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.     

The polymerase subunit of DNA polymerase III of Escherichia coli. II. Purification of the alpha subunit, devoid of nuclease activities

The alpha subunit (140 kDa) of DNA polymerase III (pol III) holoenzyme has been purified to near-homogeneity from a plasmid-carrying Escherichia coli strain which overproduced the alpha subunit about 20-fold. Pol III core (containing only the alpha, epsilon, and theta subunits), produced at twice the normal level, was also purified in good yield. The isolated alpha subunit has DNA polymerase activity, which is completely inhibited by 10 mM N-ethylmaleimide or 150 mM KCl as observed in the pol III core or holoenzyme. The alpha subunit has an apparent turnover number of 7.7 nucleotides polymerized per s, compared to 20 for pol III core, and is more thermolabile. The alpha subunit lacks the 3'----5' exonuclease (proofreading) activity of pol III core; neither alpha subunit nor core (nor holoenzyme) possesses any of the previously reported 5'----3' exonuclease activity. Thus, the alpha polypeptide is the polymerase subunit and epsilon (27 kDa) is the proofreading subunit (Scheuermann, R. H., and Echols, H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 7747-7751). Together with the theta polypeptide (10 kDa), of unknown function, they form a pol III core with greater stability and catalytic efficiency.     

Total reconstitution of DNA polymerase III holoenzyme reveals dual accessory protein clamps

DNA polymerase III holoenzyme (holoenzyme) is the 10-subunit replicase of the Escherichia coli chromosome. In this report, pure preparations of delta, delta', and a gamma chi psi complex are resolved from the five protein gamma complex subassembly. Using these subunits and other holoenzyme subunits isolated from overproducing plasmid strains of E. coli, the rapid and highly processive holoenzyme has been reconstituted from only five pure single subunits: alpha, epsilon, gamma, delta, and beta. The preceding report showed that of the three subunits in the core polymerase, only a complex of alpha (DNA polymerase) and epsilon (3'-5' exonuclease) are required to assemble a processive holoenzyme on a template containing a preinitiation complex (Studwell, P.S., and O'Donnell, M. (1990) J. Biol. Chem. 265, 1171-1178). This report shows that of the five proteins in the gamma complex only a heterodimer of gamma and delta is required with the beta subunit to form the ATP-activated preinitiation complex with a primed template. Surprisingly, the delta' subunit does not form an active complex with gamma but forms a fully active heterodimer complex with the tau subunit (as does delta). Hence, the tau delta' and gamma delta heterodimers are fully active in the preinitiation complex reaction with beta and primed DNA. Holoenzymes reconstituted using the alpha epsilon complex, beta subunit, and either gamma delta or tau delta' are fully processive in DNA synthesis, and upon completing the template they rapidly cycle to a new primed template endowed with a preinitiation complex clamp. Since the holoenzyme molecule contains all of these accessory subunits (gamma, delta, tau, delta', and beta) in all likelihood it has the capacity to form two preinitiation complex clamps simultaneously at two primer termini. Two primer binding components within one holoenzyme may mediate its rapid cycling to multiple primers on the lagging strand and also provides functional evidence for the hypothesis of holoenzyme as a dimeric polymerase capable of simultaneous replication of both leading and lagging strands of a replication fork.     

Specificity and enzymatic mechanism of the editing exonuclease of Escherichia coli DNA polymerase III.

Exonucleolytic editing is a major contributor to the fidelity of DNA replication by the multisubunit DNA polymerase (pol) III holoenzyme. To investigate the source of editing specificity, we have studied the isolated exonuclease subunit, epsilon, and the pol III core subassembly, which carries the epsilon, theta, and alpha (polymerase) subunits. Using oligonucleotides with specific terminal mismatches, we have found that both epsilon and pol III core preferentially excise a mispaired 3' terminus and therefore have intrinsic editing specificity. For both epsilon and pol III core, exonuclease activity is much more effective with single-strand DNA; with a double-strand DNA, the exonuclease is strongly temperature-dependent. We conclude that the epsilon subunit of pol III holoenzyme is itself a specific editing exonuclease and that the source of specificity is the greater melting capacity of a mispaired 3' terminus.     

Cytochrome c oxidase from bakers' yeast. V. Arrangement of the subunits in the isolated and membrane-bound enzyme.

