Analysis of the ATPase Subassembly Which Initiates Processive DNA Synthesis by DNA Polymerase I 11 Holoenzyme *

The y complex (y66’xJ.) subassembly of DNA polymerase I11 holoenzyme transfers the /3 subunit onto primed DNA in a reaction which requires ATP hydrolysis. Once on DNA, B is a “sliding clamp” which tethers the polymerase to DNA for highly processive synthesis. We have examined B and the y complex to identify which subunit@) hydrolyzes ATP. We find the y complex is a DNA dependent ATPase. The /3 subunit, which lacks ATPase activity, enhances the y complex ATPase when primed DNA is used as an effector. Hence, the y complex recognizes DNA and couples ATP hydrolysis to clamp B onto primed DNA. Study of y complex subunits showed no single subunit contained significant ATPase activity. However, the heterodimers, y6 and yb‘, were both DNA-dependent ATPases. Only the y6 ATPase was stimulated by B and was functional in transferring the B from solution to primed DNA. Similarity in ATPase activity of DNA polymerase I11 holoenzyme accessory proteins to accessory proteins of phage T4 DNA polymerase and mammalian DNA polymerase 6 suggests the basic strategy of chromosome duplication has been conserved throughout evolution.

tion complex, the holoenzyme is rapid in synthesis (>500 nucleotides/s) and replicates the entire template without dissociating from the DNA even once (i.e. it is highly processive) The remarkable processivity of the holoenzyme requires its accessory proteins (4). The three subunit core subassembly of the holoenzyme contains the DNA polymerase subunit ( a ) (7), the proofreading 3'-5' exonuclease subunit (6) (8), and the 6' subunit (9). The core polymerase synthesizes DNA at a rate of approximately 20 nucleotides/s (10) and is only processive for approximately 11 nucleotides (4). However, the highly processive character of the holoenzyme can be reconstituted upon mixing the core polymerase with both the / 3 subunit and the 5-protein y complex (y66'xll/ subunits) (3,11,12).
Reconstitution of the processive polymerase activity of the holoenzyme can be divided into two distinct stages (3,11,12).
In the first stage, the y complex and p subunit hydrolyze ATP to form a preinitiation complex on primed DNA. In the second stage, the core polymerase assembles with the preinitiation complex to form the highly processive enzyme. Thus, it is the preinitiation complex which confers such remarkable processivity onto the holoenzyme. Further study of the first stage in which the preinitiation complex forms showed the y complex acts catalytically to transfer the / 3 subunit from solution to primed DNA (2,11,33). Only the y and 6 subunits of the y complex are essential to transfer p onto primed DNA (20). Once on DNA, the p subunit slides freely along the duplex portion of the primed template (33). The / 3 subunit also directly binds to a, the DNA polymerase subunit (33). Hence, the preinitiation complex is a "sliding clamp" of the p subunit on DNA, which tethers the polymerase to the primed template and thereby confers onto it highly processive synthesis.
To further our understanding of highly processive synthesis, in this report, we examine the p subunit and y complex to identify which subunit(s) hydrolyze the ATP. (4)(5)(6).

MATERIALS AND METHODS
Sources-Radioactive nucleotides were from DuPont-New England Nuclear; unlabeled nucleotides were from Pharmacia LKB Biotechnology Inc.; DNA modification enzymes were from New England Biolabs; RNAs were from Sigma; and pure proteins were prepared as described SSB (16), CY (7), e (S), CYC complex (17), y (IS), p (E)), yx$ (20), 6 (20), 6' (20). The y complex was purified as described (21) with the following modifications: chromatography on Mono Q was performed in place of the DEAE-Trisacryl step, and the second heparin-agarose chromatography step was replaced by chromatography on an ATP-agarose column and a phosphocellulose column. The concentration of p was determined by absorbance using an e2"(, value of 17,900 M" cm" (19). The concentration of y was determined by amino acid analysis (Protein Microchemistry Facility, Biological Chemistry Department, University of Michigan). Concentrations of a, e, SSB, y complex, and yx$ were determined by the method of Bradford (22) using BSA as a standard. The concentrations of 6 and of Polymerase ZII Holoenzyme 6' were determined by comparison of their Coomassie Blue staining intensity (using a laser densitometer) in a SDS-polyacrylamide gel with standard curves of y, 8, and BSA of known concentration analyzed in the same gel (the relative staining intensities per microgram of y, 0, and BSA were all within 30%). The concentration of ATP was determined by absorbance at 259 nm (ezb9 = 15,400 M" cm").
