Purification and Properties of the Escherichia coli Deoxyribonucleic Acid-unwinding Protein

The DNA-unwinding protein from Escherichia coli has been purified to homogeneity. It is a single polypeptide of 22,000 daltons; the native molecular weight is 90,000. The effect of the protein on the activity of the three DNA polymerases of E. coli has been studied. The activities of DNA polymerases I and III are significantly reduced, whereas DNA polymerase II activity is enhanced in the presence of unwinding protein. The rate of transcription catalyzed by E. coli RNA polymerase in the presence of the protein is reduced when single-stranded, but not double-stranded, DNAs are employed as templates. Using fd DNA as template in a DNA synthetic reaction that is dependent on both RNA polymerase and DNA polymerase II, the unwinding protein was found to be essential for the synthesis of a DNA product that is equal in size to the template. With the use of crude cell-free extracts of E. coli, it was shown that DNA polymerase II can convert bacteriophage fd DNA to the replicative form. These experiments suggest a possible physiological role for DNA polymerase II and the unwinding protein of E. coli.


SUMMARY
The DNA-unwinding protein from Escherichia coli has been purified to homogeneity.
It is a single polypeptide of 22,000 daltons; the native molecular weight is 90,000. The effect of the protein on the activity of the three DNA polymerases of E. coli has been studied.
The activities of DNA polymerases I and III are significantly reduced, whereas DNA polymerase II activity is enhanced in the presence of unwinding protein.
The rate of transcription catalyzed by E. coli RNA polymerase in the presence of the protein is reduced when single-stranded, but not double-stranded, DNAs are employed as templates.
Using fd DNA as template in a DNA synthetic reaction that is dependent on both RNA polymerase and DNA polymerase II, the unwinding protein was found to be essential for the synthesis of a DNA product that is equal in size to the template.
With the use of crude cell-free extracts of E. coli, it was shown that DNA polymerase II can convert bacteriophage fd DNA to the replicative form. These experiments suggest a possible physiological role for DNA polymerase II and the unwinding protein of E. coli.
Proteins that bind strongly to Di\'A have been isolated from several sources, including bacterial (l), phage-infected bacteria (2-4), and mammalian cells (5). These proteins have in common the ability to reduce the denaturation temperature of DNA, presumably by binding to what would otherwise be transient single strands and preventing them from renaturing.
So evidence for the binding of these proteins to completely doublestranded DNA has been reported.
Furthermore, these proteins cause a DNA strand to exist in an extended, fully hyperchromic form (6). In addition to this interaction with DNA, some of these proteins have a specific effect on the rate of DNA synthesis * This work was supported in part by Grant 5-ROl-GM"0363-02 from the National Institutes of Health and by Grant B36649 from the National Science Foundation.
1 The description of this protein has been previously reported (1). For brevity, the "DNA-unwinding protein" will be referred to as "binding protein." catalyzed by certain DNA polymerases.
In particular, the bacteriophage T4-gene 32 protein interacts with the T4-induced DNA polymerasc (7) ; the bacteriophage Tli-induced binding protein stimulates the T7-induced DNA polymerasc (4), and the Escherichia coli binding protein stimulates E. coli DNA polymerase II (1). Heterologous systems such as the T4-gene 32 protein and the E'. coli DNA polymerases or the E. coli protein and E. coli DNA polymerases I and III show no such stimulation and in general are slightly inhibitory.
The reported purification (1) of the E. coli protein resulted in a preparation that was contaminated Ivith exonuclease I (8, 9) and with ribonuclease H (a lluclease that degrades the RNA of RXADNA hybrids) (10). We report here a modified purification procedure of the protein that results in a homogeneous product free of contaminating nucleascs and which is fully active in its stimulatory properties.
As previously reported (l), the protein stimulates DNA polymerase 11 activity on native I)NA templates previously digested extensively with cxonuclease III. This stimulation has now been shown to be specific for DKv,LI polymerase II under a variety of l)NA synthetic conditions. The activities of DNA polymerase 1 and DNA polymerase III are reduced under all conditions tested. Furthermore, the binding protein inhibits E. coli RNA polymerase-catalyzed RNA synthesis transcribed from single strand DNA but has little, if any, effect on transcription from native DNA. III the primary synthetic step of phage fd rep!ication, the single-stranded DNA is converted to double strand form (RF*), a reaction shown to be catalyzed by RNA polymerase and a DNA polymerase (11). The reconstruction of this synthetic step has been attempted with the use of purified RNA polymerase, DNA polymerases, and binding protein.
