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.     

DNA footprinting studies of the complex formed by the T4 DNA polymerase holoenzyme at a primer-template junction

We have used DNA footprinting techniques to analyze the interactions of five DNA replication proteins at a primer-template junction: the bacteriophage T4 DNA polymerase (the gene 43 protein), its three accessory proteins (the gene 44/62 and 45 proteins), and the gene 32 protein, which is the T4 helix-destabilizing (or single-stranded DNA-binding) protein. The 177-nucleotide-long DNA substrate consisted of a perfect 52-base pair hairpin helix with a protruding single-stranded 5' tail. As expected, the DNA polymerase binds near the 3' end of this molecule (at the primer-template junction) and protects the adjacent double-stranded region from cleavage. When the gene 32 protein binds to the single-stranded tail, it reduces the concentration of the DNA polymerase required to observe the polymerase footprint by 10-30-fold. Periodic ATP hydrolysis by the 44/62 protein is required to maintain the activity of the DNA polymerase holoenzyme (a complex of the 43, 44/62, and 45 proteins). Footprinting experiments demonstrate the formation of a weak complex between the DNA polymerase and the gene 45 protein, but there is no effect of the 44/62 protein or ATP on this enlarged footprint. We propose a model for holoenzyme function in which the complex of the three accessory proteins uses ATP hydrolysis to keep a moving polymerase tightly bound to the growing 3' end, providing a "clock" to measure polymerase stalling.     

Autogenous translational operator recognized by bacteriophage T4 DNA polymerase

The synthesis of the DNA polymerase of bacteriophage T4 is autogenously regulated. This protein (gp43), the product of gene 43, binds to a segment of its mRNA that overlaps its ribosome binding site, and thereby blocks translation. We have determined the Kd of the gp43-operator interaction to be 1.0 x 10(-9) M. The minimum operator sequence to which gp43 binds consists of 36 nucleotides that include a hairpin (containing a 5 base-pair helix and an 8 nucleotide loop) and a single-stranded segment that contains the Shine-Dalgarno sequence of the ribosome binding site. In the distantly related bacteriophage RB69 there is a remarkable conservation of this hairpin and loop sequence at the ribosome binding site of its DNA polymerase gene. We have constructed phage operator mutants that overproduce gp43 in vivo, yet are unchanged for in vivo replication rates and phage yield. We present data that show that the replicative and autoregulatory functions are mutually exclusive activities of this polymerase, and suggest a model for gp43 synthesis that links autoregulation to replicative demand.     

Biochemical characterization of interactions between DNA polymerase and single-stranded DNA-binding protein in bacteriophage RB69

The organization and proper assembly of proteins to the primer-template junction during DNA replication is essential for accurate and processive DNA synthesis. DNA replication in RB69 (a T4-like bacteriophage) is similar to those of eukaryotes and archaea and has been a prototype for studies on DNA replication and assembly of the functional replisome. To examine protein-protein interactions at the DNA replication fork, we have established solution conditions for the formation of a discrete and homogeneous complex of RB69 DNA polymerase (gp43), primer-template DNA, and RB69 single-stranded DNA-binding protein (gp32) using equilibrium fluorescence and light scattering. We have characterized the interaction between DNA polymerase and single-stranded DNA-binding protein and measured a 60-fold increase in the overall affinity of RB69 single-stranded DNA-binding protein (SSB) for template strand DNA in the presence of DNA polymerase that is the result of specific protein-protein interactions. Our data further suggest that the cooperative binding of the RB69 DNA polymerase and SSB to the primer-template junction is a simple but functionally important means of regulatory assembly of replication proteins at the site of action. We have also shown that a functional domain of RB69 single-stranded DNA-binding protein suggested previously to be the site of RB69 DNA polymerase-SSB interactions is dispensable. The data from these studies have been used to model the RB69 DNA polymerase-SSB interaction at the primer-template junction.     

