Processivity of the gene 41 DNA helicase at the bacteriophage T4 DNA replication fork.

The gene 41 protein is the DNA helicase associated with the bacteriophage T4 DNA replication fork. This protein is a major component of the primosome, being essential for coordinated leading and lagging strand DNA synthesis. Models suggest that such DNA helicases are loaded only onto DNA at origins of replication, and that they remain with the ensuing replication fork until replication is terminated. To test this idea, we have measured the extent of processivity of the 41 protein in the context of an in vitro DNA replication system composed of eight purified proteins (the gene 43, 44/62, 45, 32, 41, 59, and 61 proteins). After starting DNA replication in the presence of these proteins, we diluted the 41 helicase enough to prevent any association of new helicase molecules and analyzed the replication products. We measured an association half-life of 11 min, revealing that the 41 protein is processive enough to finish replicating the entire 169-kilobase T4 genome at the observed replication rate of ∼400 nucleotides/s. This processivity of the 41 protein does not require the 59 protein, the protein that catalyzes 41 protein assembly onto 32 protein-covered single-stranded DNA. The stability we measure for the 41 protein as part of the replication fork is greater than estimated for it alone on single-stranded DNA. We suggest that the 41 protein interacts with the polymerase holoenzyme at the fork, both stabilizing the other protein components and being stabilized thereby.

The gene 41 protein is the DNA helicase associated with the bacteriophage T4 DNA replication fork. This protein is a major component of the primosome, being essential for coordinated leading and lagging strand DNA synthesis. Models suggest that such DNA helicases are loaded only onto DNA at origins of replication, and that they remain with the ensuing replication fork until replication is terminated. To test this idea, we have measured the extent of processivity of the 41 protein in the context of an in vitro DNA replication system composed of eight purified proteins (the gene 43, 44/62, 45, 32, 41, 59, and 61 proteins). After starting DNA replication in the presence of these proteins, we diluted the 41 helicase enough to prevent any association of new helicase molecules and analyzed the replication products. We measured an association half-life of 11 min, revealing that the 41 protein is processive enough to finish replicating the entire 169-kilobase T4 genome at the observed replication rate of ϳ400 nucleotides/s. This processivity of the 41 protein does not require the 59 protein, the protein that catalyzes 41 protein assembly onto 32 protein-covered single-stranded DNA. The stability we measure for the 41 protein as part of the replication fork is greater than estimated for it alone on single-stranded DNA. We suggest that the 41 protein interacts with the polymerase holoenzyme at the fork, both stabilizing the other protein components and being stabilized thereby.
DNA is replicated during bacteriophage T4 infection by a protein complex that is composed entirely of viral proteins. This "protein machine" serves as a universal model for the apparatus that catalyzes DNA replication (1)(2)(3), and it is composed of several proteins that have distinct activities. The gene 43, 44, 62, and 45 proteins combine to form the DNA polymerase holoenzyme. Two molecules of this holoenzyme are required during replication, one on the leading strand and one on the lagging strand. The gene 43 protein is the DNA polymerase while the other three proteins are "accessory" proteins, forming an ATP-dependent sliding clamp that greatly increases the processivity of the polymerase (4,5). The gene 32 protein binds in cooperative clusters to single-stranded DNA and is required for any replication of double-stranded DNA in the absence of a helicase (6,7).
The DNA helicase associated with the T4 DNA replication fork is the gene 41 protein (7). A ring-shaped hexamer (8), the 41 protein unwinds the double helix ahead of the DNA polymerase on the leading strand, translocating in the 5Ј33Ј direction along the opposite strand (the lagging strand template). The 41 protein promotes DNA synthesis on the leading strand at a rate of ϳ400 nucleotides/s in vitro (9), close to the rate observed in vivo (10). The newest addition to the T4 in vitro DNA replication system, the gene 59 protein, greatly facilitates the loading of the 41 helicase onto 32 protein-covered singlestranded regions of the double-stranded DNA template (9,(11)(12)(13). The T4 gene 41 and 61 proteins together form the primosome. Although the 41 protein is required for the gene 61 DNA primase to recognize its priming sites efficiently (14), it is the primase alone that synthesizes the RNA primer (15)(16)(17).
Gene 41 mutations strongly reduce the amount of DNA replication in the phage-infected cell (18) and eliminate synthesis on the lagging strand (19). DNA helicases that are part of the primosome, including this one, have been shown to stay with a single replication fork for a substantial period before dissociating (20). However, in this report, we present the first quantitation of the processivity of a DNA helicase that is associated with a DNA replication fork, demonstrating that the 41 protein remains with the replication fork long enough to replicate the entire 169-kb 1 T4 chromosome without dissociating.