U'e report here that, in the presence of binding protein, DNA polymerase 11 is capable of performing the SS --f RF conversion.
In experiments in which crude cell-free extracts prepared from mutants defective in each or all of the three known E. co2i I)N;X polymerases were used, this conversion of SS --f RF of fd DNA has been examined.
In the absence of DNA polymerase 111, DNA polymerase 11 has been shown to perform this replicative step. A possible biological role for DN,4 polymerase II, and binding protein, is thus suggested.  x g for 15 min (Fraction II). Fraction II was dialyzed for 12 hours against four changes of 4 liters of 0.02 M Tris-HCl (pH 8.1)-5 mM EDTA.
The precipitate that formed was removed by centrifugation (30,000 x g for 15 min) (Fraction III).

DNA-cellulose
Chromatography-Fraction III was passed through a column (1.5 x 13 cm) of denatured DNA-cellulose at a flow rate of 5 ml per hour.
Fractions (5 ml) were collected and monitored by absorption at 280 nm. The 0.6 M eluate removes most of the low affinity proteins; the 1 M eluate removes most of the exonuclease I and much of the ribonuclease H. The 2 M eluate contains most of the binding protein, together with a protein of molecular weight 65,000 and minor proteins including the above two nucleases.
(Variable amounts of binding protein are found in the 1 M NaCl eluate, depending on the batch of DNA-cellulose. If the amounts are considerable and the polyacrylamide gel electrophoretograms of the 1 and 2 M eluates are comparable, they may be pooled and further purified together (Fraction IV).) DEAE-Sephadex Chromatography-Fraction IV was dialyzed against 0.02 M Tris-HCl (pH 8.1) and applied to a column (1.5 x 20 cm) of DEAE-Sephadex A-50 equilibrated in the same buffer. A linear gradient (200 ml) of 0 to 0.7 M NaCl in buffer was applied, and 1.5-ml fractions were collected and assayed for binding protein and nucleases. A typical elution pattern is shown in Fig. 1. (Use of exonuclease I-deficient E. coli (BW46 or JC7623) results in a change in the elution pattern in that the residual exonuclease I activity elutes between ribonuclease H and the binding protein.
Good separation is, however, still obtained.) The binding protein was pooled (Fraction V) and dialyzed against 0.02 M Tris-HCl (pH 7.4). It is stable for at least 6 months at 0" or when frozen at -70".

Properties of Binding Protein
Yield-The protein has been isolated from wild type E. coli K12 and from mutants lacking DNA polymerase I (H560), recA-cells (3M455), and recB2lrecC22sbcB15 cells (JC7623). The yield o binding protein isolated from the mutants was identical with the yield from wild type cells.
Purity-Under denaturing conditions, the purified binding protein gave a single band on acrylamide gel electrophoresis when stained with Coomassie brilliant blue (Fig. 2). Sodium dodecyl sulfate-acrylamide gels were run as described under "Methods." The stained gel was monitored at 550 nm on a Gilford spectrophotometer equipped with linear transport.
The molecular weight of the protein was determined relative to the standard proteins run in parallel and is shown in the inset.

Molecular
Weight-The protein has an apparent mass of 22,000 daltons, as judged by electrophoresis (Fig. 2) on sodium dodecyl sulfate-acrylamide gels. Sedimentation through glycerol gradients under non-denaturing conditions resulted in a major peak of activity sedimenting with an apparent mass of 90,000 (~10,000) daltons (Fig. 3). Assuming that all markers as well as the protein are the same shape, the molecular weight of 90,000 for the native protein is equivalent to a tetramer of the 22,000-dalton subunit. This sedimentation behavior of the native protein has been observed in the concentration range of 75 to 750 pg per ml. The tetramer is also stable in the absence of MgClz and in the presence of 0.1 M KC1 (data not shown). Up to a concentration of 750 pg per ml of binding protein, there is no evidence of the existence of higher (greater than tetramer) aggregates, whereas binding protein activity is observed at positions approximating to the dimeric and monomeric forms (see Fig. 3).

II
Effect of temperature on activity of DNA polymerases I and II Reaction conditions are those described (27), except that 4 nmoles of poly(dA) and 2 nmoles of (pT)lo were employed.