Cytochrome c oxidase from baker's yeast contains three mitochondrially made subunits (I to III) which are relatively hydrophobic and four cytoplasmically made subunits (IV to VII) which are relatively hydrophilic (Mason, T. L., Poyton, R. O., Wharton, D.C., and Schatz, G. (1973) J. Biol. Chem. 248, 1346-1354 and Poyton, R. O., and Schatz, G. (1975) J. Biol. Chem. 250, 752-761). In order to explore the arrangement of these subunits in the holoenzyme, the reactivity of each subunit with a variety of "surface probes" was tested with isolated cytochrome c oxidase, with cytochrome c oxidase incorporated into liposomes, and with mitochondrially bound cytochrome c oxidase. The surface probes included iodination with lactoperoxidase and coupling with p-diazonium benzenesulfonate. In addition, external subunits were identified by linking them to bovine serum albumin carrying a covalently bound isocyanate group. In the membrane-bound enzyme, Subunit I was almost completely inaccessible and Subunit II was partly inaccessible to all surface probes. All of the other subunits were accessible. Similar results were obtained with the solubilized enzyme, except that the differences in reactivity between the individual subunits were less clear-cut. The results obtained with liposome-bound cytochrome c oxidase resembled those obtained with the mitochondrially bound enzyme. These data suggest that the two largest mitochondrially made subunits are localized in the interior of the enzyme and that they are genuine components of cytochrome c oxidase.     

Discovery and characterization of the cryptic psi subunit of the pseudomonad DNA replicase

We previously reconstituted a minimal DNA replicase from Pseudomonas aeruginosa consisting of alpha and epsilon (polymerase and editing nuclease), beta (processivity factor), and the essential tau, delta, and delta' components of the clamp loader complex (Jarvis, T., Beaudry, A., Bullard, J., Janjic, N., and McHenry, C. (2005) J. Biol. Chem. 280, 7890-7900). In Escherichia coli DNA polymerase III holoenzyme, chi and Psi are tightly associated clamp loader accessory subunits. The addition of E. coli chiPsi to the minimal P. aeruginosa replicase stimulated its activity, suggesting the existence of chi and Psi counterparts in P. aeruginosa. The P. aeruginosa chi subunit was recognizable from sequence similarity, but Psi was not. Here we report purification of an endogenous replication complex from P. aeruginosa. Identification of the components led to the discovery of the cryptic Psi subunit, encoded by holD. P. aeruginosa chi and Psi were co-expressed and purified as a 1:1 complex. P. aeruginosa chiPsi increased the specific activity of tau(3)deltadelta' 25-fold and enabled the holoenzyme to function under physiological salt conditions. A synergistic effect between chiPsi and single-stranded DNA binding protein was observed. Sequence similarity to P. aeruginosa Psi allowed us to identify Psi subunits from several other Pseudomonads and to predict probable translational start sites for this protein family. This represents the first identification of a highly divergent branch of the Psi family and confirms the existence of Psi in several organisms in which Psi was not identifiable based on sequence similarity alone.     

The DNA polymerase-primase from drosophila melanogaster embryos. Rate and fidelity of polymerization on single-stranded DNA templates

The DNA polymerase activity of the near homogeneous, multisubunit DNA polymerase-primase from Drosophila melanogaster embryos has been compared to Escherichia coli DNA polymerase III core, DNA polymerase III, and DNA polymerase III holoenzyme. The rate of deoxynucleotide incorporation by the Drosophila polymerase on singly primed phi X174 DNA is similar to that observed with equivalent levels of DNA polymerase III holoenzyme in the absence of E. coli single-stranded DNA binding protein. However, analysis of the DNA products indicates that the Drosophila polymerase is less processive than DNA polymerase III holoenzyme, and closely resembles DNA polymerase III. The Drosophila polymerase-primase contains neither 3'-5' exonuclease nor RNase H-like activities, and catalyzes no significant pyrophosphate exchange. There is a low level of DNA-dependent ATPase activity which can be eliminated by a second glycerol gradient sedimentation (Kaguni, L.S., Rossignol, J.-M., Conaway, R.C., and Lehman, I.R. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2221-2225). Although lacking a 3'-5' exonuclease, the replication fidelity of the D. melanogaster polymerase is similar to that of E. coli DNA polymerase III holoenzyme which possesses such an activity.     