EXPERIMENTAL PROCEDURES
Reagents, Enzymes, and Substrate DNA-Nucleoside triphosphates were obtained from Pharmacia Biotech Inc., human serum albumin from Worthington, and [␣-32 P]dCTP from Amersham Corp. The bacteriophage T4 gene 32, 41, 43, 44/62, 45, 59, and 61 proteins were purified to homogeneity as described elsewhere (9,21). All proteins were free of detectable nuclease activity. The 44/62 proteins were prepared from T4-infected cells, whereas the other proteins were the products of cloned T4 genes expressed on plasmids in Escherichia coli. The purified template DNA used for replication was circular double-stranded DNA, isolated after growing plasmid pJMC110 in E. coli (22).
DNA Synthesis-A specific DNA 3Ј end, produced by treating plasmid DNA with bacteriophage fd gene 2 protein, serves as the primer to start strand-displacement DNA replication on a double-stranded circular DNA template (9,22). Reactions contained assay buffer (33 mM Tris acetate, pH 7.8, 66 mM potassium acetate, and 10 mM magnesium acetate), 1 mM dithiothreitol, 0.1 mg/ml human serum albumin, deoxyribonucleoside triphosphates (0.3 mM each of dATP, dGTP, and dTTP, plus 0.08 mM [␣-32 P]dCTP at a specific activity of 1200 Ci/mol), and ribonucleoside triphosphates (1.25 mM ATP and 0.5 mM GTP). The DNA concentration before dilution was 0.8 g/ml (0.26 nM plasmid). Unless otherwise noted, the 32 protein concentration was 40 g/ml and the concentrations of the DNA polymerase holoenzyme components were: 3 g/ml 43 protein, 16 g/ml 44/62 proteins, and 10 g/ml 45 protein. The gene 41, 59, and 61 proteins were used at the concentrations specified in each experiment. In reactions containing the 61 protein, 0.25 mM UTP and CTP were added. * This work was supported in part by National Institutes of Health Grant GM24020. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Alkaline Agarose Gel Electrophoresis-DNA synthesis was stopped by the addition of alkaline electrophoresis loading buffer to produce a final concentration of 8 mM NaOH, 20 mM Na 3 EDTA, 10% w/v sucrose, and 0.04% w/v bromcreosol green. The radioactive DNA products were analyzed by electrophoresis through alkaline agarose gels, as described elsewhere (23). Following electrophoresis, gels were soaked in two changes of 7% trichloroacetic acid for 30 min each to remove residual [␣-32 P]dCTP. The gels were dried under vacuum onto Whatman DE81 DEAE-cellulose paper and autoradiographed at Ϫ80°C using an intensifying screen. In addition, the dried gels were analyzed using a Phos-phorImager SF with ImageQuant software, IQ 3.3␤ C15 (Molecular Dynamics, Sunnyvale, CA). The sizes of the radioactively labeled DNA strands were determined by comparison with 5Ј-end-labeled 32 P standards (T7 DNA at 40 kb and restriction fragments of DNA of known size).

RESULTS
Our experimental approach to assess the processivity of the 41 protein relies on establishing a moving replication fork in vitro and then diluting the mixture enough to prevent all further 41 protein associations with the DNA. Double-stranded DNA, nicked at a unique sequence by the bacteriophage fd gene 2 protein (22), served as both the primer and template for this DNA replication reaction. Synthesis begins at the 3Ј OH side of the nick. Initially, a double-stranded rolling circle with a 5Ј single-stranded tail is produced by the polymerase holoenzyme in a relatively slow strand-displacement reaction requiring the 32 protein. The 5Ј single-stranded tail then provides a "loading zone" onto which the 59 protein assembles the 41 helicase (9). After a brief period of DNA synthesis in the presence of all of these proteins, the samples are diluted 10-fold into solutions that maintain the original protein and reagent concentrations, except for DNA and the 41 and 59 proteins. Before dilution, the 59 protein very rapidly loads the 41 helicase onto the DNA template, whereas after dilution no more loading occurs. Therefore, by observing the course of DNA synthesis after dilution, we could determine the degree of 41 protein processivity at the replication fork (how long it stays on the DNA without dissociating).
