The T4 DNA polymerase accessory proteins form an ATP-dependent complex on a primer-template junction

The DNA polymerase holoenzyme of bacteriophage T4 contains, besides the DNA polymerase itself (the gene 43 protein), a complex of the protein products of T4 genes 44 and 62 (a DNA-dependent ATPase) and of gene 45. Together, the 44/62 and 45 proteins form an ATP-dependent "sliding clamp" that holds a moving DNA polymerase molecule at the 3' terminus of a growing DNA chain. We have used a unique DNA fragment that forms a short hairpin helix with a single-stranded 5' tail (a "primer-template junction") to map the binding sites for these polymerase accessory proteins by DNA footprinting techniques. In the absence of the DNA polymerase, the accessory proteins protect from DNase I cleavage 19-20 nucleotides just behind the 3' end of the primer strand and 27-28 nucleotides on the complementary portion of the template strand. Detection of this DNA-protein complex requires the 44/62 and 45 proteins plus the nonhydrolyzable ATP analogue adenosine 5'-O-(thiotriphosphate). The complex is not detected in the presence of ATP. We suggest that ATP hydrolysis by the 44/62 protein normally activates the accessory proteins at a primer-template junction, permitting the DNA polymerase to bind and thus form the complete holoenzyme. However, when the polymerase is missing, as in these experiments, ATP hydrolysis is instead followed by a release (or loosening) of the accessory protein complex.     

Single-molecule investigation of the T4 bacteriophage DNA polymerase holoenzyme: multiple pathways of holoenzyme formation

In T4 bacteriophage, the DNA polymerase holoenzyme is responsible for accurate and processive DNA synthesis. The holoenzyme consists of DNA polymerase gp43 and clamp protein gp45. To form a productive holoenzyme complex, clamp loader protein gp44/62 is required for the loading of gp45, along with MgATP, and also for the subsequent binding of polymerase to the loaded clamp. Recently published evidence suggests that holoenzyme assembly in the T4 replisome may take place via more than one pathway [Zhuang, Z., Berdis, A. J., and Benkovic, S. J. (2006) Biochemistry 45, 7976-7989]. To demonstrate unequivocally whether there are multiple pathways leading to the formation of a productive holoenzyme, single-molecule fluorescence microscopy has been used to study the potential clamp loading and holoenzyme assembly pathways on a single-molecule DNA substrate. The results obtained reveal four pathways that foster the formation of a functional holoenzyme on DNA: (1) clamp loader-clamp complex binding to DNA followed by polymerase, (2) clamp loader binding to DNA followed by clamp and then polymerase, (3) clamp binding to DNA followed by clamp loader and then polymerase, and (4) polymerase binding to DNA followed by the clamp loader-clamp complex. In all cases, MgATP is required. The possible physiological significance of the various assembly pathways is discussed in the context of replication initiation and lagging strand synthesis during various stages of T4 phage replication.     

Building a replisome solution structure by elucidation of protein-protein interactions in the bacteriophage T4 DNA polymerase holoenzyme

Assembly of DNA replication systems requires the coordinated actions of many proteins. The multiprotein complexes formed as intermediates on the pathway to the final DNA polymerase holoenzyme have been shown to have distinct structures relative to the ground-state structures of the individual proteins. By using a variety of solution-phase techniques, we have elucidated additional information about the solution structure of the bacteriophage T4 holoenzyme. Photocross-linking and mass spectrometry were used to demonstrate interactions between I107C of the sliding clamp and the DNA polymerase. Fluorescence resonance energy transfer, analytical ultracentrifugation, and isothermal titration calorimetry measurements were used to demonstrate that the C terminus of the DNA polymerase can interact at two distinct locations on the sliding clamp. Both of these binding modes may be used during holoenzyme assembly, but only one of these binding modes is found in the final holoenzyme. Present and previous solution interaction data were used to build a model of the holoenzyme that is consistent with these data.     

