Single-Molecule FRET to Measure Conformational Dynamics of DNA Mismatch Repair Proteins.

Single-molecule FRET measurements have a unique sensitivity to protein conformational dynamics. The FRET signals can either be interpreted quantitatively to provide estimates of absolute distance in a molecule configuration or can be qualitatively interpreted as distinct states, from which quantitative kinetic schemes for conformational transitions can be deduced. Here we describe methods utilizing single-molecule FRET to reveal the conformational dynamics of the proteins responsible for DNA mismatch repair. Experimental details about the proteins, DNA substrates, fluorescent labeling, and data analysis are included. The complementarity of single molecule and ensemble kinetic methods is discussed as well.

Mechanism of beta clamp opening by the delta subunit of Escherichia coli DNA polymerase III holoenzyme.

The beta sliding clamp encircles the primer-template and tethers DNA polymerase III holoenzyme to DNA for processive replication of the Escherichia coli genome. The clamp is formed via hydrophobic and ionic interactions between two semicircular beta monomers. This report demonstrates that the beta dimer is a stable closed ring and is not monomerized when the gamma complex clamp loader (gamma(3)delta(1)delta(1)chi(1)psi(1)) assembles the beta ring around DNA. delta is the subunit of the gamma complex that binds beta and opens the ring; it also does not appear to monomerize beta. Point mutations were introduced at the beta dimer interface to test its structural integrity and gain insight into its interaction with delta. Mutation of two residues at the dimer interface of beta, I272A/L273A, yields a stable beta monomer. We find that delta binds the beta monomer mutant at least 50-fold tighter than the beta dimer. These findings suggest that when delta interacts with the beta clamp, it binds one beta subunit with high affinity and utilizes some of that binding energy to perform work on the dimeric clamp, probably cracking one dimer interface open.

The delta subunit of DNA polymerase III holoenzyme serves as a sliding clamp unloader in Escherichia coli.

In Escherichia coli, the circular beta sliding clamp facilitates processive DNA replication by tethering the polymerase to primer-template DNA. When synthesis is complete, polymerase dissociates from beta and DNA and cycles to a new start site, a primed template loaded with beta. DNA polymerase cycles frequently during lagging strand replication while synthesizing 1-2-kilobase Okazaki fragments. The clamps left behind remain stable on DNA (t(12) approximately 115 min) and must be removed rapidly for reuse at numerous primed sites on the lagging strand. Here we show that delta, a single subunit of DNA polymerase III holoenzyme, opens beta and slips it off DNA (k(unloading) = 0.011 s(-)(1)) at a rate similar to that of the multisubunit gamma complex clamp loader by itself (0.015 s(-)(1)) or within polymerase (pol) III* (0.0065 s(-)(1)). Moreover, unlike gamma complex and pol III*, delta does not require ATP to catalyze clamp unloading. Quantitation of gamma complex subunits (gamma, delta, delta', chi, psi) in E. coli cells reveals an excess of delta, free from gamma complex and pol III*. Since pol III* and gamma complex occur in much lower quantities and perform several DNA metabolic functions in replication and repair, the delta subunit probably aids beta clamp recycling during DNA replication.

Molecular mechanism and energetics of clamp assembly in Escherichia coli. The role of ATP hydrolysis when gamma complex loads beta on DNA.

Escherichia coli DNA polymerase III holoenzyme is a multisubunit composite containing the beta sliding clamp and clamp loading gamma complex. The gamma complex requires ATP to load beta onto DNA. A two-color fluorescence spectroscopic approach was utilized to study this system, wherein both assembly (red fluorescence; X-rhodamine labeled DNA anisotropy assay) and ATP hydrolysis (green fluorescence; phosphate binding protein assay) were simultaneously measured with millisecond timing resolution. The two temporally correlated stopped-flow signals revealed that a preassembled beta. gamma complex composite rapidly binds primer/template DNA in an ATP hydrolysis independent step. Once bound, two molecules of ATP are rapidly hydrolyzed (approximately 34 s(-1)). Following hydrolysis, gamma complex dissociates from the DNA ( approximately 22 s(-1)). Once dissociated, the next cycle of loading is severely compromised, resulting in steady-state ATP hydrolysis rates with a maximum of only approximately 3 s(-1). Two single-site beta dimer interface mutants were examined which had impaired steady-state rates of ATP hydrolysis. The pre-steady-state correlated kinetics of these mutants revealed a pattern essentially identical to wild type. The anisotropy data showed that these mutants decrease the steady-state rates of ATP hydrolysis by causing a buildup of "stuck" binary-ternary complexes on the primer/template DNA.