Escherichia coli thioredoxin stabilizes complexes of bacteriophage T7 DNA polymerase and primed templates

The DNA polymerase activity induced after bacteriophage T7 infection of Escherichia coli is found in a complex of two proteins, the T7 gene 5 protein and a host protein, thioredoxin. Gene 5 protein is a DNA polymerase and a 3' to 5' exonuclease. Thioredoxin binds tightly to the gene 5 protein and increases the processivity of polymerization some 1000-fold. Gene 5 protein forms a short-lived complex with the primer-template, poly(dA).oligo(dT), in the absence of Mg2+ and nucleotides. Thioredoxin increases the half-life of the preformed primer-template-polymerase complex from less than a second to approximately 5 min. The dissociation is accelerated by excess single-stranded DNA in an apparent second order reaction, indicating direct transfer of polymerase between DNA fragments. Thioredoxin also reduces the equilibrium dissociation constant, Kd, of the gene 5 protein -poly(dA).oligo(dT) complex 20- to 80-fold. The salt dependence of Kd indicates that thioredoxin stabilizes the primer-template-polymerase complex mainly through additional charge-charge interactions, increasing the estimated number of interactions from 2 to 7. The affinity of gene 5 protein for single-stranded DNA is at least 1000-fold higher than for double-stranded DNA and is little affected by thioredoxin. Under conditions of steady state synthesis the effect of thioredoxin on the polymerization rate is determined by two competing factors, an increase in processivity and a decrease of the dissociation rate of polymerase and replicated template.     

Structural and enzymatic studies of the T4 DNA replication system. II. ATPase properties of the polymerase accessory protein complex

In this paper we report a detailed enzymatic characterization of the interaction of the polymerase accessory protein complex of the T4 DNA replication system with the various nucleic acid cofactors that activate the ATPase of the complex. We show that the ATPase activity of the T4 coded gene 44/62 protein complex is stimulated synergistically by binding of DNA and T4 gene 45 protein and that the level of ATPase activation appears to be directly correlated with the binding of nucleic acid cofactor. Binding of any partially or completely single-stranded DNA to the complete accessory protein complex increases the catalytic activity (as measured by Vmax) while decreasing the binding affinity for the ATP substrate. While single-stranded DNA is a moderately effective cofactor, we find that the optimal nucleic acid-binding site for the complex is the primer-template junction, rather than single-stranded DNA ends as previously reported in the literature. Gene 45 protein plays an essential role in directing the specificity of binding to primer-template sites, lowering the Km for primer-template sites almost 1000-fold, and increasing Vmax 100-fold, compared with the analogous values for gene 44/62 protein alone. The most effective primer-template site for binding and enzymatic activation has the physiologically relevant recessed 3'-OH configuration and an optimal size in excess of 18 base pairs of duplex DNA. We find that the chemical nature of the primer terminus (i.e. 3'-OH or 3'-H) does not affect the extent of ATPase activation and that binding of the polymerase accessory protein complex to DNA cofactors is salt concentration dependent but appreciably less so when the activating DNA is a primer-template junction. Finally, we show that the gene 32 protein (T4 coded single-stranded DNA-binding protein) can compete with the polymerase accessory protein complex for single-stranded DNA but not for the primer-template junction activation sites. The implications of these results for the structure and function of the polymerase accessory protein complex within the T4 DNA replication system are discussed.     

Interaction of a DNA-binding protein, the product of gene D5 of bacteriophage T5, with double-stranded DNA: effects on T5 DNA polymerase functions in vitro.

The gene D5 product (gpD5) of bacteriophage T5 is a DNA-binding protein that binds preferentially to double-stranded DNA and is essential for T5 DNA replication, yet it inhibits DNA synthesis in vitro. Mechanisms of inhibition were studied by using nicked DNA and primed single-stranded DNA as a primer-template. Inhibition of T5 DNA polymerase activity by gpD5 occurred when double-stranded regions of DNA were saturated with gpD5. The 3' leads to 5' exonuclease associated with T5 DNA polymerase was not very active with nicked DNA, but inhibition of hydrolysis of substituents at 3'-hydroxyl termini by gpD5 could be observed. T5 DNA polymerase appears to be capable of binding to the 3' termini even when double-stranded regions are saturated with gpD5. The interaction of gpD5 with the polymerases at the primer terminus is apparently the primary cause of inhibition of polymerization.     