The theta subunit of Escherichia coli DNA polymerase III: a role in stabilizing the epsilon proofreading subunit

The function of the theta subunit of Escherichia coli DNA polymerase III holoenzyme is not well established. theta is a tightly bound component of the DNA polymerase III core, which contains the alpha subunit (polymerase), the epsilon subunit (3'-->5' exonuclease), and the theta subunit, in the linear order alpha-epsilon-theta. Previous studies have shown that the theta subunit is not essential, as strains carrying a deletion of the holE gene (which encodes theta) proved fully viable. No significant phenotypic effects of the holE deletion could be detected, as the strain displayed normal cell health, morphology, and mutation rates. On the other hand, in vitro experiments have indicated the efficiency of the 3'-exonuclease activity of epsilon to be modestly enhanced by the presence of theta. Here, we report a series of genetic experiments that suggest that theta has a stabilizing role for the epsilon proofreading subunit. The observations include (i) defined DeltaholE mutator effects in mismatch-repair-defective mutL backgrounds, (ii) strong DeltaholE mutator effects in certain proofreading-impaired dnaQ strains, and (iii) yeast two- and three-hybrid experiments demonstrating enhancement of alpha-epsilon interactions by the presence of theta. theta appears conserved among gram-negative organisms which have an exonuclease subunit that exists as a separate protein (i.e., not part of the polymerase polypeptide), and the presence of theta might be uniquely beneficial in those instances where the proofreading 3'-exonuclease is not part of the polymerase polypeptide.     

Interaction between RNA polymerase and RapA, a bacterial homolog of the SWI/SNF protein family

Recently, we identified a novel Escherichia coli RNA polymerase (RNAP)-associated protein, an ATPase, called RapA (Sukhodolets, M. V. , and Jin, D. J. (1998) J. Biol. Chem. 273, 7018-7023). RapA is a bacterial homolog of SWI2/SNF2. We showed that RapA forms a stable complex with RNAP holoenzyme and that binding to RNAP holoenzyme stimulates the ATPase activity of RapA. We have further analyzed the interactions between purified RapA and the two forms of RNAP: core RNAP and RNAP holoenzyme. We found that RapA interacts with either form of RNAP. However, RapA exhibits higher affinity for core RNAP than for RNAP holoenzyme. Chemical cross-linking of the RNAP-RapA complex indicated that the RapA-binding sites are located at the interface between the alpha and beta' subunits of RNAP. Contrary to previously reported results (Muzzin, O., Campbell, E., A., Xia, L., Severinova, E., Darst, S. A., and Severinov, K. (1998) J. Biol. Chem. 273, 15157-15161), our in vivo analysis of a rapA null mutant suggested that RapA is not likely to be directly involved in DNA repair.     

The DnaX-binding subunits delta' and psi are bound to gamma and not tau in the DNA polymerase III holoenzyme

The DnaX complex subassembly of the DNA polymerase III holoenzyme is comprised of the DnaX proteins tau and gamma and the auxiliary subunits delta, delta', chi, and psi, which together load the beta processivity factor onto primed DNA in an ATP-dependent reaction. delta' and psi bind directly to DnaX whereas delta and chi bind to delta' and psi, respectively (Onrust, R., Finkelstein, J., Naktinis, V., Turner, J., Fang, L., and O'Donnell, M. (1995) J. Biol. Chem. 270, 13348-13357). Until now, it has been unclear which DnaX protein, tau or gamma, in holoenzyme binds the auxiliary subunits delta, delta', chi,and psi. Treatment of purified holoenzyme with the homobifunctional cross-linker bis(sulfosuccinimidyl)suberate produces covalently cross-linked gamma-delta' and gamma-psi complexes identified by Western blot analysis. Immunodetection of cross-linked species with anti-delta' and anti-psi antibodies revealed that no tau-delta' or tau-psi cross-links had formed, suggesting that the delta' and psi subunits reside only on gamma within holoenzyme.     

