Characterization of the Escherichia coli XPD/Rad3 iron-sulfur helicase YoaA in complex with the DNA polymerase III clamp loader subunit chi (χ).

Escherichia coli YoaA aids in the resolution of DNA damage that halts DNA synthesis in vivo in conjunction with χ, an accessory subunit of DNA polymerase III. YoaA and χ form a discrete complex separate from the DNA polymerase III holoenzyme, but little is known about how YoaA and χ work together to help the replication fork overcome damage. Although YoaA is predicted to be an iron-sulfur helicase in the XPD/Rad3 helicase family based on sequence analysis, the biochemical activities of YoaA have not been described. Here, we characterize YoaA and show that purified YoaA contains iron. YoaA and χ form a complex that is stable through three chromatographic steps, including gel filtration chromatography. When overexpressed in the absence of χ, YoaA is mostly insoluble. In addition, we show the YoaA-χ complex has DNA-dependent ATPase activity. Our measurement of the YoaA-χ helicase activity illustrates for the first time YoaA-χ translocates on ssDNA in the 5' to 3' direction and requires a 5' single-stranded overhang, or ssDNA gap, for DNA/DNA unwinding. Furthermore, YoaA-χ preferentially unwinds forked duplex DNA that contains both 3' and 5' single-stranded overhangs versus duplex DNA with only a 5' overhang. Finally, we demonstrate YoaA-χ can unwind damaged DNA that contains an abasic site or damage on 3' ends that stall replication extension. These results are the first biochemical evidence demonstrating YoaA is a bona fide iron-sulfur helicase, and we further propose the physiologically relevant form of the helicase is YoaA-χ.

Cellular and molecular characterization of two novel asparagine synthetase gene mutations linked to asparagine synthetase deficiency.

Asparagine synthetase (ASNS) catalyzes synthesis of asparagine (Asn) and Glu from Asp and Gln in an ATP-dependent reaction. Asparagine synthetase deficiency (ASNSD) results from biallelic mutations in the ASNS gene. Affected children exhibit congenital microcephaly, continued brain atrophy, seizures, and often premature mortality. However, the underlying mechanisms are unclear. This report describes a compound heterozygotic ASNSD child with two novel mutations in the ASNS gene, c.1118G>T (paternal) and c.1556G>A (maternal), that lead to G373V or R519H ASNS variants. Structural mapping suggested that neither variant participates directly in catalysis. Growth of cultured fibroblasts from either parent was unaffected in Asn-free medium, whereas growth of the child's cells was suppressed by about 50%. Analysis of Asn levels unexpectedly revealed that extracellular rather than intracellular Asn correlated with the reduced proliferation during incubation of the child's cells in Asn-free medium. Our attempts to ectopically express the G373V variant in either HEK293T or JRS cells resulted in minimal protein production, suggesting instability. Protein expression and purification from HEK293T cells revealed reduced activity for the R519H variant relative to WT ASNS. Expression of WT ASNS in ASNS-null JRS cells resulted in nearly complete rescue of growth in Asn-free medium, whereas we observed no proliferation for the cells expressing either the G373V or R519H variant. These results support the conclusion that the coexpression of the G373V and R519H ASNS variants leads to significantly reduced Asn synthesis, which negatively impacts cellular growth. These observations are consistent with the ASNSD phenotype.

Potassium Glutamate and Glycine Betaine Induce Self-Assembly of the PCNA and ß-Sliding Clamps.

Sliding clamps are oligomeric ring-shaped proteins that increase the efficiency of DNA replication. The stability of the Escherichia coli ß-clamp, a homodimer, is particularly remarkable. The dissociation equilibrium constant of the ß-clamp is of the order of 10 pM in buffers of moderate ionic strength. Coulombic electrostatic interactions have been shown to contribute to this remarkable stability. Increasing NaCl concentration in the assay buffer results in decreased dimer stability and faster subunit dissociation kinetics in a way consistent with simple charge-screening models. Here, we examine non-Coulombic ionic effects on the oligomerization properties of sliding clamps. We determined relative diffusion coefficients of two sliding clamps using fluorescence correlation spectroscopy. Replacing NaCl by KGlu, the primary cytoplasmic salt in E. coli, results in a decrease of the diffusion coefficient of these proteins consistent with the formation of protein assemblies. The UV-vis spectrum of the ß-clamp labeled with tetramethylrhodamine shows the characteristic absorption band of dimers of rhodamine when KGlu is present in the buffer. This suggests that KGlu induces the formation of assemblies that involve two or more rings stacked face-to-face. Results can be quantitatively explained on the basis of unfavorable interactions between KGlu and the functional groups on the protein surface, which drive biomolecular processes that bury exposed surface. Similar results were obtained with the Saccharomyces cerevisiae PCNA sliding clamp, suggesting that KGlu effects are not specific to the ß-clamp. Clamp association is also promoted by glycine betaine, a zwitterionic compound that accumulates intracellularly when E. coli is exposed to high concentrations of extracellular solute. Possible biological implications are discussed.