Sliding clamps: a (tail)ored fit.

New structural information on the architecture of a DNA replisome provides insights into a number of DNA metabolic processes and their modulation by circular 'sliding damps', which form rings around DNA that play an Important role in processive processes such as replication.

A model for Escherichia coli DNA polymerase III holoenzyme assembly at primer/template ends. DNA triggers a change in binding specificity of the gamma complex clamp loader.

The gamma complex of the Escherichia coli DNA polymerase III holoenzyme assembles the beta sliding clamp onto DNA in an ATP hydrolysis-driven reaction. Interactions between gamma complex and primer/template DNA are investigated using fluorescence depolarization to measure binding of gamma complex to different DNA substrates under steady-state and presteady-state conditions. Surprisingly, gamma complex has a much higher affinity for single-stranded DNA (K(d) in the nM range) than for a primed template (K(d) in the microM range) under steady-state conditions. However, when examined on a millisecond time scale, we find that gamma complex initially binds very rapidly and with high affinity to primer/template DNA but is converted subsequently to a much lower affinity DNA binding state. Presteady-state data reveals an effective dissociation constant of 1.5 nM for the initial binding of gamma complex to DNA and a dissociation constant of 5.7 microM for the low affinity DNA binding state. Experiments using nonhydrolyzable ATPgammaS show that ATP binding converts gamma complex from a low affinity "inactive" to high affinity "active" DNA binding state while ATP hydrolysis has the reverse effect, thus allowing cycling between active and inactive DNA binding forms at steady-state. We propose that a DNA-triggered switch between active and inactive states of gamma complex provides a two-tiered mechanism enabling gamma complex to recognize primed template sites and load beta, while preventing gamma complex from competing with DNA polymerase III core for binding a newly loaded beta.DNA complex.

A tale of toroids in DNA metabolism.

A strikingly large number of the proteins involved in DNA metabolism adopt a toroidal -- or ring-shaped -- quaternary structure, even though they have completely unrelated functions. Given that these proteins all use DNA as a substrate, their convergence to one shape is probably not a coincidence. Ring-forming proteins may have been selected during evolution for advantages conferred by the toroidal shape on their interactions with DNA.

Division of labor--sequential ATP hydrolysis drives assembly of a DNA polymerase sliding clamp around DNA.

The beta sliding clamp encircles DNA and enables processive replication of the Escherichia coli genome by DNA polymerase III holoenzyme. The clamp loader, gamma complex, assembles beta around DNA in an ATP-fueled reaction. Previous studies have shown that gamma complex opens the beta ring and also interacts with DNA on binding ATP. Here, a rapid kinetic analysis demonstrates that gamma complex hydrolyzes two ATP molecules sequentially when placing beta around DNA. The first ATP is hydrolyzed fast, at 25-30 s(-1), while the second ATP hydrolysis is limited to the steady-state rate of 2 s(-1). This step-wise reaction depends on both primed DNA and beta. DNA alone promotes rapid hydrolysis of two ATP molecules, while beta alone permits hydrolysis of only one ATP. These results suggest that beta inserts a slow step between the two ATP hydrolysis events in clamp assembly, during which the clamp loader may perform work on the clamp. Moreover, one ATP hydrolysis is sufficient for release of beta from the gamma complex. This implies that DNA-dependent hydrolysis of the other ATP is coupled to a separate function, perhaps involving work on DNA. A model is presented in which sequential ATP hydrolysis drives distinct events in the clamp-assembly pathway. We also discuss underlying principles of this step-wise mechanism that may apply to the workings of other ATP-fueled biological machines.

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.

ATP binding to the Escherichia coli clamp loader powers opening of the ring-shaped clamp of DNA polymerase III holoenzyme.