Multiple ATP binding is required to stabilize the "activated" (clamp open) clamp loader of the T4 DNA replication complex

Most DNA replication systems include a sliding clamp that encircles the genomic DNA and links the polymerase to the template to control polymerase processivity. A loading complex is required to open the clamp and place it onto the DNA. In phage T4 this complex consists of a trimeric clamp of gp45 subunits and a pentameric loader assembly of four gp44 and one gp62 subunit(s), with clamp loading driven by ATP binding. We measure this binding as a function of input ligand concentration and show that four ATPs bind to the gp44/62 complex with equal affinity. In contrast, the ATPase rate profile of the clamp-clamp loader complex exhibits a marked peak at an input ATP concentration close to the overall Kd (approximately 30 microm), with further increases in bound ATP decreasing the ATPase rate to a much lower level. Thus the progressive binding of the four ATPs triggers a conformational change in the complex that markedly inhibits ATPase activity. This inhibition is related to ring opening by using a clamp that is covalently cross-linked across its subunit interfaces and thus rendered incapable of opening. Binding of this clamp abolishes substrate inhibition of the ATPase but leaves ATP binding unchanged. We show that four ATP ligands must bind to the T4 clamp loader before the loader can be fully "activated" and the clamp opened, and that ATP hydrolysis is required only for release of the loader complex after clamp loading onto the replication fork has been completed.     

Replication factors required for SV40 DNA replication in vitro. I. DNA structure-specific recognition of a primer-template junction by eukaryotic DNA polymerases and their accessory proteins.

Eukaryotic DNA polymerase delta and its accessory proteins are essential for SV40 DNA replication in vitro. A multi-subunit protein complex, replication factor C (RF-C), which is composed of subunits with apparent molecular weights of 140,000, 41,000, and 37,000, has primer/template binding and DNA-dependent ATPase activities. UV-cross-linking experiments demonstrated that the Mr = 140,000 subunit recognizes and binds to the primer-template DNA, whereas the Mr = 41,000 polypeptide binds ATP. Assembly of a replication complex at a primer-template junction has been studied in detail with synthetic, hairpin DNAs. Following glutaraldehyde fixation, a gel shift assay demonstrated that RF-C alone forms a weak binding complex with the hairpin DNA. Addition of ATP or its nonhydrolyzable analogue, ATP gamma S, increased specific binding to the DNA. Footprinting experiments revealed that RF-C recognizes the primer-template junction, covering 15 bases of the primer DNA from the 3'-end and 20 bases of the template DNA. Another replication factor, proliferating cell nuclear antigen (PCNA) binds to RF-C and the primer-template DNA forming a primer recognition complex and extends the protected region on the duplex DNA. This RF-C.PCNA complex has significant single-stranded DNA binding activity in addition to binding to a primer-template junction. However, addition of another replication factor, RF-A, completely blocked the nonspecific, single-stranded DNA binding by the RF-C.PCNA complex. RF-A therefore functions as a specificity factor for primer recognition. In the absence of RF-C, DNA polymerase delta (pol delta) and PCNA form a complex at the primer-template junction, protecting exactly the same site as the primer recognition complex. Addition of RF-C to this complex produced a higher order complex which is unstable unless its formation is coupled with translocation of pol delta. These results suggest that the sequential binding of RF-C, PCNA, and pol delta to a primer-template junction might directly account for the initiation of leading strand DNA synthesis at a replication origin. We demonstrate this directly in an accompanying paper (Tsurimoto, T., and Stillman, B. (1991) J. Biol. Chem. 266, 1961-1968).     

