Structural basis for recruitment of translesion DNA polymerase Pol IV/DinB to the beta-clamp

Y-family DNA polymerases can extend primer strands across template strand lesions that stall replicative polymerases. The poor processivity and fidelity of these enzymes, key to their biological role, requires that their access to the primer-template junction is both facilitated and regulated in order to minimize mutations. These features are believed to be provided by interaction with processivity factors, beta-clamp or proliferating cell nuclear antigen (PCNA), which are also essential for the function of replicative DNA polymerases. The basis for this interaction is revealed by the crystal structure of the complex between the 'little finger' domain of the Y-family DNA polymerase Pol IV and the beta-clamp processivity factor, both from Escherichia coli. The main interaction involves a C-terminal peptide of Pol IV, and is similar to interactions seen between isolated peptides and other processivity factors. However, this first structure of an entire domain of a binding partner with an assembled clamp reveals a substantial secondary interface, which maintains the polymerase in an inactive orientation, and may regulate the switch between replicative and Y-family DNA polymerases in response to a template strand lesion.     

Structural and biochemical analysis of sliding clamp/ligand interactions suggest a competition between replicative and translesion DNA polymerases

Most DNA polymerases interact with their cognate processive replication factor through a small peptide, this interaction being absolutely required for their function in vivo. We have solved the crystal structure of a complex between the beta sliding clamp of Escherichia coli and the 16 residue C-terminal peptide of Pol IV (P16). The seven C-terminal residues bind to a pocket located at the surface of one beta monomer. This region was previously identified as the binding site of another beta clamp binding protein, the delta subunit of the gamma complex. We show that peptide P16 competitively prevents beta-clamp-mediated stimulation of both Pol IV and alpha subunit DNA polymerase activities, suggesting that the site of interaction of the alpha subunit with beta is identical with, or overlaps that of Pol IV. This common binding site for delta, Pol IV and alpha subunit is shown to be formed by residues that are highly conserved among many bacterial beta homologs, thus defining an evolutionarily conserved hydrophobic crevice for sliding clamp ligands and a new target for antibiotic drug design.     

Defining the position of the switches between replicative and bypass DNA polymerases

Cells contain specialized DNA polymerases that are able to copy past lesions with an associated risk of generating mutations, the major cause of cancer. Here, we reconstitute translesion synthesis (TLS) using the replicative (Pol III) and major bypass (Pol V) DNA polymerases from Escherichia coli in the presence of accessory factors. When the replicative polymerase disconnects from the template in the vicinity of a lesion, Pol V binds the blocked replication intermediate and forms a stable complex by means of a dual interaction with the tip of the RecA filament and the beta-clamp, the processivity factor donated by the blocked Pol III holoenzyme. Both interactions are required to confer to Pol V the processivity that will allow it synthesize, in a single binding event, a TLS patch long enough to support further extension by Pol III. In the absence of these accessory factors, the patch synthesized by Pol V is too short, being degraded by the Pol III-associated exonuclease activity that senses the distortion induced by the lesion, thus leading to an aborted bypass process.     

The biochemical requirements of DNA polymerase V-mediated translesion synthesis revisited

In addition to replicative DNA polymerases, cells contain specialized DNA polymerases involved in processes such as lesion tolerance, mutagenesis and immunoglobulin diversity. In Escherichia coli, DNA polymerase V (Pol V), encoded by the umuDC locus, is involved in translesion synthesis (TLS) and mutagenesis. Genetic studies have established that mutagenesis requires both UmuC and a proteolytic product of UmuD (UmuD'). In addition, RecA protein and the replication processivity factor, the beta-clamp, were genetically found to be essential co-factors for mutagenesis. Here, we have reconstituted Pol V-mediated bypass of three common replication-blocking lesions, namely the two major UV-induced lesions and a guanine adduct formed by a chemical carcinogen (G-AAF) under conditions that fulfil these in vivo requirements. Two co-factors are essential for efficient Pol V-mediated lesion bypass: (i) a DNA substrate onto which the beta-clamp is stably loaded; and (ii) an extended single-stranded RecA/ATP filament assembled downstream from the lesion site. For efficient bypass, Pol V needs to interact simultaneously with the beta-clamp and the 3' tip of the RecA filament. Formation of an extended RecA/ATP filament and stable loading of the beta-clamp are best achieved on long single-stranded circular DNA templates. In contrast to previously published data, the single-stranded DNA-binding protein (SSB) is not absolutely required for Pol V-mediated lesion bypass provided ATP, instead of ATPgammaS, activates the RecA filament. Further discrepancies with the existing literature are explainable by the use of either inadequate DNA substrates or a UmuC fusion protein instead of native Pol V.     