The Gene 41 DNA Helicase at the Replication Fork Is Highly Processive-In our initial experiments, rolling circle DNA replication was performed in the absence of primase (61 protein), so that only leading strand DNA synthesis occurred (Fig. 1A). The template was first incubated with the DNA polymerase holoenzyme and 32 protein for 1 min, and the slowly elongating leading strand was labeled with [␣-32 P]dCTP. This pulse labeling of DNA was terminated by the addition of excess dCTP at the same time that the 41 and 59 proteins were added to begin rapid leading strand synthesis. The rate of replication fork movement was then determined by measuring the increase in the length of the pulse-labeled DNA products with time, using a denaturing agarose gel.
Without the 41 and 59 proteins, replication forks produced DNA on the leading strand at a rate of about 10 nucleotides/s (Fig. 1B, lanes A-C). When the 41 and 59 proteins were present at 5 and 0.3 g/ml, respectively, the rate of the DNA replication fork increased 40-fold to 380 nucleotides/s (Fig. 1B, lanes D-F). This rapid rate of replication was not decreased by a dilution of the 41 and 59 proteins; it even increased slightly to 430 nucle-FIG. 1. Helicase-led replication forks are processive. A, schematic of the assay used to determine helicase processivity. DNA replication is initiated at a site-specific nick in a circular double-stranded plasmid DNA (step 1). The gene 43, 44/62, 45 proteins and the 32 protein (80 g/ml) catalyze a relatively slow strand-displacement reaction that produces a 5Ј single-stranded tail 500-1000 nucleotides in length; during this step the DNA is labeled by the incorporation of [␣-32 P]dCTP (labeled DNA is shown in gray) (step 2). After 1 min, 41 protein, 59 protein, and a 12.5-fold increase of dCTP (1 mM dCTP) were added to the reaction and the 32 protein concentration was lowered to 40 g/ml (step 3). Under these conditions, little additional radiolabel is incorporated into the growing DNA product and the 41 protein is loaded onto the 5Ј single-stranded tail (9). At a 41 protein concentration of 5 g/ml, the molar ratio of 41 protein hexamers:DNA template molecules is 60:1. After 25 s, the reaction was diluted 10-fold (step 4). The dilution mix included each of the components of the original reaction mixture except DNA, with the 41 and 59 proteins either omitted or included. The 32 protein concentration was maintained at 40 g/ml. Aliquots were taken at 25 s intervals after dilution, and the products were run on a denaturing 0.5% agarose gel. B, autoradiograph of the dried gel. The pre-and post-dilution concentrations of the 41 and 59 proteins were as indicated. C, quantitation of data in panel B. The percentage of replication forks that contained a helicase was determined by dividing the radioactivity in the fast-moving band (representing forks with helicase) by the total radioactivity in the lane, as determined with a Phosphor-Imager. Data from lanes D-I are shown; in lanes A-C and J-L no forks with helicase were detected. otides/s (Fig. 1B, lanes G-I). The percentage of replication forks that contained a helicase could be determined by comparing the amount of radioactivity in the fast-moving band (representing forks with helicase) with the total amount of radioactivity in each lane. As shown in Fig. 1C, the percentage of replication forks that contained a helicase after dilution of the 41 and 59 proteins remained constant throughout the 75-s incubation. This result was expected, inasmuch as controls show that none of the slow-moving replication forks can acquire a DNA helicase at the diluted concentration of the 41 and 59 proteins (0.5 and 0.03 g/ml, respectively; Fig. 1B, lanes J-L). We conclude that, once loaded, a 41 protein molecule can function for at least 75 s at a single replication fork without dissociating.
Half-life of the 41 Protein at the DNA Replication Fork-To determine the half-life of the gene 41 helicase at a complete replication fork, we performed a rolling circle replication reaction in which the primase (61 protein) was included, resulting in both leading and lagging strand DNA synthesis. In addition, we needed a way to measure 41 protein association for much longer times after dilution. Since dilution of the 41 and 59 proteins does not decrease the rate of movement of replication forks with a helicase (see Fig. 1), any change in the rate of dCTP incorporation into the leading strand after dilution of these two proteins can be attributed to a slowing of forks due to helicase dissociation. An experiment like that in Fig. 1 was therefore performed, except that after the 10-fold dilution, the dCTP incorporation was measured between 2 and 16 min of incubation. When products were analyzed by electrophoresis through a denaturing agarose gel, the very long leading strand products (Ͼ40 kb) migrated through the gel as a sharp band and were easily separated from any lagging strand products (data not shown). The total amount of [␣-32 P]dCTP incorporated into the leading strand at each time point was quantitated and used to determine the effect of 41 and 59 protein dilution.