Deep Analysis of Residue Constraints (DARC): identifying determinants of protein functional specificity.

Protein functional constraints are manifest as superfamily and functional-subgroup conserved residues, and as pairwise correlations. Deep Analysis of Residue Constraints (DARC) aids the visualization of these constraints, characterizes how they correlate with each other and with structure, and estimates statistical significance. This can identify determinants of protein functional specificity, as we illustrate for bacterial DNA clamp loader ATPases. These load ring-shaped sliding clamps onto DNA to keep polymerase attached during replication and contain one δ, three γ, and one δ' AAA+ subunits semi-circularly arranged in the order δ-γ1-γ2-γ3-δ'. Only γ is active, though both γ and δ' functionally influence an adjacent γ subunit. DARC identifies, as functionally-congruent features linking allosterically the ATP, DNA, and clamp binding sites: residues distinctive of γ and of γ/δ' that mutually interact in trans, centered on the catalytic base; several γ/δ'-residues and six γ/δ'-covariant residue pairs within the DNA binding N-termini of helices α2 and α3; and γ/δ'-residues associated with the α2 C-terminus and the clamp-binding loop. Most notable is a trans-acting γ/δ' hydroxyl group that 99% of other AAA+ proteins lack. Mutation of this hydroxyl to a methyl group impedes clamp binding and opening, DNA binding, and ATP hydrolysis-implying a remarkably clamp-loader-specific function.

Mechanism of opening a sliding clamp.

Clamp loaders load ring-shaped sliding clamps onto DNA where the clamps serve as processivity factors for DNA polymerases. In the first stage of clamp loading, clamp loaders bind and stabilize clamps in an open conformation, and in the second stage, clamp loaders place the open clamps around DNA so that the clamps encircle DNA. Here, the mechanism of the initial clamp opening stage is investigated. Mutations were introduced into the Escherichia coli ß-sliding clamp that destabilize the dimer interface to determine whether the formation of an open clamp loader-clamp complex is dependent on spontaneous clamp opening events. In other work, we showed that mutation of a positively charged Arg residue at the ß-dimer interface and high NaCl concentrations destabilize the clamp, but neither facilitates the formation of an open clamp loader-clamp complex in experiments presented here. Clamp opening reactions could be fit to a minimal three-step 'bind-open-lock' model in which the clamp loader binds a closed clamp, the clamp opens, and subsequent conformational rearrangements 'lock' the clamp loader-clamp complex in a stable open conformation. Our results support a model in which the E. coli clamp loader actively opens the ß-sliding clamp.

Solution structure of an "open" E. coli Pol III clamp loader sliding clamp complex.

Sliding clamps are opened and loaded onto primer template junctions by clamp loaders, and once loaded on DNA, confer processivity to replicative polymerases. Previously determined crystal structures of eukaryotic and T4 clamp loader-clamp complexes have captured the sliding clamps in either closed or only partially open interface conformations. In these solution structure studies, we have captured for the first time the clamp loader-sliding clamp complex from Escherichia coli using size exclusion chromatography coupled to small angle X-ray scattering (SEC-SAXS). The data suggests the sliding clamp is in an open conformation which is wide enough to permit duplex DNA binding. The data also provides information about spatial arrangement of the sliding clamp with respect to the clamp loader subunits and is compared to complex crystal structures determined from other organisms.

Kinetic analysis of PCNA clamp binding and release in the clamp loading reaction catalyzed by Saccharomyces cerevisiae replication factor C.