The values shown represent the percentage of activity relative to "gapped" DNA at 25", obtained after 5 min of incubation, and reflect initial rates of reaction. The binding protein appears to be free of detectable nucleolytic activity as assayed under conditions described under "Methods." There was no change in the sedimentation profile of 35 S polio virus [32P]RNA (300 cpm per pmole, 3 nmoles used) nor in that of F22 13H]DNA (native or denatured) (30 cpm per pmole, 2 nmoles used) when incubated for 1 hour at 37" in the presence of binding protein of equal or IO-fold greater weight. In addition, under the same conditions, there was no release of acid-soluble radioactivity from these templates. The purified protein is also free of detectable ribonuclease H activity, assayed under standard conditions. E$ect on DNA Polymerase Activity-The binding protein stimulation of DNA polymerase II in the standard assay would appear to be due to the reversal of a conformational change in the poly(dA) which occurs at low temperature, as poly(dA)-(PT)~~ at a base ratio of 2:l is a good template at 25" (Table II). DNA polymerase II has in fact a &lo of 250 on poly(dA)-(PT)*~ between 25' and 15", whereas with "gapped" DNA (20) it has Qlo of 3. DNA polymerase I does not show this phenomenon and has a more normal &lo over this temperature range. Addition of the binding protein causes a 20-to 50-fold stimulation of activity at the low temperature ( Fig. 4). Excess of the binding protein over that required to give maximal activity has no further effect. No inhibition was observed even with a 5fold excess of protein.
In contrast to the effects on DNA polymerase II, binding protein has only slight effects on the activity of DNA polymerase I, slightly stimulating at subsaturating levels and slightly inhibiting activity when in excess. DNA polymerase III had no activity onpoly(dA)-(pT)lo under these conditions; the binding protein did not render the template active.
The principal effect of the binding protein in its stimulation of DNA polymerase II activity would appear to be on the DNA template, for maximal stimulation is achieved at a fixed DNA to protein ratio and is independent of polymerase concentration (Fig. 4). This is in keeping with the results reported for the properties of the bacteriophage T7-induced binding protein (4).
In order to measure directly the effect of the binding protein on DNA synthesis catalyzed by DNA polymerase III, it was necessary to use a DNA which was active as a template.
Exonuclease III-treated bacteriophage P22 DNA was employed. The template activity of exonuclease III-treated duplex DNA is dependent on the amount of digestion allowed, as neither DNA polymerases II nor III can repair long, single-stranded regions effectively, even at 37" (27). DNA polymerase I activity is inhibited by 45'9',',, and DNA polymerase III activity by 5Ooj, at a comparable level of binding protein.
This inhibition of DNA polymerase I activity was not observed when poly(dA)-(pT)lo template (Figure 4) was used. Effect on RNA Polymerase Actitity-The primary effect of the binding protein on E. coli RNA polymerase activity is to inhibit the rate of transcription from single-stranded DNA.
As is seen in Fig. 6, RNA polymerase activity on fd DNA is inhibited by 50% at a weight ratio of binding protein to DNA of 8: 1, an amount sufficient to saturate the DNA (1). In contrast, transcription from duplex P22 DNA is only slightly affected at this level. The inhibition would appear to be on elongation of the RNA product.
Sucrose gradient analysis of the fd DNA-directed product (Fig. 7) shows that the RNA of high molecular weight (227 S) is selectively prevented from accumulating when   7. Sucrose gradient analysis of the RNA products synthesized in the presence and absence of binding protein.
The reaction was performed, as described under "Methods," for 30 min at 37". Binding protein (0.65 pg) was employed (a ratio of protein to DNA of 7:l (w/w)). Centrifugation was for 3 hours at 15" in an SW 50.1 rotor at 45,000 rpm. Fractions were collected from the bottom onto filter paper discs, dried, washed four times with 50/, trichloroacetic acid and then with ethanol (twice), dried, and counted.
The arrow marks the position of an internal fd ["C]DNA marker.
O-O, RNA synthesized in the absence of binding protein; m---m, RNA synthesized in the presence of binding protein.
binding protein is included in the reaction (the products of the latter reaction sediment with an X value of 4 to 8).
Effect on RNA-Primed DNA Synthesis-DNA synthesis on single-stranded DNA initiated with an RNA primer has been reported for all three DNA polymerases of E. coli (28,29), but there was no characterization of the reaction products other than acid insolubility.