As shown in Fig. 2A, without dilution of these proteins (q), the rate of dCTP incorporation was linear for 16 min, revealing that there was no measurable decrease in the number of replication forks with helicase. However, after a 10-fold 41 and 59 protein dilution (Fig. 2, panels A and B; Ⅺ) there was a small but detectable decrease in the rate of dCTP incorporation throughout the 16 min time course. This indicated a very slow decrease in the number of functional replication forks due to a loss of helicase. The rate of fork loss obeyed the first-order rate law: where k ϭ rate constant, t ϭ time after dilution , F ϭ number of forks at a given time. F 0 ϭ initial number of forks at t ϭ 0. If y ϭ total dCTP incorporated into DNA, then where R ϭ rate of fork. After substituting [F] 0 e Ϫkt for [F], integrating, and assuming that at t ϭ 0, y ϭ 0, we find that The equation of the best fit line shown in Fig. 2B fit the following equation: As time t becomes very large, y approaches a maximum incorporation of 3.09 pmol. To find the half-life, we solved the equation for t when y ϭ one half of 3.09 pmol, revealing a half-time for 41 protein association of 11 min at 37°C. Electron Microscopy of Very Long DNA Products after Dilution of the 41 and 59 Proteins-We wanted to prove directly that, once loaded, the helicase-led replication fork is processive enough to replicate the length of the T4 genome (169 kb). We   FIG. 2. The 41 helicase has a half-life of 11 min at the replication fork. DNA replication reactions were initiated and diluted as described in Fig. 1 and under "Experimental Procedures," with the following changes. DNA was continuously labeled after dilution by adding [␣-32 P]dCTP at the time of dilution; the pre-dilution loading time for 41 protein was 1 min; and 61 protein (0.15 g/ml) was added with the 41 and 59 proteins to include lagging strand synthesis. A, the 41 and 59 proteins were maintained at concentrations of 5 and 0.3 g/ml, respectively, after the dilution step (q) or diluted 10-fold to concentrations of 0.5 g/ml and 0.03 g/ml (Ⅺ). The leading and lagging strand products were separated on a denaturing 0.6% agarose gel (see "Experimental Procedures"). The amount of leading strand synthesis in each lane was determined using a PhosphorImager: the quantity of radioactivity present in the leading strand was determined by comparison to a spot of [␣-32 P]dCTP of known activity that was exposed alongside the dried gel. The best fit line for incorporation of dCTP was linear for the reaction in which the 41 and 59 proteins remained undiluted. B, the incorporation of dCTP after dilution of the 41 and 59 proteins (Ⅺ) was exponential with a half-life of 11 min after dilution. Because the initial concentration of 41 protein (5 g/ml) was low enough to require a few minutes for complete 41 protein loading, the number of forks with helicase in the reaction that was not diluted was 3-4 times higher than that obtained with only 1 min of loading time before dilution. performed a DNA replication reaction in which the 41 and 59 proteins were diluted 10-fold, stopped after 8 min, and the samples then immediately spread for electron microscopy. Although these very long DNA products were tangled and prone to breakage, 20% of the molecules had 5Ј double-stranded tails in the range of 165-197 kb (data not shown).
The Gene 59 Protein Is Not Required for the Processivity of the Gene 41 Protein at the Replication Fork-Although the 59 protein has no measurable effect on the rate of replication fork movement once the 41 protein has become engaged (9), the 59 protein could remain associated with the DNA replication fork and affect its processivity. To test this possibility, we raised the 41 protein concentration in our assays to levels that allow some loading of this helicase onto DNA in the absence of the 59 protein. Under these conditions, we were able to load the 41 protein onto only ϳ10% of the 5Ј-tailed templates (Fig. 3, lanes  G-I), in contrast to the Ͼ80% of templates that obtain a 41 protein molecule when 59 protein is present (Fig. 3, lanes D-F). However, when such a DNA replication reaction with 41 protein and no 59 protein was diluted 10-fold, those helicasedriven replication forks present were as processive as those in the undiluted control reaction (Fig. 3, compare lanes M-O with  lanes G-I). These replication forks also move as rapidly and processively as forks formed in the presence of 59 protein (Fig.  3, compare lanes D-F with lanes M-O). We conclude that, as far as can be determined from a 75-s incubation, the 59 protein is not required for the high processivity of the 41 helicase at the replication fork. DISCUSSION In this report we measured the degree of processivity of the 41 protein DNA helicase during DNA replication by diluting this protein after establishing a moving replication fork. We have shown that, at a rate of ϳ400 nucleotides/s and a half-life of 11 min, the 41 protein is processive enough to participate in replicating the entire 169-kb T4 genome without dissociating. In addition, the 59 protein is not needed for the high processivity of the 41 protein. We cannot rule out the possibility that the 59 protein travels with the replication fork, but it seems likely that the most important role for this protein is in the initiation phase of DNA replication, when the protein machine is first being assembled.