Tracking sliding clamp opening and closing during bacteriophage T4 DNA polymerase holoenzyme assembly

The bacteriophage T4 DNA polymerase holoenzyme, consisting of the DNA polymerase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62), is loaded onto DNA in an ATP-dependent, multistep reaction. The trimeric, ring-shaped gp45 is loaded onto DNA such that the DNA passes through the center of the ring. gp43 binds to this complex, thereby forming a topological link with the DNA and increasing its processivity. Using stopped-flow fluorescence-resonance energy transfer, we have investigated opening and closing of the gp45 ring during the holoenzyme assembly process. Two amino acids that lie on opposite sides of the gp45 subunit interface, W91 and V162C labeled with coumarin, were used as the fluorescence donor and acceptor, respectively. Free in solution, gp45 has two closed subunit interfaces with W91 to V162-coumarin distances of 19 A and one open subunit interface with a W91 to V162C-coumarin distance of 40 A. Making the assumption that the distance across the two closed subunit interfaces is unchanged during the holoenzyme assembly process, we have found that the distance across the open subunit interface is first increased to greater than 45 A and is then decreased to 30 A during a 10-step assembly mechanism. The gp45 ring is not completely closed in the holoenzyme complex, consistent with previous evidence suggesting that the C-terminus of gp43 is inserted into the gp45 subunit interface. Unexpectedly, ATP-hydrolysis events are coupled to only a fraction of the total distance change, with conformational changes linked to binding DNA and gp43 coupled to the majority of the total distance change. Using the nonhydrolyzable ATP analogue ATP-gamma-S results in formation of a nonproductive gp45 x gp44/62 complex; however, adding an excess of ATP to this nonproductive complex results in rapid ATP/ATP-gamma-S exchange to yield a productive gp45 x gp44/62 complex within seconds.     

Protein-DNA cross-linking demonstrates stepwise ATP-dependent assembly of T4 DNA polymerase and its accessory proteins on the primer-template.

T4 DNA polymerase, the 44/62 and 45 polymerase accessory proteins, and 32 single-stranded DNA-binding protein catalyze ATP-dependent DNA synthesis. Using DNA primers with cross-linkable residues at specific positions, we obtained structural data that reveal how these proteins assemble on the primer-template. With the nonhydrolyzable ATP analog ATP gamma S, assembly of the 44/62 and 45 proteins on the primer requires 32 protein but not polymerase. ATP hydrolysis changes the position and intensity of cross-linking to each of the accessory proteins and allows cross-linking of polymerase. Our data indicate that the initial binding of the three accessory proteins and ATP to a 32 protein-covered primer-template is followed by ATP hydrolysis, binding of polymerase, and movement of the accessory proteins to yield a complex capable of processive DNA synthesis.     

Recognition and use of the unusual X-DNA as a primer-template by Klenow DNA polymerase enzyme

Based on CD spectra, 2-amino-2'-deoxyadenosine-containing synthetic alternating DNA, poly(amino2dA-dt) undergoes a conformational transition from a B-form to a non-Z zig-zag form of DNA, called X, even under conditions where enzymes can work. Kinetic parameters of the E. coli Klenow DNA polymerase enzyme-catalyzed copying of both the B- and X-forms of poly(amino2dA-dT) have been determined. Binding affinity of X-DNA to the enzyme proved to be even higher than that of the B-DNA; primer-chain extension of X-poly(amino2dA-dT) was however hindered as compared to its B-form. This differential utilization of X-DNA versus B-DNA by a DNA polymerase is an in vitro enzymatic evidence of an unusual DNA conformation.     

Constitution of the twin polymerase of DNA polymerase III holoenzyme

It is speculated that DNA polymerases which duplicate chromosomes are dimeric to provide concurrent replication of both leading and lagging strands. DNA polymerase III holoenzyme (holoenzyme), is the 10-subunit replicase of the Escherichia coli chromosome. A complex of the alpha (DNA polymerase) and epsilon (3'-5' exonuclease) subunits of the holoenzyme contains only one of each protein. Presumably, one of the eight other subunit(s) functions to dimerize the alpha epsilon polymerase within the holoenzyme. Based on dimeric subassemblies of the holoenzyme, two subunits have been elected as possible agents of polymerase dimerization, one of which is the tau subunit (McHenry, C. S. (1982) J. Biol. Chem. 257, 2657-2663). Here, we have used pure alpha, epsilon, and tau subunits in binding studies to determine whether tau can dimerize the polymerase. We find tau binds directly to alpha. Whereas alpha is monomeric, tau is a dimer in its native state and thereby serves as an efficient scaffold to dimerize the polymerase. The epsilon subunit does not associate directly with tau but becomes dimerized in the alpha epsilon tau complex by virtue of its interaction with alpha. We have analyzed the dimeric alpha epsilon tau complex by different physical methods to increase the confidence that this complex truly contains a dimeric polymerase. The tau subunit is comprised of the NH2-terminal two-thirds of tau but does not bind to alpha epsilon, identifying the COOH-terminal region of tau as essential to its polymerase dimerization function. The significance of these results with respect to the organization of subunits within the holoenzyme is discussed.     