As the binding protein has a marked effect on both DNA polymerase and RNA polymerase activities, it was of interest to determine its effect on DNA synthesis that is dependent on RNA synthesis for primer formation. Table  III shows the general properties of the DNA synthetic reaction which is dependent on the presence of RNA polymerase and the four ribotriphosphates as well as a DNA polymerase. As reported by Hurwitz et al. (28), there was little synthesis by DNA ing amounts (protein to nucleic acid, 7:l (w/w)).

Conditions
Complete system + DNA polymerase I. .
- Reactions were as described under "Methods" and were for 1 hour at 37" after DNA polymerase addition.
polymerase III, even in the presence of the binding protein, and this reaction has not yet been studied further.
DNA polymerase II showed considerable activity in an RNA-primed reaction and was stimulated about 3-fold when optimal amounts of binding protein were added.
Conversely, DNA polymerase I was inhibited a-fold by the binding protein at the same concentration. Whether this inhibition is due to direct inhibition of DNA polymerase activity or to the inhibition of RNA primers, or both, is not known.
Similarly, the stimulation of DNA polymerase II activity may be a combination of primer inhibition and a large enhancement of DNA synthesis. Fig. 8 shows the rate of DNA polymerase-catalyzed synthesis in the presence of RNA polymerase and binding protein.
DNA synthesis is maximal when the template DNA is saturated with binding protein at a protein to DNA ratio of 8:l (w/w) (1). Higher concentrations of binding protein cause an inhibition in the rate of DNA synthesis. Since inhibition of DNA polymerase II activity was not noted when a primer was provided, the inhibition observed in the coupled reaction is therefore presumably due to a reduction in the number of RNA primers available for DNA synthesis. DNA polymerase I activity in the coupled system was inhibited over the complete concentration range of binding protein tested. In addition to the quantitative effects of binding protein on the DNA and RNA polymerase-coupled system, it was of interest to examine the nature of the DEA product synthesized in the presence and absence of binding protein.
Sucrose gradient analysis of the DNA polymerase II-catalyzed product generated in the coupled system is shown in Fig. 9a. A much greater proportion of the product is of unit size3 when synthesis proceeds in the absence of the binding protein.
The DNA polymerase Icatalyzed product (Fig. 9b) appears to be independent of the presence of binding protein, even though the latter caused a 70% reduction in the rat.e of incorporation.
Further characterization of the RNA polymerase-DNA polymerase II-catalyzed reaction product revealed that the unit length material is composed of a DNA chain containing a small percentage of randomly inserted ribonucleotides.
Unit size reaction product isolated from the sucrose gradient (Fig. 9a) was centrifuged to equilibrium in a neutral Cs&O4 density gradient.
The DNA product had a density equal to that of purified DNA isolated from bacteriophage fd. Further characterization of this material revealed, however, that it was not composed of pure DNA.
Alkaline sucrose gradients of the reaction product did not reveal the presence of any unit length material (a similar reduction in size of the reaction product, following alkaline treatment, was also noted with the DNA polymerase I-catalyzed material). These results suggest that, although the product is predominantly DNA, a small number of ribonucleotides were present in the product.4 3 "Unit size" refers to the size of the product expected if one complete complement of the template is synthesized. 4 We have considered the possibility that the neutral sucrose gradient analysis as well as the buoyant density analysis could be 6095 The pooled product was dialyzed and centrifuged to equilibrium in a CstSOd gradient. The material banding at the density of fd DNA was pooled, dialyzed, and subjected to alkaline digestion (0.3 M KOH, 37", for 18 hours). The product was neutralized with HClO, and the supernatant was subjected to paper electrophoresis at pH 3.5 in pyridine acetate buffer. Ribomonophosphates were detected by internal optical density markers, cut out, and counted.
Moles of unit length product were calculated in each experiment by assuming a base composition of fd DNA of 24.4yo dA, 19.9% dA, 21.7% dC, and 34.1% T (30). To determine whether ribonucleotides, covalently inserted in the DNA chain, caused the alkali lability of the product, &*Plabeled dNTPs were employed for the synthetic reaction.
The unit length product isolated from a sucrose gradient was digested with alkali and the products were subjected to electrophoresis. Table IV shows the results of this experiment.
Transfer of 32P from DNA to ribonucleotides was essentially random, occurring from all four deoxyribonculeotide triphosphates and transferring to all four ribonucleotide monophosphates.
The average chain length of DNA calculated from this base transfer experiment was only 100 nucleotides (and yet was 27 S, or 5000 nucleotides, by sedimentation).