Mechanism of polymerase collision release from sliding clamps on the lagging strand

Replicative polymerases are tethered to DNA by sliding clamps for processive DNA synthesis. Despite attachment to a sliding clamp, the polymerase on the lagging strand must cycle on and off DNA for each Okazaki fragment. In the ‘collision release’ model, the lagging strand polymerase collides with the 5′ terminus of an earlier completed fragment, which triggers it to release from DNA and from the clamp. This report examines the mechanism of collision release by the Escherichia coli Pol III polymerase. We find that collision with a 5′ terminus does not trigger polymerase release. Instead, the loss of ssDNA on filling in a fragment triggers polymerase to release from the clamp and DNA. Two ssDNA‐binding elements are involved, the τ subunit of the clamp loader complex and an OB domain within the DNA polymerase itself. The τ subunit acts as a switch to enhance polymerase binding at a primed site but not at a nick. The OB domain acts as a sensor that regulates the affinity of Pol III to the clamp in the presence of ssDNA.     

Crystal structure of the catalytic alpha subunit of E. coli replicative DNA polymerase III

Bacterial replicative DNA polymerases such as Polymerase III (Pol III) share no sequence similarity with other polymerases. The crystal structure, determined at 2.3 A resolution, of a large fragment of Pol III (residues 1-917), reveals a unique chain fold with localized similarity in the catalytic domain to DNA polymerase beta and related nucleotidyltransferases. The structure of Pol III is strikingly different from those of members of the canonical DNA polymerase families, which include eukaryotic replicative polymerases, suggesting that the DNA replication machinery in bacteria arose independently. A structural element near the active site in Pol III that is not present in nucleotidyltransferases but which resembles an element at the active sites of some canonical DNA polymerases suggests that, at a more distant level, all DNA polymerases may share a common ancestor. The structure also suggests a model for interaction of Pol III with the sliding clamp and DNA.     

Translesion synthesis in Escherichia coli: lessons from the NarI mutation hot spot

Duplication of DNA containing damaged bases is a challenge to DNA polymerases that normally replicate with high speed, high accuracy and high processivity undamaged templates only. When a replicative DNA polymerase encounters a chemically altered base that it is unable to copy, a process called translesion synthesis (TLS) takes place during which the replicative polymerase is transiently replaced by a so-called specialized or lesion bypass polymerase. In addition to the central players that are the replicative and translesion DNA polymerases, TLS pathways involve accessory factors such as the general replication processivity factor (i.e. the beta-clamp in prokaryotes and PCNA in eukaryotes). In Escherichia coli, besides the beta-clamp, RecA plays a fundamental role as a co-factor of Pol V the major bypass polymerase in this organism. An integrated view of TLS pathways necessarily requires both genetic and biochemical studies. In this review we will attempt to summarize the insights into TLS gained over the last 25 years by studying a frameshift mutation hot spot, the NarI site. This site was initially discovered by serendipity when establishing a forward mutation spectrum induced by a chemical hepatocarcinogen, N-2-acetylaminofluorene (AAF). Indeed, this chemical carcinogen covalently binds to DNA forming adducts with guanine residues. When bound to G* in the NarI site, 5'-GGCG*CC-, AAF induces the loss of the G*pC dinucleotide at a frequency that is approximately 10(7)-fold higher than the spontaneous frequency. In vivo studies showed that the NarI mutation hot spot is neither restricted to the NarI sequence itself, nor to the carcinogen AAF. Instead, the hot spot requires a sequence containing at least two GpC repeats and any of a family of aromatic amides and nitro aromatic compounds that form a large class of human carcinogens. Genetic analysis initially revealed that the NarI frameshift pathway is SOS dependent but umuDC (i.e. Pol V) independent. More recently, DNA Pol II was identified as the enzyme responsible of this frameshift pathway. Concurrently the AAF adduct in the NarI site can be bypassed in an error-free way by Pol V. The NarI site thus offers a unique possibility to study the interplay between two specialized DNA polymerases, Pol II and Pol V, that can both extend replication intermediates formed when the replicative Pol III dissociates in the vicinity of the damage. Full reconstitution of the two pathways led us to highlight a key feature for TLS pathways, namely that it is critical the specialized DNA polymerase synthesizes, during the course of a single binding event, a patch of DNA synthesis (TLS patch) that is long enough as to "hide the lesion induced distortion" from the proofreading activity upon reloading of the replicative DNA polymerase (or any exonuclease that may get access to the primer when the specialized DNA polymerase detaches). The beta-clamp, to which all DNA polymerases bind, plays a critical role in allowing the specialized DNA polymerases to synthesize TLS patches that are long enough to resist such "external proofreading" activities.     