DNAs"M13mpl8 ssDNA and 6x174 were phenol-extracted from phage purified by two successive bandings (one downward and one upward) in cesium chloride gradients as described (23). DNA oligonucleotides were synthesized on an Applied Biosystems 381A DNA synthesizer. The M13mp18 ssDNA was primed with a DNA 30-mer (map position 6817-6846) as described (17). MP13mp18 form I DNA was purified by banding twice in cesium chloride equilibrium density gradients (24).
The DNA 317-mer was excised from wild type M13 ssDNA as follows. Two DNA 15-mers were hybridized to M13 ssDNA at the Sau96I site and a ClaI site (map positions 5719-5733 and 6035-6049, respectively). The two DNA 15-mers (110 pg each) were annealed to 486 pg of M13 ssDNA in 540 p1 of 10 mM Tris-HC1 (pH 7.5), 10 mM MgCl,, 1 mM DTT by heating to 95 "C and slow cooling to room temperature. Both Sau96I (240 units, 60 pl) and ClaI (300 units, 60 pl) were incubated with the DNA for 18 h at 37 "C, after which agarose gel analysis showed complete digestion by both enzymes. The digest was quenched with 12 pl of 10% SDS. The 317-mer was resolved from the large M13 DNA remnant and the DNA oligonucleotides by gel filtration over a 25-ml fast protein liquid chromatography Superose 6 column in 10 mM Tris-HC1 (pH 7.5), 1.0 mM EDTA, 100 mM NaCl.
The 317-mer (4 pg/ml) was primed using a 20-fold molar excess each of a synthetic DNA 40-mer (map position 5753-5792) and a synthetic DNA 30-mer (map position 5975-6004) as described (17) followed by gel filtration to remove excess DNA oligonucleotides.
Poly(dA) .oligo(dT) was prepared by annealing oligo(dT)20 with ( d A ) , , ( vents the 3'-5' exonuclease activity in the e subunit of the ae polymerase from digesting the DNA oligonucleotide primer. A pulse of DNA synthesis was initiated upon rapid addition of dATP and [a-'*P]TTP to 60 and 20 p~, respectively, then quenched after 20 seconds by spotting onto Whatman DE81 filters. The radiolabel incorporated into M13mp18 DNA was quantitated as described (25).

The y Complex Is a DNA-dependent ATPase Stimulated by
@-In the absence of DNA, neither the y complex (Table I), , nor a mixture of the two (Table I) demonstrated ATPase activity. This lack of ATPase activity in the absence of DNA is consistent with previous studies, which showed the holoenzyme hydrolyzes ATP upon binding to a singly primed G4 bacteriophage ssDNA genome coated with the E. coli SSB protein (17). Therefore, we added primed DNA to the ATPase assays. To mimic templates known to serve as substrates for the holoenzyme we used ssDNA of natural sequence and coated it with SSB. Since only a few molecules of ATP are hydrolyzed per primed DNA in the holoenzyme initiation reaction (17), we increased the primer density of the substrate by doubly priming a 317-mer ssDNA excised from the M13 genome. The y complex displayed ATPase activity in the presence of the primed SSB-coated 317-mer ( Fig. 1) and even showed slight ATPase activity with the unprimed 317-mer (Fig. 1). The ATPase activity was not likely a contaminant in the y complex preparation as indicated by the coincidence of thermal inactivation rates of y complex ATPase activity and y complex replication activity (with @ and at) (Fig. 2). Furthermore, ATPase activity was present in every y complex preparation and comigrated with the y complex during its purification (not shown). The y complex hydrolyzed dATP at a rate similar to that for ATP and was unable to hydrolyze any of the other (deoxy)ribonucleoside triphosphates (Table 11). This is the same nucleoside triphosphate specificity (ATP and dATP) as displayed by the holoenzyme in initiation of processive DNA synthesis (4).
The / 3 subunit showed no ATPase activity on the primed 317-mer ( Fig. 1) or on the unprimed 317-mer (not shown).
However, addition of /3 to the y complex resulted in approximately 3-fold more ATPase activity (Fig. 1). We presume the p subunit stimulated the ATPase activity of the y complex.
However, it remains possible that a latent ATPase activity intrinsic to p becomes active upon interaction with the y complex and DNA. The p stimulation of the y complex ATPase was specific to the primed 317-mer ( Fig. 1); p did not stimulate the y complex ATPase on the unprimed 317-mer ( Fig. 1). Stimulation by ,6 of the y complex ATPase was observed over a wide range of y complex concentration (over a 60-fold range, not shown).