The Escherichia coli gamma complex serves as a clamp loader, catalyzing ATP-dependent assembly of beta protein clamps onto primed DNA templates during DNA replication. These ring-shaped clamps tether DNA polymerase III holoenzyme to the template, facilitating rapid and processive DNA synthesis. This report focuses on the role of ATP binding and hydrolysis catalyzed by the gamma complex during clamp loading. We show that the energy from ATP binding to gamma complex powers several initial events in the clamp loading pathway. The gamma complex (gamma2 delta delta'chi psi) binds two ATP molecules (one per gamma subunit in the complex) with high affinity (Kd = 1-2. 5 x 10(-6) M) or two adenosine 5'-O-(3-thiotriphosphate)(ATPgammaS) molecules with slightly lower affinity (Kd = 5-6.5 x 10(-6) M). Experiments performed prior to the first ATP turnover (kcat = 4 x 10(-3) s-1 at 4 degreesC), or in the presence of ATPgammaS (kcat = 1 x 10(-4) s-1 at 37 degreesC), demonstrate that upon interaction with ATP the gamma complex undergoes a change in conformation. This ATP-bound gamma complex binds beta and opens the ring at the dimer interface. Still prior to ATP hydrolysis, the composite of gamma complex and the open beta ring binds with high affinity to primer-template DNA. Thus ATP binding powers all the steps in the clamp loading pathway leading up to the assembly of a gamma complex. open beta ring.DNA intermediate, setting the stage for ring closing and turnover of the clamp loader, steps that may be linked to subsequent hydrolysis of ATP.

Toroidal proteins: running rings around DNA.

Recent structural data indicate that the toroidal form is quite common among DNA-binding enzymes. Is this abundance of ring-shaped proteins a coincidence, or does it reflect convergence to a winning quaternary structure?

The dTTPase mechanism of T7 DNA helicase resembles the binding change mechanism of the F1-ATPase.

Bacteriophage T7 DNA helicase is a ring-shaped hexamer that catalyzes duplex DNA unwinding using dTTP hydrolysis as an energy source. Of the six potential nucleotide binding sites on the hexamer, we have found that three are noncatalytic sites and three are catalytic sites. The noncatalytic sites bind nucleotides with a high affinity, but dTTPs bound to these sites do not dissociate or hydrolyze through many dTTPase turnovers at the catalytic sites. The catalytic sites show strong cooperativity which leads to sequential binding and hydrolysis of dTTP. The elucidated dTTPase mechanism of the catalytic sites of T7 helicase is remarkably similar to the binding change mechanism of the ATP synthase. Based on the similarity, a general mechanism for hexameric helicases is proposed. In this mechanism, an F1-ATPase-like rotational movement around the single-stranded DNA, which is bound through the central hole of the hexamer, is proposed to lead to unidirectional translocation along single-stranded DNA and duplex DNA unwinding.

Cooperative interactions of nucleotide ligands are linked to oligomerization and DNA binding in bacteriophage T7 gene 4 helicases.

The equilibrium nucleotide binding and oligomerization of bacteriophage T7 gene 4 helicases have been investigated using thymidine 5'-triphosphate (dTTP), deoxythymidine 5'-(beta, gamma-methylenetriphosphate)(dTMP-PCP), thymidine 5'-diphosphate (dTDP), adenosine 5'-triphosphate (ATP), and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S). In the presence of nucleotide ligands, T7 helicases self-assemble into hexamers with six potential nucleotide binding sites that are nonequivalent both in the absence and in the presence of single-stranded DNA. All nucleotides tested bind with high affinity to three sites (K(d) = 5 x 10(-6) M, dTTP; 6 x 10(-7) M, dTMP-PCP; 4 x 10(-6) M, dTDP; 3 x 10(-5) M, ATP; 2 x 10(-6) M, ATP gamma S), while binding to the remaining sites is undetectable. Interestingly, nucleotide binding to the high-affinity sites exhibits positive cooperativity which is sensitive to protein concentration. This effect is a result of ligand binding-linked oligomerization wherein helicase oligomer equilibrium changes as a function of both nucleotide and protein concentration. A study of DNA binding shows that 1-2 NTPs bound per hexamer are sufficient for stoichiometric interaction between the helicase and DNA. Thus, the ring-shaped helicase hexamers assemble around DNA with one, two, or three NTPs bound to each hexamer. This study also examines the preferred use of dTTP for T7 helicase-catalyzed DNA unwinding by comparison with ATP, the more commonly used nucleotide ligand. ATP binds to the helicase with 6-fold weaker affinity than dTTP and promotes hexamerization as well as DNA binding. Nevertheless, DNA unwinding with ATP is at least 100-fold slower than with dTTP. Thus, the difference in ATP and dTTP utilization probably lies in a highly specific step in the coupling of NTP hydrolysis to DNA unwinding.