The C-terminal domain of dnaQ contains the polymerase binding site

The Escherichia coli dnaQ gene encodes the 3'-->5' exonucleolytic proofreading (epsilon) subunit of DNA polymerase III (Pol III). Genetic analysis of dnaQ mutants has suggested that epsilon might consist of two domains, an N-terminal domain containing the exonuclease and a C-terminal domain essential for binding the polymerase (alpha) subunit. We have created truncated forms of dnaQ resulting in epsilon subunits that contain either the N-terminal or the C-terminal domain. Using the yeast two-hybrid system, we analyzed the interactions of the single-domain epsilon subunits with the alpha and theta subunits of the Pol III core. The DnaQ991 protein, consisting of the N-terminal 186 amino acids, was defective in binding to the alpha subunit while retaining normal binding to the theta subunit. In contrast, the NDelta186 protein, consisting of the C-terminal 57 amino acids, exhibited normal binding to the alpha subunit but was defective in binding to the theta subunit. A strain carrying the dnaQ991 allele exhibited a strong, recessive mutator phenotype, as expected from a defective alpha binding mutant. The data are consistent with the existence of two functional domains in epsilon, with the C-terminal domain responsible for polymerase binding.     

T7-induced DNA polymerase. Characterization of associated exonuclease activities and resolution into biologically active subunits.

Bacteriophage T7-induced DNA polymerase has been isolated by a procedure suitable for large scale use and which yields near homogeneous enzyme. In addition to previously described DNA polymerase activity and 3' to 5' exonucleolytic activity on single stranded DNA (Grippo, P., and Richardson, C. C. (1971) J. Biol. Chem. 246, 6867-6873), the enzyme also possesses a highly active exonuclease which hydrolyzes duplex substrates with 3' to 5' directionality. The native polymerase has been dissociated using 6 M guanidine HCl and resolved into biologically active subunits: T7 gene 5 protein and Escherichia coli thioredoxin. The phage-specified subunit obtained by this procedure is deficient in DNA polymerase and double strand exonuclease activities, with deficiencies in these activities being apparent at the level of a single turnover. However, it possesses near normal levels of a single strand hydrolytic activity which is identical to that associated with the native polymerase with respect to substrate specificity and suppression of hydrolysis by low levels of deoxyribonucleoside 5'-triphosphates. Thioredoxin forms a molecular complex with the T7 gene 5 protein, and addition of the host protein restores restores DNA polymerase and double strand exonuclease activities to near normal levels.     

Two functional domains of the epsilon subunit of DNA polymerase III

The epsilon subunit is the 3'-->5' proofreading exonuclease that associates with the alpha and theta subunits in the E. coli DNA polymerase III. Two fragments of the epsilon protein were prepared, and binding of these epsilon fragments with alpha and theta was investigated using gel filtration chromatography and exonuclease stimulation assays. The N-terminal fragment of epsilon, containing amino acids 2-186 (epsilon186), is a relatively protease-resistant core domain of the exonuclease. The purified recombinant epsilon186 protein catalyzes the cleavage of 3' terminal nucleotides, demonstrating that the exonuclease domain of epsilon is present in the N-terminal region of the protein. The absence of the C-terminal 57 amino acids of epsilon in the epsilon186 protein reduces the binding affinity of epsilon186 for alpha by at least 400-fold relative to the binding affinity of epsilon for alpha. In addition, stimulation of the epsilon186 exonuclease by alpha using a partial duplex DNA is about 50-fold lower than stimulation of the epsilon exonuclease by alpha. These results indicate that the C-terminal region of epsilon is required in the epsilonalpha association. To directly demonstrate that the C-terminal region of epsilon contains the alpha-association domain fusion protein, constructs containing the maltose-binding protein (MBP) and fragments of the C-terminal region of epsilon were prepared. Gel filtration analysis demonstrates that the alpha-association domain of epsilon is contained within the C-terminal 40 amino acids of epsilon. Also, the epsilon186 protein forms a tight complex with theta, demonstrating that the association of theta with epsilon is localized to the N-terminal region of epsilon. Association of epsilon186 and theta is further supported by the stimulation of the epsilon186 exonuclease in the presence of theta. These data support the concept that epsilon contains a catalytic domain located within the N-terminal region and an alpha-association domain located within the C-terminal region of the protein.     

