a trap of Pol III* in solution. The β monomer trap (105 pmol in 2 μl buffer A) (top gel) or 2 μl of buffer A (bottom gel) was added to reactions containing β assembled on DNA in a preincubation with γ complex. After 5 s, DNA synthesis was initiated upon adding 100 fmol Pol III* and the remaining dNTPs. Reactions were quenched after 10, 20, 40, and 60 s.
(B) Pol IV was added to stalled Pol III*-β for 10 s followed by addition of 105 pmol β monomer mutant (top gel right) or an equal volume of buffer A (bottom gel right). After 5 s, replication was initiated and then quenched after 10, 20, 40, or 60 s. Control reactions in the absence of Pol IV are shown to the left.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 Coordinated Polymerase Switching on the β Clamp Toolbelt
Diagram 1: Pol IV and Pol III bind simultaneously to the β dimer. Pol III* has control of the p/t during normal replication. Diagram 2: When Pol III* stalls, Pol IV gains control of the primed DNA. Diagram 3: Pol IV-β extends the p/t past the stall site. Diagram 4: Pol III* regains control of the p/t from the moving Pol IV and resumes rapid and high-fidelity processive synthesis.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions