A Sliding-Clamp Toolbelt Binds High- and Low-Fidelity DNA Polymerases SimultaneouslyIntroductionResultsPol IV Rapidly Gains Control of beta and p/t from a Stalled Pol IIIPol IV and Pol III* Form a Ternary Complex with betaPol III* Rapidly Regains the p/t from a Moving Pol IVA Moving Pol III*-beta Is Refractory to Pol IVPol III and Pol IV Remain Bound to the beta Toolbelt During Polymerase SwitchingDiscussionTwo DNA Polymerases Bind beta at the Same TimeRegulation within the beta Toolbeltbeta Trafficking with Other FactorsExperimental ProceduresMaterialsPrimed M13mp18 ssDNA Extension AssaysStability of Stalled Pol III* with betaEffect of Pol IV on Stalled S. aureus Pol CFRET Measurementsbeta Monomer Trap ExperimentsAcknowledgmentsReferencesMolecular Cell, Vol. 19, 805–815, September 16, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.molcel.2005.08.011A Sliding-Clamp Toolbelt Binds High-and Low-Fidelity DNA Polymerases SimultaneouslyChiara Indiani,1Peter McInerney,1Roxana Georgescu,1Myron F. Goodman,2and Mike O’Donnell1,*1Laboratory of DNA ReplicationThe Rockefeller University1230 York AvenueNew York, New York 100212Department of Biological Sciences and ChemistryUniversity of Southern CaliforniaLos Angeles, California 90089SummaryThis report demonstrates that the ␤ sliding clamp ofE. coli binds two different DNA polymerases at thesame time. One is the high-fidelity Pol III chromo-somal replicase and the other is Pol IV, a low-fidelitylesion bypass Y family polymerase. Further, polymer-ase switching on the primed template junction is reg-ulated in a fashion that limits the action of the low-fidelity Pol IV. Under conditions that cause Pol III tostall on DNA, Pol IV takes control of the primed tem-plate. After the stall is relieved, Pol III rapidly regainscontrol of the primed template junction from Pol IVand retains it while it is moving, becoming resistantto further Pol IV takeover events. These polymerasedynamics within the ␤ toolbelt complex restrict theaction of the error-prone Pol IV to only the area onDNA where it is required.IntroductionRing-shaped DNA sliding clamps are opened andclosed around DNA by multiprotein clamp loaders, afterwhich they are used by numerous different proteins inboth prokaryotic (β clamp) and eukaryotic (PCNAclamp) organisms. For example, the E. coli β clamptethers the replicase, DNA polymerase (Pol) III holoen-zyme, to DNA for rapid (>500 ntd/s) and processivesynthesis (reviewed in Johnson and O’Donnell, 2005a).Additionally, β functions with the three damage induc-ible polymerases (Pol II, Pol IV, and Pol V) and with PolI(Bonner et al., 1992; Hughes et al., 1991; Kobayashiet al., 2002; Tang et al., 2000; Tippin et al., 2004;Wagner et al., 2000). The β clamp also binds ligase, MutS, and probably several other proteins as well (Dalrym-ple et al., 2001; Lopez de Saro and O’Donnell, 2001).Likewise, the eukaryotic PCNA sliding clamp is knownto interact with a wide variety of DNA polymerases andrepair proteins (Warbrick, 2000).The E. coli Pol III* replicase contains within its multi-component structure a clamp loader (γ1τ2δδ’χψ) thatbinds two molecules of Pol III core through the two τsubunits. At the replication fork, the clamp loader as-sembles β clamps onto the leading and lagging strandsfor use by the two Pol III cores. During DNA damage,*Correspondence: [email protected] damaged base on the leading strand will halt forkprogression by the high-fidelity Pol III. At this point, alow-fidelity polymerase like Pol IV and Pol V, both mem-bers of the Y family, presumably trade places with thestalled Pol III on β for lesion bypass, after which thehigh-fidelity Pol III may resume synthesis (Fujii andFuchs, 2004; Goodman, 2002; Lenne-Samuel et al.,2002; Lopez de Saro et al., 2003a; Maor-Shoshani etal., 2003; Napolitano et al., 2000). Although the exactroles of Pols II, IV, and V are still unclear, studies indi-cate that Pol V is responsible for the bulk of mutageniclesion bypass (Maor-Shoshani et al., 2003). However, allthree enzymes increase cell fitness (Yeiser et al., 2002),and Pol IV is mutagenic under some conditions (Bull etal., 2001; Kim et al., 1997; Kuban et al., 2004; Napo-litano et al., 2000; Tang et al., 2000; Tippin et al., 2004;Wagner and Nohmi, 2000).The detailed structure of the β binding site is revealedby the crystal structure of β in complex with the clamploader δ subunit, which shows that δ binds to a hy-drophobic pocket on the surface of β (Jeruzalmi et al.,2001). Studies of Pols II, III, and IV reveal that they tooattach to β via this same hydrophobic pocket, and theydo so via a short sequence in the extreme C terminusof the polymerases (Bunting et al., 2003; Burnouf et al.,2004; Dalrymple et al., 2001; Goodman, 2002). Recentstudies indicate that Pols I, II, III, IV, and V competewith one another and with the δ subunit for the clamp(Burnouf et al., 2004; Lopez de Saro et al., 2003a).Hence, mechanisms must exist that regulate the trafficflow of different polymerases on the clamp.Our previous studies of protein trafficking on β haveillustrated two distinct mechanisms by which proteinstrade places on the clamp. Traffic flow on β betweenthe clamp loader and the Pol III core is mediated bydirect competition for β, and the competition is regu-lated by ATP hydrolysis (Naktinis et al., 1996). ATP bind-ing is sufficient for clamp loading, but the clamp loaderremains bound to β, blocking Pol III (Hingorani andO’Donnell, 1998; Turner et al., 1999). ATP hydrolysisejects the clamp loader, freeing β to function with PolIII (Ason et al., 2003). A second β trafficking event oc-curs on the lagging strand. Upon the completion ofeach Okazaki fragment, Pol III detaches from β, leavingthe clamp on DNA where it may be used by Pol I andligase in Okazaki fragment maturation (O’Donnell, 1987;Stukenberg et al., 1994). This removal of Pol III from βis assisted by the τ subunit of the clamp loader, whichsevers the Pol III-β connection when replication of eachfragment is complete (Lopez de Saro et al., 2003b).The E. coli DNA polymerases may operate on β bycompeting with one another as suggested by the factthat all the DNA polymerases bind the same peptidebinding site on β. However, because the βclamp is adimer of identical protomers, it contains two peptidebinding pockets and therefore may act like a moleculartoolbelt to bind different DNA polymerases simulta-neously (Fujii and Fuchs, 2004; Johnson