Nuclear magnetic resonance solution structure of the Escherichia coli DNA polymerase III theta subunit

The catalytic core of Escherichia coli DNA polymerase III holoenzyme contains three subunits: alpha, epsilon, and theta. The alpha subunit contains the polymerase, and the epsilon subunit contains the exonucleolytic proofreading function. The small (8-kDa) theta subunit binds only to epsilon. Its function is not well understood, although it was shown to exert a small stabilizing effect on the epsilon proofreading function. In order to help elucidate its function, we undertook a determination of its solution structure. In aqueous solution, theta yielded poor-quality nuclear magnetic resonance spectra, presumably due to conformational exchange and/or protein aggregation. Based on our recently determined structure of the theta homolog from bacteriophage P1, named HOT, we constructed a homology model of theta. This model suggested that the unfavorable behavior of theta might arise from exposed hydrophobic residues, particularly toward the end of alpha-helix 3. In gel filtration studies, theta elutes later than expected, indicating that aggregation is potentially responsible for these problems. To address this issue, we recorded 1H-15N heteronuclear single quantum correlation (HSQC) spectra in water-alcohol mixed solvents and observed substantially improved dispersion and uniformity of peak intensities, facilitating a structural determination under these conditions. The structure of theta in 60/40 (vol/vol) water-methanol is similar to that of HOT but differs significantly from a previously reported theta structure. The new theta structure is expected to provide additional insight into its physiological role and its effect on the epsilon proofreading subunit.     

DNA and RNA-DNA annealing activity associated with the tau subunit of the Escherichia coli DNA polymerase III holoenzyme.

The DNA polymerase III (pol III)holoenzyme is the 10 subunit replicase of Escherichia coli. The 71 kDa tau subunit, encoded by dnaX, dimerizes the core polymerase (alpha epsilon theta) to form pol III'[(alpha epsilon theta)2 tau 2]. tau is also a single-stranded DNA-dependent ATPase and can substitute for the gamma subunit during initiation complex formation. We show here that tau also possesses a DNA-DNA and RNA-DNA annealing activity that is stimulated by Mg2+, but neither requires ATP nor is inhibited by non-hydrolyzable ATP analogs. This suggests the tau may act to stabilize the primer-template interaction during DNA replication.     

The beta subunit of the Escherichia coli DNA polymerase III holoenzyme interacts functionally with the catalytic core in the absence of other subunits

We have previously demonstrated that the addition of a stoichiometric excess of the beta subunit of Escherichia coli DNA polymerase III holoenzyme to DNA polymerase III or holoenzyme itself can lead to an ATP-independent increase in the processivity of these enzyme forms (Crute, J. J., LaDuca, R. J., Johanson, K. O., McHenry, C. S., and Bambara, R. A. (1983) J. Biol. Chem. 258, 11344-11349). Here, we show that the beta subunit can interact directly with the catalytic core of the holoenzyme, DNA polymerase III, generating a new form of the enzyme with enhanced catalytic and processive capabilities. The addition of saturating levels of the beta subunit to the core DNA polymerase III enzyme results in as much as a 7-fold stimulation of synthetic activity. Two populations of DNA products were generated by the DNA polymerase III X beta enzyme complex. Short products resulting from the addition of 5-10 nucleotides/primer fragment were generated by DNA polymerase III in the presence and absence of added beta subunit. A second population of much longer products was generated only in beta-supplemented DNA polymerase III reactions. The DNA polymerase III-beta reaction was inhibited by single-stranded DNA binding protein and was unaffected by ATP, distinguishing it from the holoenzyme-catalyzed reaction. Complex formation of the DNA polymerase III core enzyme with beta increased the residence time of the enzyme on synthetic DNA templates. Our results demonstrate that the beta stimulation of DNA polymerase III can be attributed to a more efficient and highly processive elongation capability of the DNA polymerase III X beta complex. They also prove that at least part of beta's normal contribution to the DNA polymerase III holoenzyme reaction takes place through interaction with DNA polymerase III core enzyme components to produce the essential complex necessary for efficient elongation in vivo.     