On the specificity of interaction between the Saccharomyces cerevisiae clamp loader replication factor C and primed DNA templates during DNA replication

Replication factor C (RFC) catalyzes assembly of circular proliferating cell nuclear antigen clamps around primed DNA, enabling processive synthesis by DNA polymerase during DNA replication and repair. In order to perform this function efficiently, RFC must rapidly recognize primed DNA as the substrate for clamp assembly, particularly during lagging strand synthesis. Earlier reports as well as quantitative DNA binding experiments from this study indicate, however, that RFC interacts with primer-template as well as single- and double-stranded DNA (ssDNA and dsDNA, respectively) with similar high affinity (apparent K(d) approximately 10 nm). How then can RFC distinguish primed DNA sites from excess ssDNA and dsDNA at the replication fork? Further analysis reveals that despite its high affinity for various DNA structures, RFC selects primer-template DNA even in the presence of a 50-fold excess of ssDNA and dsDNA. The interaction between ssDNA or dsDNA and RFC is far less stable than between primed DNA and RFC (k(off) > 0.2 s(-1) versus 0.025 s(-1), respectively). We propose that the ability to rapidly bind and release single- and double-stranded DNA coupled with selective, stable binding to primer-template DNA allows RFC to scan DNA efficiently for primed sites where it can pause to initiate clamp assembly.     

The Rev1 translesion synthesis polymerase has multiple distinct DNA binding modes

Rev1 is a eukaryotic DNA polymerase of the Y family involved in translesion synthesis (TLS), a major damage tolerance pathway that allows DNA replication at damaged templates. Uniquely amongst the Y family polymerases, the N-terminal part of Rev1, dubbed the BRCA1 C-terminal homology (BRCT) region, includes a BRCT domain. While most BRCT domains mediate protein-protein interactions, Rev1 contains a predicted α-helix N-terminal to the BRCT domain and in human Replication Factor C (RFC) such a BRCT region endows the protein with DNA binding capacity. Here, we studied the DNA binding properties of yeast and mouse Rev1. Our results show that the BRCT region of Rev1 specifically binds to a 5' phosphorylated, recessed, primer-template junction. This DNA binding depends on the extra α-helix, N-terminal to the BRCT domain. Surprisingly, a stretch of 20 amino acids N-terminal to the predicted α-helix is also critical for high-affinity DNA binding. In addition to 5' primer-template junction binding, Rev1 efficiently binds to a recessed 3' primer-template junction. These dual DNA binding characteristics are discussed in view of the proposed recruitment of Rev1 by 5' primer-template junctions, downstream of stalled replication forks.     

Molecular mechanisms of the functional coupling of the helicase (gp41) and polymerase (gp43) of bacteriophage T4 within the DNA replication fork

Processive strand-displacement DNA synthesis with the T4 replication system requires functional "coupling" between the DNA polymerase (gp43) and the helicase (gp41). To define the physical basis of this functional coupling, we have used analytical ultracentrifugation to show that gp43 is a monomeric species at physiological protein concentrations and that gp41 and gp43 do not physically interact in the absence of DNA, suggesting that the functional coupling between gp41 and gp43 depends significantly on interactions modulated by the replication fork DNA. Results from strand-displacement DNA synthesis show that a minimal gp41-gp43 replication complex can perform strand-displacement synthesis at approximately 90 nts/s in a solution containing poly(ethylene glycol) to drive helicase loading. In contrast, neither the Klenow fragment of Escherichia coli DNA polymerase I nor the T7 DNA polymerase, both of which are nonprocessive polymerases, can carry out strand-displacement DNA synthesis with gp41, suggesting that the functional helicase-polymerase coupling may require the homologous system. However, we show that a heterologous helicase-polymerase pair can work if the polymerase is processive. Strand-displacement DNA synthesis using the gp41 helicase with the T4 DNA polymerase holoenzyme or the phage T7 DNA polymerase-thioredoxin complex, both of which are processive, proceeds at the rate of approximately 250 nts/s. However, replication fork assembly is less efficient with the heterologous helicase-polymerase pair. Therefore, a processive (homologous or heterologous) "trailing" DNA polymerase is sufficient to improve gp41 processivity and unwinding activity in the elongation stage of the helicase reaction, and specific T4 helicase-polymerase coupling becomes significant only in the assembly (or initiation) stage.     