DNA helicases that are associated with the replication fork have been found to be processive in other replication systems as well. Richardson and co-workers found that the bacteriophage T7 gene 4 protein, the single polypeptide that is both the helicase and primase in the T7 system, is resistant to dilution in vitro and therefore highly processive (24,25). In E. coli, the replicative helicase is DnaB. Both DnaB and DnaG (primase) are needed to make the primers utilized for Okazaki fragment synthesis on the lagging strand template (20). Marians and co-workers found that limiting concentrations of DnaB did not affect the size of Okazaki fragments, yet limiting the primase increased their size, indicating that the helicase functions processively but the primase does not (26).
Is the 41 protein stabilized by other proteins in the replication machinery? The extent of processivity of the 41 helicase alone translocating along naked single-stranded DNA or 32 protein-covered single-stranded DNA is under debate. A halflife of 1 min was reported by Liu and Alberts, with an estimated translocation rate of 400 nucleotides/s (27). However, Young et al. (28) reported an average distance translocated per 41 protein binding event of only 60 -700 nucleotides. No matter which assessment is correct, the half-life we measure for the 41 helicase of 11 min when it is part of the replication fork is considerably longer than the half-life of the 41 protein on single-stranded DNA. One explanation for the increase in halflife is that the components of the DNA polymerase holoenzyme stabilize the helicase. There is evidence for protein-protein interactions between the polymerase accessory proteins (44/62 and 45 proteins) and the 41 protein. Richardson and Nossal found that the polymerase accessory proteins partially relieved the 32 protein-induced inhibition of primosome-catalyzed pentaribonucleotide synthesis on a single-stranded DNA template (29). Since both the 41 and 61 proteins are needed to synthesize pentaribonucleotides, they reasoned that the accessory proteins were able to load the 41 helicase onto 32-covered singlestranded DNA, albeit not as efficiently as the 59 protein. In addition, they found that a proteolytic fragment of the 41 protein that is missing the C terminus was unable to interact with the accessory proteins (30). The activities of this fragment were normal on DNA in the absence of 32 protein. Barry and Alberts (9) reported supporting results: the 44/62 and 45 proteins increased the 41 protein single-stranded DNA-dependent GTPase activity 4-fold over the activity of 41 protein alone on 32 protein-covered single-stranded DNA.
In addition to interacting with the 44/62 and 45 proteins, the 41 helicase may also interact with the DNA polymerase (43 protein). Spacciapoli and Nossal found that a mutant form of 43 protein (A737V) had an absolute requirement for the 59 protein to maintain replication forks with a helicase (13). They suggested that a domain in the polymerase interacts with the helicase; if this domain is not functional, then the helicase requires the 59 protein for continuous reloading or for stabilization. In addition, there is ample evidence that two 43 protein FIG. 3. The 59 protein is not required for the processivity of the 41 helicase. DNA replication reactions were initiated and diluted as described in Fig. 1 and under "Experimental Procedures" with the following changes to increase the amount 41 protein loaded onto the template. The pre-dilution loading time was 40 s (compared to 25 s); in the absence of the 59 protein the 41 protein concentration was increased to 60 g/ml; and the concentrations of the accessory proteins were increased to 40 g/ml 44/62 proteins and 25 g/ml 45 protein. The preand post-dilution concentrations of the 41 and 59 proteins were as indicated. Aliquots were taken at 25 s intervals after dilution, and the products were analyzed on a denaturing 0.5% agarose gel (see "Experimental Procedures"). molecules are present at the replication fork (2). The simplest model of the replication fork would include a 43 protein dimer. However, sedimentation equilibrium analysis unambiguously shows that the 43 protein exists as a monomer in solution under the conditions used in our in vitro replication experiments. 2 This suggests the 43 protein must interact with DNA and/or other components of the DNA replication machine to hold the two 43 protein molecules at the replication fork.
The interactions of the 41 protein with other replication proteins, together with the increased half-life of the helicase on DNA as part of the replication complex, point to the helicase as a central protein around which the other proteins assemble. The highly processive nature of the 41 helicase makes it a prime candidate for the cornerstone of the replication machinery, perhaps providing a physical link between the two polymerase molecules at the replication fork.