Motion of a DNA sliding clamp observed by single molecule fluorescence spectroscopy

DNA sliding clamps attach to polymerases and slide along DNA to allow rapid, processive replication of DNA. These clamps contain many positively charged residues that could curtail the sliding due to attractive interactions with the negatively charged DNA. By single-molecule spectroscopy we have observed a fluorescently labeled sliding clamp (polymerase III beta subunit or beta clamp) loaded onto freely diffusing, single-stranded M13 circular DNA annealed with fluorescently labeled DNA oligomers of up to 90 bases. We find that the diffusion constant for the beta clamp diffusing along DNA is on the order of 10(-14) m(2)/s, at least 3 orders of magnitude less than that for diffusion through water alone. We also find evidence that the beta clamp remains at the 3' end in the presence of Escherichia coli single-stranded-binding protein. These results may imply that the clamp not only acts to hold the polymerase on the DNA but also prevents excessive drifting along the DNA.     

Lethality of bypass polymerases in Escherichia coli cells with a defective clamp loader complex of DNA polymerase III

Escherichia coli DNA polymerase III (Pol III) is one of the best studied replicative DNA polymerases. Here we report the properties of an E. coli mutant that lacks one of the subunits of the Pol III clamp loader complex, Psi (psi), as a result of the complete inactivation of the holD gene. We show that, in this mutant, chronic induction of the SOS response in a RecFOR-dependent way leads to lethality at high temperature. The SOS-induced proteins that are lethal in the holD mutant are the specialized DNA polymerases Pol II and Pol IV, combined with the division inhibitor SfiA. Prevention of SOS induction or inactivation of Pol II, Pol IV and SfiA encoding genes allows growth of the holD mutant, although at a reduced rate compared to a wild-type cell. In contrast, the SOS-induced Pol V DNA polymerase does not participate to the lethality of the holD mutant. We conclude that: (i) Psi is essential for efficient replication of the E. coli chromosome; (ii) SOS-induction of specialized DNA polymerases can be lethal in cells in which the replicative polymerase is defective, and (iii) specialized DNA polymerases differ in respect to their access to inactivated replication forks.     

Protein trafficking on sliding clamps

The sliding clamps of chromosomal replicases are acted upon by both the clamp loader and DNA polymerase. Several other proteins and polymerases also interact with the clamp. These proteins bind the clamp at the same spot and use it in sequential fashion. First the clamp loader must bind the clamp in order to load it onto DNA, but directly thereafter the clamp loader must clear away from the clamp so it can be used by the replicative DNA polymerase. At the end of replication, the replicase is ejected from the clamp, which presumably allows the clamp to interact with yet other proteins after its use by the replicase. This paper describes how different proteins in the Escherichia coli replicase, DNA polymerase III holoenzyme, coordinate their traffic flow on the clamp. The mechanism by which traffic flow on the beta clamp is directed is based on competition of the proteins for the clamp, where DNA structure modulates the competition. It seems likely that the principles will generalize to a traffic flow of other factors on these circular clamp proteins.     

Purification and characterization of an inducible Escherichia coli DNA polymerase capable of insertion and bypass at abasic lesions in DNA