DNA polymerases require a sliding clamp to achieve processive DNA synthesis. The toroidal clamps are loaded onto DNA by clamp loaders, members of the AAA+family of ATPases. These enzymes utilize the energy of ATP binding and hydrolysis to perform a variety of cellular functions. In this study, a clamp loader-clamp binding assay was developed to measure the rates of ATP-dependent clamp binding and ATP-hydrolysis-dependent clamp release for the Saccharomyces cerevisiae clamp loader (RFC) and clamp (PCNA). Pre-steady-state kinetics of PCNA binding showed that although ATP binding to RFC increases affinity for PCNA, ATP binding rates and ATP-dependent conformational changes in RFC are fast relative to PCNA binding rates. Interestingly, RFC binds PCNA faster than the Escherichia coli γ complex clamp loader binds the ß-clamp. In the process of loading clamps on DNA, RFC maintains contact with PCNA while PCNA closes, as the observed rate of PCNA closing is faster than the rate of PCNA release, precluding the possibility of an open clamp dissociating from DNA. Rates of clamp closing and release are not dependent on the rate of the DNA binding step and are also slower than reported rates of ATP hydrolysis, showing that these rates reflect unique intramolecular reaction steps in the clamp loading pathway.

The interplay of primer-template DNA phosphorylation status and single-stranded DNA binding proteins in directing clamp loaders to the appropriate polarity of DNA.

Sliding clamps are loaded onto DNA by clamp loaders to serve the critical role of coordinating various enzymes on DNA. Clamp loaders must quickly and efficiently load clamps at primer/template (p/t) junctions containing a duplex region with a free 3'OH (3'DNA), but it is unclear how clamp loaders target these sites. To measure the Escherichia coli and Saccharomyces cerevisiae clamp loader specificity toward 3'DNA, fluorescent ß and PCNA clamps were used to measure clamp closing triggered by DNA substrates of differing polarity, testing the role of both the 5'phosphate (5'P) and the presence of single-stranded binding proteins (SSBs). SSBs inhibit clamp loading by both clamp loaders on the incorrect polarity of DNA (5'DNA). The 5'P groups contribute selectivity to differing degrees for the two clamp loaders, suggesting variations in the mechanism by which clamp loaders target 3'DNA. Interestingly, the χ subunit of the E. coli clamp loader is not required for SSB to inhibit clamp loading on phosphorylated 5'DNA, showing that χ·SSB interactions are dispensable. These studies highlight a common role for SSBs in directing clamp loaders to 3'DNA, as well as uncover nuances in the mechanisms by which SSBs perform this vital role.

Human AP endonuclease inefficiently removes abasic sites within G4 structures compared to duplex DNA.

Excision repair processes are essential to maintain genome stability. A decrease in efficiency and fidelity of these pathways at regions of the genome that can assume non-canonical DNA structures has been proposed as a possible mechanism to explain the increased mutagenesis and consequent diseased state frequently associated with these sites. Here we describe the development of a FRET-based approach to monitor the presence of G quadruplex (G4) DNA, a non-canonical DNA structure formed in runs of guanines, in damage-containing single-stranded and double-stranded DNA. Using this approach, we directly show for the first time that the presence within the G4 structure of an abasic site, the most common lesion spontaneously generated during cellular metabolism, decreases the efficiency of human AP endonuclease activity and that this effect is mostly the result of a decreased enzymatic activity and not of decreased binding of the enzyme to the damaged site. This approach can be generally applied to dissecting the biochemistry of DNA repair at non-canonical DNA structures.

The ß sliding clamp closes around DNA prior to release by the Escherichia coli clamp loader γ complex.

Escherichia coli γ complex clamp loader functions to load the ß sliding clamp onto sites of DNA replication and repair. The clamp loader uses the energy of ATP binding and hydrolysis to drive conformational changes allowing for ß binding and opening, DNA binding, and then release of the ß·DNA complex. Although much work has been done studying the sliding clamp and clamp loader mechanism, kinetic analysis of the events following ߷γ complex·DNA formation is not complete. Using fluorescent clamp closing and release assays, we show that ß closing is faster than ß release, indicating that γ complex closes ß before releasing it around DNA. Using a fluorescent ATP hydrolysis assay, we show that there is a burst of ATP hydrolysis before ß closing and that ß release may be the rate-limiting step in the overall clamp loading reaction. The combined use of these fluorescent assays provides a unique perspective into the E. coli clamp loader by providing a measure of the relative timing of different events in the clamp loading reaction, helping to elucidate the complicated clamp loading mechanism.