Bacteriophage T7 helicase/primase proteins form rings around single-stranded DNA that suggest a general structure for hexameric helicases.

Most helicases studied to date have been characterized as oligomeric, but the relation between their structure and function has not been understood. The bacteriophage T7 gene 4 helicase/primase proteins act in T7 DNA replication. We have used electron microscopy, three-dimensional reconstruction, and protein crosslinking to demonstrate that both proteins form hexameric rings around single-stranded DNA. Each subunit has two lobes, so the hexamer appears to be two-tiered, with a small ring stacked on a large ring. The single-stranded DNA passes through the central hole of the hexamer, and the data exclude substantial wrapping of the DNA about or within the protein ring. Further, the hexamer binds DNA with a defined polarity as the smaller ring of the hexamer points toward the 5' end of the DNA. The similarity in three-dimensional structure of the T7 gene 4 proteins to that of the Escherichia coli RuvB helicase suggests that polar rings assembled around DNA may be a general feature of numerous hexameric helicases involved in DNA replication, transcription, recombination, and repair.

Nucleotide binding studies of bacteriophage T7 DNA helicase-primase protein.

Bacteriophage T7 DNA helicase protein is a hexameric protein that contains identical subunits arranged in a ring-like structure. Single-stranded DNA binds through the hole of the ring, and the helicase protein translocates and unwinds duplex DNA using nucleoside triphosphate (NTP) hydrolysis. In our efforts to understand how NTP hydrolysis may be coupled to movement of the helicase on the DNA, we have quantitated the equilibrium binding of deoxythymidine triphosphate and thymidine 5'-(beta,gamma-methylenetriphosphate) using nitrocellulose binding assays. Even though the helicase consists of six identical subunits, each hexamer was found to bind only three NTP molecules. These results indicate half-site binding or negative cooperativity in NTP binding by the hexamer. Interestingly, binding of three NTP molecules to the hexamer was sufficient for stoichiometric binding of a single-stranded oligodeoxynucleotide. Similar negative cooperativity in NTP binding has also been observed for other helicases, suggesting that it may be a general feature of hexameric helicases. The significance of half-site binding, however, is not understood at the present time.

The K318A mutant of bacteriophage T7 DNA primase-helicase protein is deficient in helicase but not primase activity and inhibits primase-helicase protein wild-type activities by heterooligomer formation.

Lysine 318 in the conserved sequence SXXXGXGKS of bacteriophage T7 gene 4A' protein was mutated to an alanine to understand the effect of this substitution on the helicase and primase activities. The dTTPase activity of 4A'/K318A mutant protein was much lower than that of 4A', and both Km and kcat values were affected. The Km of the mutant protein was 3-5-fold higher, and the kcat was about 100-fold lower, than that of 4A'. The mutation did not affect the ability of 4A'/K318A to assemble into hexamers or bind DNA in the presence of MgdTTP. Interestingly, the mutant protein does not bind DNA in the presence of MgdTMP-PCP. The reduced dTTPase activity, however, decreased the helicase activity of the mutant protein to an undetectable level, whereas its primase activity was only 1.5-2.5-fold lower. When 4A'/K318A mutant protein was mixed with 4A', heterooligomers were formed and the helicase and the DNA-dependent dTTPase activities of 4A' were inhibited, but the DNA-independent activity actually increased. The extent of decrease in activities upon heterooligomer formation depended both on the length of time 4A' and 4A'/K318A proteins were incubated and on the concentration of the mutant protein. In addition, the decrease in the dTTPase activity was observed only when the two proteins were incubated in the absence of MgdTTP and DNA, conditions under which both proteins form unstable hexamers. Even though 4A'/K318A does not bind a 30-mer DNA in the presence of MgdTMP-PCP, heterooligomers were capable of binding DNA with the same stoichiometry as 4A'. Protein-DNA cross-linking experiments with (dT)30 and poly(5-BrdU) showed that DNA interacts with five and perhaps all six subunits of 4A'. Therefore, unless heterooligomer restores the ability of the mutant protein to bind DNA in the presence of MgdTMP-PCP, these results suggest that the DNA can bind 4A' by interacting with a few subunits. However, a fully active hexamer is required for both the helicase and the single-stranded M13 DNA-dependent dTTPase activities.