We have investigated the ability of DNA polymerases from SOS-induced and uninduced Escherichia coli to incorporate nucleotides at a well-defined abasic (apurinic/apyrimidinic) DNA template site and to extend these chains from this unpaired 3' terminus. A DNA polymerase activity has been purified from E. coli, deleted for DNA polymerase I, that appears to be induced 7-fold in cells following treatment with nalidixic acid. Induction of this polymerase (designated DNA polymerase X) appears to be part of the SOS response of E. coli since it cannot be induced in strains containing a noncleavable form of the LexA repressor (Ind-). The enzyme is able to incorporate nucleotides efficiently opposite the abasic template lesion and to continue DNA synthesis. Although we observe an approximate 2-fold induction of DNA polymerase III in cells treated with nalidixic acid, several lines of evidence argue that DNA polymerase X is unrelated to DNA polymerase III (pol III). In contrast to pol X, pol III shows almost no detectable ability to incorporate at or extend beyond the abasic site; incorporation efficiency at the abasic lesion is at least 100-fold larger for pol X compared to pol III holoenzyme, pol III core, or pol III* (the polymerase III holoenzyme subassembly lacking the beta subunit). Pol X does not cross-react with polyclonal antibody directed against pol III holoenzyme complex or with monoclonal antibody prepared to the alpha subunit of pol III. Despite these structural and biochemical differences, pol X appears to interact specifically with the beta subunit of the pol III holoenzyme in the presence of single-stranded binding protein. Pol X has a molecular mass of 84 kDa. Our results indicate that this novel activity is likely to be identical to DNA polymerase II of E. coli.     

Distinctive genetic features exhibited by the Y-family DNA polymerases in Bacillus subtilis

Translesional DNA polymerases form a large family of structurally related proteins, known as the Y-polymerases. Bacillus subtilis encodes two Y-polymerases, referred herewith as Pol Y1 and Pol Y2. Pol Y1 was expressed constitutively and did not mediate UV mutagenesis. Pol Y1 overexpression increased spontaneous mutagenesis. This effect depended on Pol Y1 polymerase activity, Pol Y1 interaction with the beta-clamp, and did not require the presence of the RecA protein. In addition, Pol Y1 overexpression delayed cell growth at low temperature. The growth delay was mediated by Pol Y1 interaction with the beta-clamp but not by its polymerase activity, suggesting that an excess of Pol Y1 in the cell could sequester the beta-clamp. In contrast, Pol Y2 was expressed during the SOS response, and, in its absence, UV-induced mutagenesis was abolished. Upon Pol Y2 overproduction, both UV-induced and spontaneous mutagenesis were stimulated, and both depended on the Pol Y2 polymerase activity. However, UV mutagenesis did not appear to require the interaction of Pol Y2 with the beta-clamp whereas spontaneous mutagenesis did. In addition, Pol Y2-mediated spontaneous mutagenesis required the presence of RecA. Together, these results show that the regulation and the genetic requirements of the two B. subtilis Y-polymerases are different, indicating that they fulfil distinct biological roles. Remarkably, Pol Y1 appears to exhibit a mutator activity similar to that of Escherichia coli Pol IV, as well as an E. coli UmuD-related function in growth delay. Pol Y2 exhibits an E. coli Pol V-like mutator activity, but probably acts as a single polypeptide to bypass UV lesions. Thus, B. subtilis Pol Y1 and Pol Y2 exhibit distinctive features from the E. coli Y-polymerases, indicating that different bacteria have adapted different solutions to deal with the lesions in their genetic material.     

The Escherichia coli dnaN159 mutant displays altered DNA polymerase usage and chronic SOS induction

The Escherichia coli beta sliding clamp, which is encoded by the dnaN gene, is reported to interact with a variety of proteins involved in different aspects of DNA metabolism. Recent findings indicate that many of these partner proteins interact with a common surface on the beta clamp, suggesting that competition between these partners for binding to the clamp might help to coordinate both the nature and order of the events that take place at a replication fork. The purpose of the experiments discussed in this report was to test a prediction of this model, namely, that a mutant beta clamp protein impaired for interactions with the replicative DNA polymerase (polymerase III [Pol III]) would likewise have impaired interactions with other partner proteins and hence would display pleiotropic phenotypes. Results discussed herein indicate that the dnaN159-encoded mutant beta clamp protein (beta159) is impaired for interactions with the alpha catalytic subunit of Pol III. Moreover, the dnaN159 mutant strain displayed multiple replication and repair phenotypes, including sensitivity to UV light, an absolute dependence on the polymerase activity of Pol I for viability, enhanced Pol V-dependent mutagenesis, and altered induction of the global SOS response. Furthermore, epistasis analyses indicated that the UV sensitivity of the dnaN159 mutant was suppressed by (not epistatic with) inactivation of Pol IV (dinB gene product). Taken together, these findings suggest that in the dnaN159 mutant, DNA polymerase usage, and hence DNA replication, repair, and translesion synthesis, are altered. These findings are discussed in terms of a model to describe how the beta clamp might help to coordinate protein traffic at the replication fork.     