Therefore, there must be a minimum of 100 ribonucleotides inserted in a unit length reaction product, assuming that the ribonucleotides occur in groups of two only. Coupled RNA and DNA Synthesis in Crude Cell-free Extracts-In contrast to the product obtained in a DNA-RNA synthesiscoupled reaction employing purified components, the synthesis of alkali-stable unit-length product from single-stranded fd DNA obtained with the use of crude cell-free extracts has been reported (11,23,24,31,32). One report (23) stated that only an altered form of DNA polymerase 111, polymerase lII*, was effective in catalyzing this reaction. We undertook a study of the crude system in order to determine whether the results (i.e. alkali-labile unit length material) we have obtained are a result of using purified components (perhaps a necessary factor was not included), or whether DNA polymerase II is able to catalyze the conversion of fd DNA SS --+ RF in vitro. consistent with a shorter-than-unit-length product reannealed to the template DNA.
We consider this possibility unlikely since the amount of product synthesized was approximately equal to the amount of template employed in the initial reaction mixture. The internal marker of fd [%]DNA for gradient analysis was added prior to denaturation in an amount equal to one-half of the template employed.
Its sedimentation behavior was the same as that of an external marker of the same DNA.
Under the conditions of sedimentation (i.e. high salt), annealed marker would sediment much more slowly than single-stranded DNA of the same length; this WPS not the case (see Fig. So). We have compared the ability of three bacterial strains to catalyze bacteriophage fd DNA RF synthesis in crude cell-free extracts. The strains are all deficient in DNA polymerase 1; H560 has wild type levels of DNA polymerases II and III, BT1026 contains a temperature-sensitive DNA polymerase III and normal DNA polymerase II, and 10265 is devoid of detectable DNA polymerase 11 and contains also a temperature-sensitive DNA polymerase III (15)(16)(17)32). The three strains are otherwise isogenic. At 20", by employing conditions as described (24), all three strains yield extracts that are equally active in promoting RF formation as measured by the synthesis of unit length, alkali-stable material, and all synthesis is inhibitable by rifampicin. These results are in keeping with previous reports (11,24,31). When synthesis is carried out at 38", only extracts from H560 are completely active. The rate of synthesis seen in extracts of BT1026 is about 50% of the rate seen with H560. Synthesis in extracts of 10265 begins at the same rate as that in extracts of BT1026 but abruptly ceases after 5 min of incubation. These results are summarized in Fig. 1Oa. Analysis of the products formed indicated that, in all cases, unit length material was synthesized after 15 min of incubation. The yield of RF synthesized in H560, BT1026, and 10265 extracts were in a ratio of 1.0:0.3:0.03, respectively.
It therefore appeared that the presence of DNA polymerase II in extracts of were added to each extract before incubation (1 unit catalyzed the incorporation of 1 nmole of TTP into gapped calf thymus DNA in 5 min at 30").
BT1026 was responsible for 30% of the total product formed in vitro.
To verify this result, we supplemented extracts of strain 10265 with purified DNA polymerases I, II, III, or III*, incubated the extracts at 38", and analyzed the products. In agreement with previously reported results (24), DNA polymerase III* was active in promoting fd-dependent DNA synthesis, whereas DNA polymerase I was not. In contrast to those results, DNA polymerases II and 111 were also active in this reaction (see Fig. lob). Equal activities of the enzymes were employed in all cases, as judged by their synthetic capacity on "gapped" calf thymus DNA.
The amount of DNA polymerase III employed (0.1 unit) is equal to that amount calculated to be present in an equivalent amount of extract prepared from a wild type strain (20). The rates of synthesis observed, in those cases in which activity was enhanced by the addition of polymerase, were identical with and equal to the rate seen in extracts of H560 at 38". Analysis of the reaction product indicated that in all cases the product of the reaction was of unit length and alkali-stable (data not shown). We conclude that DNA polymerase II (as well as DNA polymerases III and III*) is capable of catalyzing fd DNA-RF synthesis in crude cell-free extracts. However, DNA polymerase II-catalyzed fd DNA-RF synthesis can be observed only in the absence of a functional dnaE (32) gene product.

6097
It is therefore suggested that the DNA polymerase II pathway functions as an independent alternative route to fd DNA RF formation.