Steady state analysis of the y complex ATPase on the primed 317-mer showed p elevated the V, , , of the ATPase (from 25 to 80 mol of ATP hydrolyzed/min/mol of y complex) and increased the K,,, for ATP 2-fold (from 26 to 49 p~) (Fig.   3). The y complex ATPase was as active at 2 nM primed 317mer as at 20 nM primed 317-mer (not shown). Hence the K, of the y complex ATPase for primed DNA is lower than 2 nM. The concentration of 3' primer ends used in these studies was above 15 nM. Various nucleic acids were examined for their ability to induce the y complex ATPase (Table I). Although poly(dA) was a poor effector of the y complex ATPase, both poly(dA). oligo(dT) and oligo(dT) were good effectors, suggesting the importance of 3' ends whether hybridized to ssDNA or not. Linear duplex DNA (RFIII) was also an effector of the y complex ATPase. Unprimed ssDNAs of natural sequence were all effectors of the y complex ATPase. Secondary structure within ssDNA was probably the inducer of the y complex ATPase since coating natural sequence ssDNAs with SSB diminished their effectiveness to near the level of poly(dA).
Addition of SSB to the primed 317-mer resulted in a %fold enhancement of y complex ATPase. (However, SSB did not enhance y complex ATPase activity on poly(dA).oligo(dT). Perhaps on natural sequence ssDNA the SSB prevents some y complex molecules from interacting nonproductively with the DNA.) RNAs were not effectors of the y complex ATPase.
The p subunit stimulated the y complex ATPase on poly(dA).oligo(dT), but not on poly(dA) alone or oligo(dT)  FIG. 2. Thermal inactivation rates of they complex ATPase and replication activities. The y complex (440 ng) was incubated at 45 "C in 80 pl of 20 mM Tris-HC1 (pH U ) , 0.1 mM EDTA, 2 mM DTT, and 20% glycerol. At the times indicated, 5 p1 was withdrawn and assayed for ATPase activity at 37 "C on poly(dA) .oligo(dT) (circles) as described under "Materials and Methods," and 0.5 p1 was assayed for replication activity (squares) as described under "Materials and Methods" except for use of 2.2 pg of LYE, 17 ng of @, and a 2min incubation at 37 "C before the 20-s pulse of DNA synthesis. "The level of detection in these assays was 0.1-0.3 mol of P, released/mol of y complex/min. subunits and yx$ complex were assayed alone and in combinations in the presence of poly(dA)-oligo(dT) (Fig. 4A). The individual y, 6, and 6' subunits were not ATPases (although use of a large amount of y in the assay showed slight DNAdependent ATPase activity'). In earlier studies we showed a y6 complex could be reconstituted upon mixing the y and 6 subunits (20). The y6 complex was active in reconstituting a processive polymerase with the at and p subunits (20). We also identified a DNA-dependent ATPase activity within y6 (26). The ATPase activity of y6 is characterized in Fig. 4. The y6 ATPase was most active on poly(dA) .oligo(dT) (Fig. 4 A ) The DNA-stimulated ATPase in the y preparation (99% pure) was only 0.05 mol of ATP hydrolyzed/mol of y/min on poly(&). oligo(dT); no ATPase activity was observed on poly(&). Its identity as y was suggested by comigration with y upon gel filtration and during salt gradient elution of Mono Q, heparin-agarose, and Afii-Gel Blue A columns. The ATPase was not contaminating y complex (as Mono Q resolves y complex from y) or 7 (which resolves from y on heparin). @ did not stimulate the putative y ATPase.
but was nearly as active on oligo(dT) (Fig. 4C). In the absence of DNA as well as in the presence of poly(dA) the ATPase activity of the y6 was negligible (Fig. 4, B and 0).
In our earlier work we found that 76' was not active in reconstituting a processive polymerase with the a€ and p subunits (20). Hence, we were somewhat surprised to find ATPase activity upon mixing y with 6' (6 and 6' are distinct polypeptides'). The 76' ATPase was most active on poly(dA). oligo(dT) (Fig. 4A), about one-third as active on oligo(dT) (Fig. 4C), and was without activity on poly(&) (Fig. 4B) and in the absence of DNA (Fig. 40).
The yx$ complex exhibited very slight DNA-dependent ATPase activity (Fig. 4A), although the preparation has not been studied for a slight ATPase contaminant (i.e. the yx$ preparation is limited in availability). Nevertheless, significant ATPase activity was only gained upon mixing the yx$ complex with either 6 or 6' (Fig. 4A). Also, mixture of both 6 and 6' with yx$ produced more ATPase activity than summation of yx$6 and yx$6', indicating formation of a yx$66' complex (Fig. 4A). The reconstituted 5 subunit y complex was approximately 1.4 times more active than y66', indicating the x$ subunits (also distinct genes)3 stimulate the ATPase activity of y66' or have ATPase activity of their own. The ATPase activity of the reconstituted y complex on poly(dA). oligo(dT) was approximately 30% more active than the y complex purified intact (Fig. 4A). This is within experimental error of the protein concentration measurements.