Conserved interactions in the Staphylococcus aureus DNA PolC chromosome replication machine

The PolC holoenzyme replicase of the Gram-positive Staphylococcus aureus pathogen has been reconstituted from pure subunits. We compared individual S. aureus replicase subunits with subunits from the Gram-negative Escherichia coli polymerase III holoenzyme for activity and interchangeability. The central organizing subunit, tau, is smaller than its Gram-negative homolog, yet retains the ability to bind single-stranded DNA and contains DNA-stimulated ATPase activity comparable with E. coli tau. S. aureus tau also stimulates PolC, although they do not form as stabile a complex as E. coli polymerase III.tau. We demonstrate that the extreme C-terminal residues of PolC bind to and function with beta clamps from different bacteria. Hence, this polymerase-clamp interaction is highly conserved. Additionally, the S. aureus delta wrench of the clamp loader binds to E. coli beta. The S. aureus clamp loader is even capable of loading E. coli and Streptococcus pyogenes beta clamps onto DNA. Interestingly, S. aureus PolC lacks functionality with heterologous beta clamps when they are loaded onto DNA by the S. aureus clamp loader, suggesting that the S. aureus clamp loader may have difficulty ejecting from heterologous clamps. Nevertheless, these overall findings underscore the conservation in structure and function of Gram-positive and Gram-negative replicases despite >1 billion years of evolutionary distance between them.     

Exchange between Escherichia coli polymerases II and III on a processivity clamp

Escherichia coli has three DNA polymerases implicated in the bypass of DNA damage, a process called translesion synthesis (TLS) that alleviates replication stalling. Although these polymerases are specialized for different DNA lesions, it is unclear if they interact differently with the replication machinery. Of the three, DNA polymerase (Pol) II remains the most enigmatic. Here we report a stable ternary complex of Pol II, the replicative polymerase Pol III core complex and the dimeric processivity clamp, β. Single-molecule experiments reveal that the interactions of Pol II and Pol III with β allow for rapid exchange during DNA synthesis. As with another TLS polymerase, Pol IV, increasing concentrations of Pol II displace the Pol III core during DNA synthesis in a minimal reconstitution of primer extension. However, in contrast to Pol IV, Pol II is inefficient at disrupting rolling-circle synthesis by the fully reconstituted Pol III replisome. Together, these data suggest a β-mediated mechanism of exchange between Pol II and Pol III that occurs outside the replication fork.     

Crystal structure of an archaebacterial DNA polymerase.

BACKGROUND: Members of the Pol II family of DNA polymerases are responsible for chromosomal replication in eukaryotes, and carry out highly processive DNA replication when attached to ring-shaped processivity clamps. The sequences of Pol II polymerases are distinct from those of members of the well-studied Pol I family of DNA polymerases. The DNA polymerase from the archaebacterium Desulfurococcus strain Tok (D. Tok Pol) is a member of the Pol II family that retains catalytic activity at elevated temperatures. RESULTS: The crystal structure of D. Tok Pol has been determined at 2.4 A resolution. The architecture of this Pol II type DNA polymerase resembles that of the DNA polymerase from the bacteriophage RB69, with which it shares less than approximately 20% sequence identity. As in RB69, the central catalytic region of the DNA polymerase is located within the 'palm' subdomain and is strikingly similar in structure to the corresponding regions of Pol I type DNA polymerases. The structural scaffold that surrounds the catalytic core in D. Tok Pol is unrelated in structure to that of Pol I type polymerases. The 3'-5' proofreading exonuclease domain of D. Tok Pol resembles the corresponding domains of RB69 Pol and Pol I type DNA polymerases. The exonuclease domain in D. Tok Pol is located in the same position relative to the polymerase domain as seen in RB69, and on the opposite side of the palm subdomain compared to its location in Pol I type polymerases. The N-terminal domain of D. Tok Pol has structural similarity to RNA-binding domains. Sequence alignments suggest that this domain is conserved in the eukaryotic DNA polymerases delta and epsilon. CONCLUSIONS: The structure of D. Tok Pol confirms that the modes of binding of the template and extrusion of newly synthesized duplex DNA are likely to be similar in both Pol II and Pol I type DNA polymerases. However, the mechanism by which the newly synthesized product transits in and out of the proofreading exonuclease domain has to be quite different. The discovery of a domain that seems to be an RNA-binding module raises the possibility that Pol II family members interact with RNA.     

The internal workings of a DNA polymerase clamp-loading machine.