Improved solubility of replication factor C (RFC) Walker A mutants.

Protein insolubility often poses a significant problem during purification protocols and in enzyme assays, especially for eukaryotic proteins expressed in a recombinant bacterial system. The limited solubility of replication factor C (RFC), the clamp loader complex from Saccharomyces cerevisiae, has been previously documented. We found that mutant forms of RFC harboring a single point mutation in the Walker A motif were even less soluble than the wild-type complex. The addition of maltose at 0.75 M to the storage and assay buffers greatly increases protein solubility and prevents the complex from falling apart. Our analysis of the clamp loading reaction is dependent on fluorescence-based assays, which are environmentally sensitive. Using wt RFC as a control, we show that the addition of maltose to the reaction buffers does not affect fluorophore responses in the assays or the enzyme activity, indicating that maltose can be used as a buffer additive for further downstream analysis of these mutants.

The Escherichia coli clamp loader can actively pry open the ß-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 ß 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.

A slow ATP-induced conformational change limits the rate of DNA binding but not the rate of beta clamp binding by the escherichia coli gamma complex clamp loader.

In Escherichia coli, the gamma complex clamp loader loads the beta-sliding clamp onto DNA. The beta clamp tethers DNA polymerase III to DNA and enhances the efficiency of replication by increasing the processivity of DNA synthesis. In the presence of ATP, gamma complex binds beta and DNA to form a ternary complex. Binding to primed template DNA triggers gamma complex to hydrolyze ATP and release the clamp onto DNA. Here, we investigated the kinetics of forming a ternary complex by measuring rates of gamma complex binding beta and DNA. A fluorescence intensity-based beta binding assay was developed in which the fluorescence of pyrene covalently attached to beta increases when bound by gamma complex. Using this assay, an association rate constant of 2.3 x 10(7) m(-1) s(-1) for gamma complex binding beta was determined. The rate of beta binding was the same in experiments in which gamma complex was preincubated with ATP before adding beta or added directly to beta and ATP. In contrast, when gamma complex is preincubated with ATP, DNA binding is faster than when gamma complex is added to DNA and ATP at the same time. Slow DNA binding in the absence of ATP preincubation is the result of a rate-limiting ATP-induced conformational change. Our results strongly suggest that the ATP-induced conformational changes that promote beta binding and DNA binding differ. The slow ATP-induced conformational change that precedes DNA binding may provide a kinetic preference for gamma complex to bind beta before DNA during the clamp loading reaction cycle.

Temporal correlation of DNA binding, ATP hydrolysis, and clamp release in the clamp loading reaction catalyzed by the Escherichia coli gamma complex.

Clamp loaders are multisubunit complexes that use the energy derived from ATP binding and hydrolysis to assemble ring-shaped sliding clamps onto DNA. Sliding clamps in turn tether DNA polymerases to the templates being copied to increase the processivity of DNA synthesis. Here, the rate of clamp release during the clamp loading reaction was measured directly for the first time using a FRET-based assay in which the E. coli gamma complex clamp loader (gamma3deltadelta'chipsi) was labeled with a fluorescent donor, and the beta-clamp was labeled with a nonfluorescent quencher. When a beta.gamma complex is added to DNA, there is a significant time lag before the clamp is released onto DNA. To establish what events take place during this time lag, the timing of clamp release was compared to the timing of DNA binding and ATP hydrolysis by measuring these reactions directly side-by-side in assays. DNA binding is relatively rapid and triggers the hydrolysis of ATP. Both events occur prior to clamp release. Interestingly, the temporal correlation data and simple modeling studies indicate that the clamp loader releases DNA prior to the clamp and that DNA release may be coupled to clamp closing. Clamp release is relatively slow and likely to be the rate-limiting step in the overall clamp loading reaction cycle.

Structure/function analysis of the interaction of adenomatous polyposis coli with DNA polymerase beta and its implications for base excision repair.