A covalent linkage between the gene 5 DNA polymerase of bacteriophage T7 and Escherichia coli thioredoxin,the processivity factor: fate of thioredoxin during DNA synthesis

Gene 5 protein (gp5) of bacteriophage T7 is a non-processive DNA polymerase, which acquires high processivity by binding to Escherichia coli thioredoxin. The gene 5 protein-thioredoxin complex (gp5/trx) polymerizes thousands of nucleotides before dissociating from a primer-template. We have engineered a disulfide linkage between the gene 5 protein and thioredoxin within the binding surface of the two proteins. The polymerase activity of the covalently linked complex (gp5-S-S-trx) is similar to that of gp5/trx on poly(dA)/oligo(dT). However, gp5-S-S-trx has only one third the polymerase activity of gp5/trx on single-stranded M13 DNA. gp5-S-S-trx has difficulty polymerizing nucleotides through sites of secondary structure on M13 DNA and stalls at these sites, resulting in lower processivity. However, gp5-S-S-trx has an identical processivity and rate of elongation when E. coli single-stranded DNA-binding protein (SSB protein) is used to remove secondary structure from M13 DNA. Upon completing synthesis on a DNA template lacking secondary structure, both complexes recycle intact, without dissociation of the processivity factor, to initiate synthesis on a new DNA template. However, a complex stalled at secondary structure becomes unstable, and both subunits dissociate from each other as the polymerase prematurely releases from M13 DNA.     

Evaluating the effects of enhanced processivity and metal ions on translesion DNA replication catalyzed by the bacteriophage T4 DNA polymerase

The fidelity of DNA replication is achieved in a multiplicative process encompassing nucleobase selection and insertion, removal of misinserted nucleotides by exonuclease activity, and enzyme dissociation from primer/templates that are misaligned due to mispairing. In this study, we have evaluated the effect of altering these kinetic processes on the dynamics of translesion DNA replication using the bacteriophage T4 replication apparatus as a model system. The effect of enhancing the processivity of the T4 DNA polymerase, gp43, on translesion DNA replication was evaluated using a defined in vitro assay system. While the T4 replicase (gp43 in complex with gp45) can perform efficient, processive replication using unmodified DNA, the T4 replicase cannot extend beyond an abasic site. This indicates that enhancing the processivity of gp43 does not increase unambiguously its ability to perform translesion DNA replication. Surprisingly, the replicase composed of an exonuclease-deficient mutant of gp43 was unable to extend beyond the abasic DNA lesion, thus indicating that molecular processes involved in DNA polymerization activity play the predominant role in preventing extension beyond the non-coding DNA lesion. Although neither T4 replicase complex could extend beyond the lesion, there were measurable differences in the stability of each complex at the DNA lesion. Specifically, the exonuclease-deficient replicase dissociates at a rate constant, k(off), of 1.1s(-1) while the wild-type replicase remains more stably associated at the site of DNA damage by virtue of a slower measured rate constant (k(off) 0.009s(-1)). The increased lifetime of the wild-type replicase suggests that idle turnover, the partitioning of the replicase from its polymerase to its exonuclease active site, may play an important role in maintaining fidelity. Further attempts to perturb the fidelity of the T4 replicase by substituting Mn(2+) for Mg(2+) did not significantly enhance DNA synthesis beyond the abasic DNA lesion. The results of these studies are interpreted with respect to current structural information of gp43 alone and complexed with gp45.     

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.     

Interaction between the T4 helicase-loading protein (gp59) and the DNA polymerase (gp43): a locking mechanism to delay replication during replisome assembly