Replicative DNA polymerases are multiprotein machines that are tethered to DNA during chain extension by sliding clamp proteins. The clamps are designed to encircle DNA completely, and they are manipulated rapidly onto DNA by the ATP-dependent activity of a clamp loader. We outline the detailed mechanism of gamma complex, a five-protein clamp loader that is part of the Escherichia coli replicase, DNA polymerase III holoenzyme. The gamma complex uses ATP to open the beta clamp and assemble it onto DNA. Surprisingly, ATP is not needed for gamma complex to crack open the beta clamp. The function of ATP is to regulate the activity of one subunit, delta, which opens the clamp simply by binding to it. The delta' subunit acts as a modulator of the interaction between delta and beta. On binding ATP, the gamma complex is activated such that the delta' subunit permits delta to bind beta and crack open the ring at one interface. The clamp loader-open clamp protein complex is now ready for an encounter with primed DNA to complete assembly of the clamp around DNA. Interaction with DNA stimulates ATP hydrolysis which ejects the gamma complex from DNA, leaving the ring to close around the duplex.     

DNA polymerase I acts in translesion synthesis mediated by the Y-polymerases in Bacillus subtilis

Translesion synthesis (TLS) across damaged DNA bases is most often carried out by the ubiquitous error-prone DNA polymerases of the Y-family. Bacillus subtilis encodes two Y-polymerases, Pol Y1 and Pol Y2, that mediate TLS resulting in spontaneous and ultraviolet light (UV)-induced mutagenesis respectively. Here we show that TLS is a bipartite dual polymerase process in B. subtilis, involving not only the Y-polymerases but also the A-family polymerase, DNA polymerase I (Pol I). Both the spontaneous and the UV-induced mutagenesis are abolished in Pol I mutants affected solely in the polymerase catalytic site. Physical interactions between Pol I and either of the Pol Y polymerases, as well as formation of a ternary complex between Pol Y1, Pol I and the beta-clamp, were detected by yeast two- and three-hybrid assays, supporting the model of a functional coupling between the A- and Y-family polymerases in TLS. We suggest that the Pol Y carries the synthesis across the lesion, and Pol I takes over to extend the synthesis until the functional replisome resumes replication. This key role of Pol I in TLS uncovers a new function of the A-family DNA polymerases.     

DNA polymerase lambda (Pol lambda), a novel eukaryotic DNA polymerase with a potential role in meiosis

A new gene (POLL) encoding a novel DNA polymerase (Pol lambda) has been identified at mouse chromosome 19. Murine Pol lambda, consisting of 573 amino acid residues, has a 32% identity to Pol beta, involved in nuclear DNA repair in eukaryotic cells. It is interesting that Pol lambda contains all the critical residues involved in DNA binding, nucleotide binding and selection, and catalysis of DNA polymerization, that are conserved in Pol beta and other DNA polymerases belonging to family X. Murine Pol lambda, overproduced in Escherichia coli, displayed intrinsic DNA polymerase activity when assessed by in situ gel analysis. Pol lambda also conserves the critical residues of Pol beta required for its intrinsic deoxyribose phosphate lyase (dRPase) activity. The first 230 amino acid residues of Pol lambda, that have no counterpart in Pol beta, contain a BRCT domain, present in a variety of cell-cycle check-point control proteins responsive to DNA damage and proteins involved in DNA repair. Northern blotting, in situ hybridization analysis and immunostaining showed high levels of Pol lambda specifically expressed in testis, being developmentally regulated and mainly associated to pachytene spermatocytes. These first evidences, although indirect, suggest a potential role of Pol lambda in DNA repair synthesis associated with meiosis.     

The Escherichia coli clamp loader can actively pry open the β-sliding clamp

Clamp loaders load ring-shaped sliding clamps onto DNA. Once loaded onto DNA, sliding clamps bind to DNA polymerases to increase the processivity of DNA synthesis. To load clamps onto DNA, an open clamp loader-clamp complex must form. An unresolved question is whether clamp loaders capture clamps that have transiently opened or whether clamp loaders bind closed clamps and actively open clamps. A simple fluorescence-based clamp opening assay was developed to address this question and to determine how ATP binding contributes to clamp opening. A direct comparison of real time binding and opening reactions revealed that the Escherichia coli γ complex binds β first and then opens the clamp. Mutation of conserved "arginine fingers" in the γ complex that interact with bound ATP decreased clamp opening activity showing that arginine fingers make an important contribution to the ATP-induced conformational changes that allow the clamp loader to pry open the clamp.