The dnaZ protein, the gamma subunit of DNA polymerase III holoenzyme of Escherichia coli

The dnaZ protein has been purified to near-homogeneity using an in vitro complementation assay that measures the restoration of activity in a crude enzyme fraction from the dnaZ mutant deficient in the replication of phi X174 DNA. Over 70-fold overproduction of the protein was obtained with a bacteriophage lambda lysogen carrying the dnaZ gene. The purified protein, under reducing and denaturing conditions, has a molecular weight of 52,000 and appears to be a dimer in its native form. The dnaZ protein is judged to be th 52,000-dalton gamma subunit of DNA polymerase III holoenzyme (McHenry, C., and Kornberg, A. (1977) J. Biol. Chem. 252, 6478-6484) for the following reasons: (i) highly purified DNA polymerase III holoenzyme contains a 52,000-dalton polypeptide and has dnaZ-complementing activity; (ii) the 52,000-dalton polypeptide is associated tightly with the DNA polymerase III holoenzyme and can be separated from the DNA polymerase III core only with severe measures; (iii) no other purified replication protein, among 14 tested, contains dnaZ protein activity; and (iv) the abundance of dnaZ protein, estimated at about 10 dimer molecules per Escherichia coli cell, is similar to that of the DNA polymerase III core. Among several circular templates tested in vitro (i.e. single stranded phi X174, G4 and M13 DNAs, and duplex phi X174 DNA), all rely on dnaZ protein for elongation by DNA polymerase III holoenzyme. The protein acts catalytically at a stoichiometry of one dimer per template.     

The interaction of DNA polymerase III and the product of the Escherichia coli mutator gene, mutD

A comparison of DNA polymerase III core enzyme (McHenry, C. S., and Crow, W. (1979) J. Biol. Chem. 254, 1748-1753) prepared from wild type Escherichia coli and a strain harboring the mutator gene, mutD5 (Degnen, G. E., and Cox, E. C. (1974) J. Bacteriol. 17, 477-487) has revealed several differences in their properties. Among these are alterations in the heat stability, divalent cation requirement, pH optimum, 3'----5'-single strand exonuclease activity, and DNA-dependent conversion of a deoxynucleoside triphosphate to its corresponding monophosphate ("turnover"). The decrease in the 3'-single strand exonuclease and turnover indicate a defect in the editing function of the mutD strain, which is at least in part responsible for the high spontaneous mutation rate in mutD. Transformation of mutD by a hybrid plasmid, pRD3, constructed from an EcoRI restriction fragment of E. coli and pBR322, cures mutD of its abnormally high mutation rate, and simultaneously restores its 3'-exonuclease activity. These observations are consistent with the notion that the mutD gene product is a subunit of DNA polymerase III, and it either contains the catalytic site for the 3'-exonuclease or modulates its activity. From a consideration of the known molecular weights of the subunits in DNA polymerase III core (McHenry C. S., and Crow, W. (1979) J. Biol. Chem. 254, 1748-1753) the molecular weights of the two proteins translated in maxicells transformed with pRD3, and from a comparison of our results with those obtained with the mutator dnaQ (Horiuchi, T., Maki, H., Maruyama, M., and Sekiguchi, M. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 3770-3774) and the work of Cox and Horner (Cox, E. C., and Horner, D. L. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2295-2299) as well as Echols et al. (Echols, H., Lu, C., and Burgers, P. M. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2189-2192) we tentatively assign the mutD gene product to the epsilon subunit of DNA polymerase III.     

Reconstitution of the activities of the RecBCD holoenzyme of Escherichia coli from the purified subunits.

The Escherichia coli RecBCD holoenzyme and the individual constituent subunits have been purified from overproducing strains. The purified RecBCD holoenzyme has a native molecular mass of approximately 330 kDa, indicative of a heterotrimer subunit assembly. The RecB, RecC, and RecD subunits can associate in vitro to give nuclease, helicase, ATPase, and Chi-specific endonuclease activities which are indistinguishable from those of the RecBCD holoenzyme. At concentrations at which the reconstituted RecB + C + D enzyme is very active, none of the individual RecB, RecC, or RecD subunits have readily detectable activities of the holoenzyme, except RecB protein which had previously been shown to exhibit DNA-dependent ATPase activity (Hickson, I. D., Robson, C. N., Atkinson, K. E., Hutton, L., and Emmerson, P. T. (1985) J. Biol. Chem. 260, 1224-1229). At higher concentrations and with shorter DNA substrates reconstituted RecBC protein exhibits low levels of helicase and exonuclease activity.