The ATPase activities of all the subunit combinations were as low on poly(dA) as in the absence of DNA (Fig. 4, B and 0, respectively). Oligo(dT) induced nearly the same level (-7040%) of ATPase activity as poly(dA)-oligo(dT) (Fig.  4C). The exceptions were the 76' and yx$6' ATPases which were only 20-30% as active on oligo(dT) relative to poly(dA). oligo(dT). Perhaps y6 interacts with ssDNA ends more efficiently than 76'. p Specifically Stimulates the ATPase Activity of the y6 Subassembly-The 766' ATPase was stimulated by @ (Fig.   5A) much like p stimulated the y complex (Fig. 1). Hence, the x+ subunits are not required for p to stimulate the y complex ATPase. The p stimulation of the 766' ATPase was specific to the poly(dA). oligo(dT) template and was not supported by poly(dA) alone or by oligo(dT) alone (Fig. 5A). Furthermore, / 3 did not stimulate the 76' ATPase on any of these DNA substrates (Fig. 5B). However, @ stimulated the y6 ATPase 10-fold specifically on the primed template, poly(dA).oligo(dT) (Fig. 5C). The greater / 3 stimulation of the y6 ATPase was due to the higher amount of @-independent ATPase in y66'. The / 3 subunit affected the yx$6, yx$6', and yx$66' ATPases in similar fashion as the 7 6 , yb', and $6' ATPases, respectively (not shown). Furthermore, 6 did not stimulate the low level ATPase of yx$ or of y (not shown). Nor did @ reveal an ATPase upon mixture with 6, b', or 66' (not shown).  cessive polymerase with /3 and the at polymerase (20). The 76' was not active in the reconstitution assay with /3 and at (20). We have found here that y6' is a DNA-dependent ATPase and 6' stimulates the y6 ATPase. This led us to examine whether the 6' subunit would stimulate y6 in the replication assay with @ and ae. To test this, the y6, y6', y66', and y complex were individually titrated into reactions containing @, at polymerase, and primed M13mp18 ssDNA coated with SSB (Fig. 6). The subunits were preincubated with the primed DNA to allow reconstitution of the processive polymerase followed by a 20-s pulse of DNA synthesis. Previous studies have shown that even the minimal polymerase, reconstituted using 76, @, and a c , is fully processive and yields completed duplex circles within 20 s (20). The y6 was active in reconstituting a processive polymerase with @ and at; the 76' was inactive (Fig. 6 ) , consistent with the previous studies (17). However, the y66' subassembly was much more active than y6 in the reconstitution assay (Fig. 6). In fact, y66' was as effective as the entire y complex. Hence 6' stimulates both the ATPase activity and the replication activity of y6. The greater amount of DNA circles replicated relative to the amount of y complex (and y66') added is explained by the M13mp18 ssDNA was preincubated with m , p, and y complex (or subassembly) for 8 min to allow reconstitution of the processive polymerase prior to initiating a 20-9 pulse of synthesis. Assays contained either y complex (circles), y66' (closed squares), yb (triangles), or 76' (open squares). The molar amount of subassembly added to the reaction was taken as the total femtomoles of 6 added to the assay. In the case of yb' the ferntomoles of 6' were used. In the case of y complex the femtomoles of 6 were calculated assuming one 6 subunit per 200-kDa y complex as determined in Ref. 21. catalytic ability of the y complex to assemble multiple @ clamps on primed DNA circles (33). Once on DNA, the @ clamp and at polymerase are fully capable of processively replicating the primed DNA circle within 20 seconds (33). Hence, this assay actually measures the efficiency of y complex subassemblies in ability to transfer @ onto primed DNA.

DISCUSSION
The studies of this report show the y complex is a DNAdependent ATPase. The best DNA effector was primed DNA of natural sequence and coated with SSB. The @ subunit showed no detectable ATPase with or without DNA. However, the y complex and primed DNA yielded approximately 3-fold more ATPase activity in the presence of @ than in its absence. Presumably, /3 stimulated the ATPase activity of the y complex, although the possibility that @ becomes an ATPase in the presence of the y complex and primed DNA can not be ruled out. Study of the y, 6,6' subunits and y x J . subassembly of the y complex showed none of these alone were significant ATPases. However, mixing experiments showed both y6 and y6' were DNA-dependent ATPases. Only the y6 ATPase was stimulated by @. Furthermore, only y6 was active in reconstitution of the processive polymerase with @ and at, although 6' stimulated y6 in the replication assay.