In vitro selection of sequence contexts which enhance bypass of abasic sites and tetrahydrofuran by T4 DNA polymerase holoenzyme

The influence of sequence context on the ability of DNA polymerase to bypass sites of base loss was addressed using an in vitro selection system. Oligonucleotides containing either an aldehydic abasic site or tetrahydrofuran surrounded by four randomized bases on both the 5' and 3' sides were used as templates for synthesis by phage T4 DNA polymerase holoenzyme proficient or deficient in the 3'-->5' proofreading exonuclease activity. Successful bypass products were purified, subcloned and the sequences of approximately 100 subclones were determined for each of the four polymerase/lesion combinations tested. Between 7 and 19 % of the bypass products contained deletions of one to three nucleotides in the randomized region. In bypass products not containing deletions, biases for and against certain nucleotides were readily noticeable across the entire randomized region. Template strands from successful bypass products of abasic sites had a high frequency of T in most of the randomized positions, while those from bypass products of tetrahydrofuran had a high frequency of G at the positions immediately to the 3' and 5' side of the lesion. Consensus sequences were shared by successful bypass products of the same lesion but not between bypass products of the two lesions. The consensus sequence for efficient bypass of tetrahydrofuran was over-represented in several frames relative to the lesion. T4 DNA polymerase inserted A opposite abasic sites 63 % of the time in the presence of proofreading and 79 % of the time in its absence, followed by G>T>C, while the insertion of A opposite tetrahydrofuran ranged between 93 % and 100 % in the presence and absence of proofreading, respectively. Finally, sequence context influenced the choice of nucleotide inserted opposite abasic sites and consensus sequences which favored the incorporation of nucleotides other than A were defined.     

Analysis of unassisted translesion replication by the DNA polymerase III holoenzyme

DNA damage-induced mutations are formed when damaged nucleotides present in single-stranded DNA are replicated. We have developed a new method for the preparation of gapped plasmids containing site-specific damaged nucleotides, as model DNA substrates for translesion replication. Using these substrates, we show that the DNA polymerase III holoenzyme from Escherichia coli can bypass a synthetic abasic site analogue with high efficiency (30% bypass in 16 min), unassisted by other proteins. The theta and tau subunits of the polymerase were not essential for bypass. No bypass was observed when the enzyme was assayed on a synthetic 60-mer oligonucleotide carrying the same lesion, and bypass on a linear gapped plasmid was 3-4-fold slower than on a circular gapped plasmid. There was no difference in the bypass when standing-start and running-start replication were compared. A comparison of translesion replication by DNA polymerase I, DNA polymerase II, the DNA polymerase III core, and the DNA polymerase III holoenzyme clearly showed that the DNA polymerase III holoenzyme was by far the most effective in performing translesion replication. This was not only due to the high processivity of the pol III holoenzyme, because increasing the processivity of pol II by adding the gamma complex and beta subunit, did not increase bypass. These results support the model that SOS regulation was imposed on a fundamentally constitutive translesion replication reaction to achieve tight control of mutagenesis.     

Escherichia coli DNA polymerase II is stimulated by DNA polymerase III holoenzyme auxiliary subunits

DNA polymerase III of Escherichia coli requires multiple auxiliary factors to enable it to serve as a replicative complex. We demonstrate that auxiliary components of the DNA polymerase III holoenzyme, the gamma delta complex and beta subunit, markedly stimulate DNA polymerase II on long single-stranded templates. DNA polymerase II activity is enhanced by single-stranded DNA binding protein, but the stimulation by gamma delta and beta can be observed either in the absence or presence of single-stranded DNA binding protein. In contrast with DNA polymerase III, the requirement of DNA polymerase II for gamma delta cannot be bypassed by large excesses of the beta subunit at low ionic strength in the absence of the single-stranded DNA binding protein. The product of the DNA polymerase II-gamma delta-beta reaction on a uniquely primed single-stranded circle is of full template length; the reconstituted enzyme apparently is incapable of strand displacement synthesis. The possible biological implications of these observations are discussed.     