Mutations in the adenomatous polyposis coli (APC) gene are associated with an early onset of colorectal carcinogenesis. Previously, we described a novel role for the APC polypeptide in base excision repair (BER). The single-nucleotide (SN) and long-patch (LP) BER pathways act to repair the abasic sites in DNA that are induced by stressors, such as spontaneous oxidation/reduction, alkylation, and hyperthermia. We have shown that APC interacts with DNA polymerase beta (Pol-beta) and flap endonuclease 1 (Fen-1) and blocks Pol-beta-directed strand-displacement synthesis. In this study, we have mapped the APC interaction site in Pol-beta and have found that Thr79, Lys81, and Arg83 of Pol-beta were critical for its interaction with APC. The Pol-beta protein (T79A/K81A/R83A) blocked strand-displacement DNA synthesis in which tetrahydrofuran was used as DNA substrate. We further showed that the APC-mediated blockage of LP-BER was due to inhibition of Fen-1 activity. Analysis of the APC-mediated blockage of SN-BER indicated that the interaction of APC with Pol-beta blocked SN-BER activity by inhibiting Pol-beta-directed deoxyribose phosphate lyase activity. Collectively, our findings indicate that APC blocked both Pol-beta-directed SN- and LP-BER pathways and increased sensitivity of cells to alkylation induced DNA damage.

A function for the psi subunit in loading the Escherichia coli DNA polymerase sliding clamp.

Crystal structures of an Escherichia coli clamp loader have provided insight into the mechanism by which this molecular machine assembles ring-shaped sliding clamps onto DNA. The contributions made to the clamp loading reaction by two subunits, chi and psi, which are not present in the crystal structures, were determined by measuring the activities of three forms of the clamp loader, gamma(3)deltadelta', gamma(3)deltadelta'psi, and gamma(3)deltadelta'psichi. The psi subunit is important for stabilizing an ATP-induced conformational state with high affinity for DNA, whereas the chi subunit does not contribute directly to clamp loading in our assays lacking single-stranded DNA-binding protein. The psi subunit also increases the affinity of the clamp loader for the clamp in assays in which ATPgammaS is substituted for ATP. Interestingly, the affinity of the gamma(3)deltadelta' complex for beta is no greater in the presence than in the absence of ATPgammaS. A role for psi in stabilizing or promoting ATP- and ATPgammaS-induced conformational changes may explain why large conformational differences were not seen in gamma(3)deltadelta' structures with and without bound ATPgammaS. The beta clamp partially compensates for the activity of psi when this subunit is not present and possibly serves as a scaffold on which the clamp loader adopts the appropriate conformation for DNA binding and clamp loading. Results from our work and others suggest that the psi subunit may introduce a temporal order to the clamp loading reaction in which clamp binding precedes DNA binding.

Dynamics of loading the Escherichia coli DNA polymerase processivity clamp.

Sliding clamps and clamp loaders are processivity factors required for efficient DNA replication. Sliding clamps are ring-shaped complexes that tether DNA polymerases to DNA to increase the processivity of synthesis. Clamp loaders assemble these ring-shaped clamps onto DNA in an ATP-dependent reaction. The overall process of clamp loading is dynamic in that protein-protein and protein-DNA interactions must actively change in a coordinated fashion to complete the mechanical clamp-loading reaction cycle. The clamp loader must initially have a high affinity for both the clamp and DNA to bring these macromolecules together, but then must release the clamp on DNA for synthesis to begin. Evidence is presented for a mechanism in which the clamp-loading reaction comprises a series of binding reactions to ATP, the clamp, DNA, and ADP, each of which promotes some change in the conformation of the clamp loader that alters interactions with the next component of the pathway. These changes in interactions must be rapid enough to allow the clamp loader to keep pace with replication fork movement. This review focuses on the measurement of dynamic and transient interactions required to assemble the Escherichia coli sliding clamp on DNA.

Multiplexed DNA sequencing-by-synthesis.