The T4 helicase-loading protein (gp59) has been proposed to coordinate leading- and lagging-strand DNA synthesis by blocking leading-strand synthesis during the primosome assembly. In this work, we unambiguously demonstrate through a series of biochemical and biophysical experiments, including single-molecule fluorescence microscopy, that the inhibition of leading-strand holoenzyme progression by gp59 is the result of a complex formed between gp59 and leading-strand polymerase (gp43) on DNA that is instrumental in preventing premature replication during the assembly of the T4 replisome. We find that both the polymerization and 3' --> 5' exonuclease activities of gp43 are totally inhibited within this complex. Chemical cross-linking of the complex followed by tryptic digestion and peptide identification through matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry identified Cys169 of gp43 and Cys215 of gp59 as residues in a region of a protein-protein contact. With the available crystal structures for both gp43 and gp59, a model of the complex was constructed based on shape complementarity, revealing that parts of the C-terminal domain from gp59 insert into the interface created by the thumb and exonuclease domains of gp43. This insertion effectively locks the polymerase into a conformation where switching between the pol and editing modes is prevented. Thus, continued assembly of the replisome through addition of the primosome components and elements of the lagging-strand holoenzyme can occur without leading-strand DNA replication.     

DNA recognition properties of the N-terminal DNA binding domain within the large subunit of replication factor C.

Replication Factor C (RFC) is a five-subunit protein complex required for eukaryotic DNA replication and repair. The large subunit within this complex contains a C-terminal DNA binding domain which provides specificity for PCNA loading at a primer-template and a second, N-terminal DNA binding domain of unknown function. We isolated the N-terminal DNA binding domain from Drosophila melanogaster and defined the region within this polypeptide required for DNA binding. The DNA determinants most efficiently recognized by both the Drosophila minimal DNA binding domain and the N-terminal half of the human large subunit consist of a double-stranded DNA containing a recessed 5' phosphate. DNA containing a recessed 5' phosphate was preferred 5-fold over hairpined DNA containing a recessed 3' hydroxyl. Combined with existing data, these DNA binding properties suggest a role for the N-terminal DNA binding domain in the recognition of phosphorylated DNA ends.     

A conformational change in E. coli DNA polymerase I (Klenow fragment) is induced in the presence of a dNTP complementary to the template base in the active site

It is well established that the insertion of a nucleotide into a growing DNA chain requires a conformational change in the structure of a DNA polymerase. These enzymes have been shown to bind a primer-template in the open conformation and then upon binding of a complementary dNTP undergo a conformational rearrangement to the closed ternary complex. This movement results in the positioning of the incoming nucleotide in the proper geometry for the nucleophilic attack by the 3'-hydroxyl of the primer. In this work, tryptic digestion experiments were performed to detect this conformational change in the structure of the exonuclease-deficient DNA polymerase I (Klenow fragment). Three distinct digestion patterns were observed: one for the polymerase alone, one for the binary complex with the primer-template, and one for the ternary polymerase-DNA-dNTP complex. The latter conformational change leads to a stable ternary closed complex formation only when the correct nucleotide is present in the reaction mixture. Positioning of nucleotides with incorrect geometry in the protein active site inhibits or eliminates formation of the closed complex. Similarly, this conformational change is inhibited when the primer terminus of the DNA molecule is altered by the presence of the 2'-hydroxyl.     

Mechanism of primer-template-dependent conversion of dNTP leads to dNMP by T5 DNA polymerase

T5 DNA polymerase catalyzes both 5' leads to 3' polymerization and 3' leads to 5' hydrolysis in a processive fashion. This knowledge has been utilized to obtain evidence indicating that the enzyme has a single primer-template binding site which can function as either polymerase or exonuclease, perhaps with the cooperation of additional or different side groups. Template-dependent conversion of dNTP leads to dNMP was observed with an excess of either primer-template or enzyme. With primer-template excess, practically all the enzymes were functional as polymerase; with enzyme excess, all primer-templates were extended during the first cycle of catalysis. These observations suggest that turnover takes place at the points of chain growth. Evidence is also provided which demonstrates that the enzyme is capable of switching its direction of catalysis from 3' leads to 5' to 5' leads to 3' without leaving the primer-template. A clear correspondence between the relative amount of hydrolysis of a terminally labeled residue on the primer and the relative amount of turnover suggests that (a) the probability of hydrolysis of a given type of residue in contact with the "active site" is constant, and (b) during each turnover episode enzyme usually takes only one step in the 3' leads to 5' direction. A simple probabilistic model of turnover is 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.