These results suggest the function of the y complex is to recognize DNA and couple ATP hydrolysis to clamp @ onto the DNA as shown in Fig. 7. In the first diagram of Fig. 7, the y complex is shown on the ss/dsDNA-primed junction with both ATP and @ bound to it. Evidence for ability of the y complex to bind ATP and primed DNA is its ATPase activity which depends on DNA and is maximally stimulated by primed DNA. Evidence the y complex on primed DNA binds @ is the stimulation of the y complex ATPase by @ in the presence of primed DNA. These experiments do not address whether the y complex first binds DNA and then p, or whether the y complex first binds @ and then DNA. These early steps are under investigation. Furthermore, the natural primed template for the y complex is a RNAaDNA heteroduplex made by primase during replication of the lagging strand. Thus, studies of the exchange of an RNA primer terminus from primase to the y complex are necessary to further understand the role of the y complex in lagging strand DNA synthesis.
In the second diagram of Fig. 7, the y complex hydrolyzes ATP to clamp @ onto DNA. The subunit that binds the ATP coupled to the @ clamp reaction must be y, 6, or @ since only these three are needed to form the preinitiation complex (20). The apparent lack of ATPase activity in @ implies either y or 6 is the ATP-binding subunit. It is known that y binds ATP tightly, making y a favorite candidate (30). Similar studies of 6 are precluded by the inability to obtain large amounts of 6. However, ultraviolet light cross-links ATP to both y and 6 subunits in the holoenzyme (28). Hence, unambiguous assignment of the subunit which binds the ATP to clamp @ to DNA will require further study. Previous studies have shown the holoenzyme binds ATP (3) but does not hydrolyze it until it is presented with a primed template (5). Hence, the ATP hydrolysis step is at the point of the ternary complex of DNA. y complex e @ and not at an earlier stage requiring only one or two of the component^.^ After transfer of p to DNA, the y complex dissociates from the paDNA clamp freeing it to transfer yet other @ molecules to DNA (2, 11,33). The @ clamp slides freely along duplex DNA (33). The p sliding clamp binds the core polymerase, thereby tethering it to the template for highly processive DNA synthesis (33).
The y complex and @ accessory proteins of the holoenzyme are functionally analogous to the accessory proteins of bacteriophage T4 polymerase and human polymerase 6. The phage T4 DNA polymerase (gene 43 protein) has three accessory proteins which utilize ATP and confer processivity onto Further evidence that ATP is coupled to formation of the preinitiation complex only at the point of the ternary complex of 0. y complex.DNA as indicated by the following experiment. ATP was incubated with either 1) y complex and primed M13mp18 ssDNA, 2) 0 and primed M13mp18 ssDNA, or 3) y complex and p. Then the ATP was removed using hexokinase and glucose. Upon addition of the missing component (either 0, y complex, or DNA) as well as core, dCTP, dGTP, [a-"'PldTTP, and dAMP-PNP (a dATP analogue which supports DNA synthesis but not the holoenzyme initiation reaction (Ref. 5)) no replication of the M13mp18 ssDNA was observed. Hence the ATP was not utilized in the preincubation of two out of three components prior to hexokinase treatment. Negative results were also obtained when the experiment was performed by preincubating just one component with ATP followed by removal of ATP then addition of the other components. In the positive control, preincubation of all the components (p, y complex, and primed M13mp18 ssDNA) with ATP, followed by removal of ATP, yielded dGTP, [a-"PIdTTP, and dAMP-PNP.
replication of the M13mp18 ssDNA upon addition of core, dCTP, the polymerase (31). The phage T4 gene 44/62 accessory protein complex, like 76, is a DNA-dependent ATPase stimulated by 3' ends and is further stimulated by the T4 gene 45 accessory protein (like @) (13). The T4 system appears to lack the ATPase analogous to y6' and the proteins analogous to x and $. Likewise the human 6 polymerase is stimulated in processive synthesis by its accessory proteins, the multiprotein activator 1 (RF-C complex), and the PCNA protein (14,32). Activator 1 (RF-C), like the y complex, has five subunits (15), is a primed DNA-stimulated ATPase, and is stimulated additionally by PCNA (accessory protein with analogous function to @) (14,15). The apparent conservation in function of polymerase accessory proteins spanning the spectrum from E. coli to humans suggests the basic solution to problems of replicating duplex DNA have gone unchanged throughout evolution.