We report a new DNA sequencing-by-synthesis method in which the sequences of DNA templates, hybridized to a surface-immobilized array of DNA primers, are determined by sensing the number of nucleotides by which the primers in each array spot are extended in sequential DNA polymerase-catalyzed nucleotide incorporation reactions, each with a single fluorescein-labeled deoxyribonucleoside triphosphate (dNTP) species. The fluorescein label is destroyed after each readout by a photostimulated reaction with diphenyliodonium chloride. A DNA polymerase with enhanced ability to incorporate, and to extend beyond, modified nucleotides is used. Self-quenching of adjacent fluorescein labels, which impedes readout of homopolymeric runs, is avoided by diluting the labeled dNTP with unlabeled reagent. Misincorporation effects have been quantified and are small; however, low-level contamination of dNTPs with other nucleotides mimics misincorporation and can produce significant false-positive signals. These impurities are removed by polymerase-catalyzed incorporation into complementary "cleaning duplexes." Here, we demonstrate the accurate sequence readout for a small array of known DNA templates, the ability to quantify homopolymeric runs, and a short sequencing example of sections of the wild-type and mutant BRCA1 gene. For a 20,000-spot array, readout rates in excess of 6000 bases per minute are projected.

Mechanism of loading the Escherichia coli DNA polymerase III sliding clamp: I. Two distinct activities for individual ATP sites in the gamma complex.

The Escherichia coli DNA polymerase III gamma complex loads the beta clamp onto DNA, and the clamp tethers the core polymerase to DNA to increase the processivity of synthesis. ATP binding and hydrolysis promote conformational changes within the gamma complex that modulate its affinity for the clamp and DNA, allowing it to accomplish the mechanical task of assembling clamps on DNA. This is the first of two reports (Snyder, A. K., Williams, C. R., Johnson, A., O'Donnell, M., and Bloom, L. B. (2004) J. Biol. Chem. 279, 4386-4393) addressing the question of how ATP binding and hydrolysis modulate specific interactions with DNA and beta. Pre-steady-state rates of ATP hydrolysis were slower when reactions were initiated by addition of ATP than when the gamma complex was equilibrated with ATP and were limited by the rate of an intramolecular reaction, possibly ATP-induced conformational changes. Kinetic modeling of assays in which the gamma complex was incubated with ATP for different periods of time prior to adding DNA to trigger hydrolysis suggests a mechanism in which a relatively slow conformational change step (kforward = 6.5 s(-1)) produces a species of the gamma complex that is activated for DNA (and beta) binding. In the absence of beta, 2 of the 3 molecules of ATP are hydrolyzed rapidly prior to releasing DNA, and the 3rd molecule is hydrolyzed slowly. In the presence of beta, all 3 molecules of ATP are hydrolyzed rapidly. These results suggest that hydrolysis of 2 molecules of ATP may be coupled to conformational changes that reduce interactions with DNA, whereas hydrolysis of the 3rd is coupled to changes that result in release of beta.

Mechanism of loading the Escherichia coli DNA polymerase III sliding clamp: II. Uncoupling the beta and DNA binding activities of the gamma complex.

Sliding clamps tether DNA polymerases to DNA to increase the processivity of synthesis. The Escherichia coli gamma complex loads the beta sliding clamp onto DNA in an ATP-dependent reaction in which ATP binding and hydrolysis modulate the affinity of the gamma complex for beta and DNA. This is the second of two reports (Williams, C. R., Snyder, A. K., Kuzmic, P., O'Donnell, M., and Bloom, L. B. (2004) J. Biol. Chem. 279, 4376-4385) addressing the question of how ATP binding and hydrolysis regulate specific interactions with DNA and beta. Mutations were made to an Arg residue in a conserved SRC motif in the delta' and gamma subunits that interacts with the ATP site of the neighboring gamma subunit. Mutation of the delta' subunit reduced the ATP-dependent beta binding activity, whereas mutation of the gamma subunits reduced the DNA binding activity of the gamma complex. The gamma complex containing the delta' mutation gave a pre-steady-state burst of ATP hydrolysis, but at a reduced rate and amplitude relative to the wild-type gamma complex. A pre-steady-state burst of ATP hydrolysis was not observed for the complex containing the gamma mutations, consistent with the reduced DNA binding activity of this complex. The differential effects of these mutations suggest that ATP binding at the gamma1 site may be coupled to conformational changes that largely modulate interactions with beta, whereas ATP binding at the gamma2 and/or gamma3 site may be coupled to conformational changes that have a major role in interactions with DNA. Additionally, these results show that the "arginine fingers" play a structural role in facilitating the formation of a conformation that has high affinity for beta and DNA.