Interactions of bacteriophage T7 DNA primase/helicase protein with single-stranded and double-stranded DNAs.

Protein-DNA interactions of bacteriophage T7 DNA primase/helicase protein 4A' with small synthetic oligodeoxynucleotides were investigated using a 20-base-paired hairpin duplex, and 10-, 30-, and 60-base-long single-stranded DNA. The effect of nucleotide cofactors on DNA binding was examined using membrane binding assays which showed that 4A' binds DNA optimally only in the presence of MgdTMP-PCP, the nonhydrolyzable analog of dTTP. About 20% of single-stranded DNA binding was observed in the presence of MgdTDP, but none was detectable in the absence of nucleotides. Native polyacrylamide gel electrophoresis showed that the DNAs bind predominantly to the hexameric form of 4A'. Larger oligomers of 4A' can bind DNA, but no DNA binding was observed to species smaller than the hexamer. Quantitative equilibrium binding studies at increasing 4A' concentrations and at increasing DNA concentrations showed tight binding of one 10-mer or 30-mer per hexamer. The 4A' hexamer can bind a second strand of DNA, but with a 50-fold weaker affinity than the first strand. The 60-mer showed tight binding to two 4A' hexamers, suggesting that a hexamer may interact with only 30-40 bases of single-stranded DNA. This was corroborated by nuclease protection experiments where the smallest length of DNA protected by 4A' or 4B protein was found to be about 30 bases. Equilibrium binding studies and competitive DNA binding data are consistent with a weaker affinity of 4A' for the duplex DNA. Only 20-25% of duplex DNA binding was observed at increasing 4A' protein in the presence of MgdTMP-PCP. About four duplex DNAs can bind each 4A' hexamer at increasing DNA concentrations, but their weaker binding was evident from their facile dissociation from 4A' in the presence of competing single-stranded DNA.

Oligomeric structure of bacteriophage T7 DNA primase/helicase proteins.

The oligomeric structure of bacteriophage T7 gene 4 helicase/primase proteins was investigated using protein cross-linking and high pressure gel-filtration chromatography. Studies were carried out with both 4A' and 4B proteins. 4A' is a M64L mutant of 4A which has similar helicase and primase activities as the wild-type mixture of 4A and 4B proteins (Patel, S. S., Rosenberg, A. H., Studier, F. W., and Johnson, K. A. (1992) J. Biol. Chem. 267, 15013-15021), and 4B is the smaller protein which has only helicase activity. Chemical cross-linking of 4A' and 4B proteins with dimethyl suberimidate resulted in cross-linked species ranging from dimers to hexamers and beyond. The cross-linking time course, however, indicated that hexamers were the predominant species to accumulate in both 4A' and 4B proteins. The effect of MgNTP and DNA binding on oligomerization of the gene 4 proteins was investigated using high pressure gel-filtration chromatography at increasing protein concentrations. In the absence of added ligands, close to 100 microM protein concentrations were required to form stable oligomers beyond dimers. However, in the presence of Mg-beta, gamma-methylene deoxythymidine triphosphate (nonhydrolyzable analog of dTTP), 4A' and 4B protein assembled into stable hexamers at protein concentrations less than 8 microM. Addition of single-stranded DNA further stabilized the hexamer structure. Therefore, in the presence of a 60-nucleotide-long single-stranded DNA, hexamers were observed at protein concentrations as low as 0.2 microM. Nuclease protection experiments indicated that the 4A' and 4B hexamers protect about 60-65 bases of single-stranded DNA.