DNA structure requirements for the Escherichia coli gamma complex clamp loader and DNA polymerase III holoenzyme

The Escherichia coli chromosomal replicase, DNA polymerase III holoenzyme, is highly processive during DNA synthesis. Underlying high processivity is a ring-shaped protein, the beta clamp, that encircles DNA and slides along it, thereby tethering the enzyme to the template. The beta clamp is assembled onto DNA by the multiprotein gamma complex clamp loader that opens and closes the beta ring around DNA in an ATP-dependent manner. This study examines the DNA structure required for clamp loading action. We found that the gamma complex assembles beta onto supercoiled DNA (replicative form I), but only at very low ionic strength, where regions of unwound DNA may exist in the duplex. Consistent with this, the gamma complex does not assemble beta onto relaxed closed circular DNA even at low ionic strength. Hence, a 3'-end is not required for clamp loading, but a single-stranded DNA (ssDNA)/double-stranded DNA (dsDNA) junction can be utilized as a substrate, a result confirmed using synthetic oligonucleotides that form forked ssDNA/dsDNA junctions on M13 ssDNA. On a flush primed template, the gamma complex exhibits polarity; it acts specifically at the 3'-ssDNA/dsDNA junction to assemble beta onto the DNA. The gamma complex can assemble beta onto a primed site as short as 10 nucleotides, corresponding to the width of the beta ring. However, a protein block placed closer than 14 base pairs (bp) upstream from the primer 3' terminus prevents the clamp loading reaction, indicating that the gamma complex and its associated beta clamp interact with approximately 14-16 bp at a ssDNA/dsDNA junction during the clamp loading operation. A protein block positioned closer than 20-22 bp from the 3' terminus prevents use of the clamp by the polymerase in chain elongation, indicating that the polymerase has an even greater spatial requirement than the gamma complex on the duplex portion of the primed site for function with beta. Interestingly, DNA secondary structure elements placed near the 3' terminus impose similar steric limits on the gamma complex and polymerase action with beta. The possible biological significance of these structural constraints is discussed.     

Characterization of the stimulatory effect of T4 gene 45 protein and the gene 4462 protein complex on DNA synthesis by T4 DNA polymerase

On a variety of single-stranded DNA templates, the overall rate of in vitro DNA synthesis catalyzed by the bacteriophage T4 DNA polymerase is increased about fourfold by addition of the T4 gene $\text{44}\text{62}$ and 45 proteins. Several different methods suggest that this stimulation reflects an increase in the average DNA polymerase “sticking distance”, or processivity, from 800 to about 3000 nucleotides per initiation event. Both the $\text{44}\text{62}$ protein complex and the 45 protein must be present to obtain this effect, and either ATP or dATP hydrolysis is required. Rapid-mixing experiments indicate that the polymerase stimulation is maximized within a few seconds after addition of these “polymerase accessory proteins.”     

DNA polymerase III holoenzyme of Escherichia coli: Components and function of a true replicative complex

Summary The DNA polymerase III holoenzyme is a complex, multisubunit enzyme that is responsible for the synthesis of most of the Escherichia coli chromosome. Through studies of the structure, function and regulation of this enzyme over the past decade, considerable progress has been made in the understanding of the features of a true replicative complex. The holoenzyme contains at least seven different subunits. Three of these, , and , compose the catalytic core. Apparently is the catalytic subunit and the product of the dnaE gene. Epsilon, encoded by dnaQ (mutD), is responsible for the proofreading 35 activity of the polymerase. The function of the B subunit remains to be established. The auxiliary subunits, , and , encoded by dnaN, dnaZ and dnaX, respectively, are required for the functioning of the polymerase on natural chromosomes. All of the proteins participate in increasing the processivity of the polymerase and in the ATP-dependent formation of an initiation complex. Tau causes the polymerase to dimerize, perhaps forming a structure that can coordinate leading and lagging strand synthesis at the replication fork. This dimeric complex may be asymmetric with properties consistent with the distinct requirements for leading and lagging strand synthesis.     

The fidelity of base selection by the polymerase subunit of DNA polymerase III holoenzyme.

In common with other DNA polymerases, DNA polymerase III holoenzyme of E. coli selects the biologically correct base pair with remarkable accuracy. DNA polymerase III is particularly useful for mechanistic studies because the polymerase and editing activities reside on separate subunits. To investigate the biochemical mechanism for base insertion fidelity, we have used a gel electrophoresis assay to measure kinetic parameters for the incorporation of correct and incorrect nucleotides by the polymerase (alpha) subunit of DNA polymerase III. As judged by this assay, base selection contributes a factor of roughly 10(4)-10(5) to the overall fidelity of genome duplication. The accuracy of base selection is determined mainly by the differential KM of the enzyme for correct vs. incorrect deoxynucleoside triphosphate. The misinsertion of G opposite template A is relatively efficient, comparable to that found for G opposite T. Based on a variety of other work, the G:A pair may require a special correction mechanism, possibly because of a syn-anti pairing approximating Watson-Crick geometry. We suggest that precise recognition of the equivalent geometry of the Watson-Crick base pairs may be the most critical feature for base selection.