Finally, because DNA polymerase II requires the binding protein to synthesize high molecular weight DNA, it is not un reasonable to implicate the latt'er protein in this react.ion, too. Therefore, it must be assumed that, in the reconstruction experiments using RNA polymerase, DNA polymeraae 11, and binding protein on id DNA, another component is essential for the synthesis of alkali-stable fd unit length product. DISCUSSIOP; The E. coli DNA-binding protein has been purified to homogeneity and is free of detectable nucleolytic activity.
Its molec ular weight, under denaturing conditions, has been determined to be 22,000.
However, by sedimentation through glycerol gradients under native conditions, the protein has an apparent molecular weight of 90,000. This corresponds to t,hc protein's existing predominantly as a tetramer of the 22,000.dalton subunit, although dimeric and monomeric forms of the protein exist. No evidence for the existence of high molecular weight aggregates has been obtained, as is seen with the bacteriophage T4gene 32 protein (7). All aggregates of the E. coli protein are active in the binding protein assay, but it is not yet known what form of the protein binds to DNA, thereby stimulating DNA polymerase 11 activity.
The properties of the protein with respect to stimulation of the DNA polymerases of E. coli are germerally in agreement with those previously report,ed (I). The protein appears to be specific for DNA polymerase II, stimulating synthesis up to 50.fold. DNA polymerase III was found to be inhibited by the protein, whereas DNA polymerase I was both inhibited or slightly stimulated, depending on the conditions employed.
Whether this stimulation of DNA polymerase I is of physiological significance is unknown; it occurs at subsaturating levels of the protein and may be analogous to the stirnulation of DNA polymerase II by the id gene 5 protein, which provides more template sequences for the polymerase (33). If the DNA is saturated with binding protein, DNA polymerase 1 activity is reduced.
RNA polymerase is also inhibited by the protein, probably on propagation and possibly also on init,iation employing a single-stranded DNA template. Little inhibition was observed on duplex DNA.
The effects of the binding protein on RNA polymerase-dependent DNA synthesis employing single strand DNA as template are complex, as would be predicted from the separate effects of the binding protein on the activities of RNA polymerase and DNA polymerase Il. The synthesis of a primer oligoribonucleotide is essential for in vitro DNA synthesis using purified enzymes and a circular, single strand DNA template.
Since the presence of binding protein inhibits the elongation of RNA chains, it may reduce the length of the potential primer to such an extent that it cannot be utilized by a DNA polymerase, thereby inhibiting DNA synthesis. Presumably, this is the mechanism by which DNA polymerase II activity is reduced by the presence of the binding protein, a phenomenon not observed when both template and primer are provided.
Once DNA synthesis has been initiated, the binding protein should have the same effects on the activities of DNA polymerases I and II as were observed on bacteriophage P22 DNA which had been extensively degraded with exonuclease III, i.e. those of inhibiting DNA polymerase I activity but of stimulating DNA polymerase II activity.
The effects therefore of the binding protein on RNA-lyzed synthesis severely (inhibition of both RNA and DNA syn thesis) but to give an over-all stimulation of DNA polymerase II-catalyzed synthesis (a combined inhibition of RNA primer synthesis but stimulation of DNA synthesis) at concentrations of the protein up to saturation (protein to nucleic acid lO:l, w/w) of the DNA.
At higher concentrations of binding protein, the synthesis of RNA primers becomes limiting and there is a reduction in the over-all incorporation of nucleotides, even by DNA polymerase II. The effects of the binding protein on DNA polymerase II activity are not only stimulation of the rate of synthesis but also alteration of the size of t'hc reaction product (which may be up to at least 5000 nucleotides in length), whereas DNA polymerase II alone is able to synthesize products of only 50 to 100 nucleotides (27). The unit length reaction product synthesized in the presence of the binding protein using fd DNA as template was not, however, pure DNA, as ribonucleotides were inserted at random in the DNA chain, causing alkali lability of the unit length product.
It has not been possible to dernonstrate ribonucleotide incorporation by DNA polymerase II,5 and DNA polymerase 1 requires XI@ for this reaction (34). RNA polymerase, however, has been shown (35) to incorporate ribonucleotides onto DNA, and presumably this reaction was functional in the RNA polymerase-DNA polymerase-coupled DNA synt,hetic reactions.
The alkali lability of the unit length product is a phenomenon concerned only with the attempted reconstruction using purified components of the reaction leading to fd DNA RF, as in crude extracts alkali-stable unit length material may be observed. We have shown that, in mut,ants defective in the three known DNA polymerases of E. coli, DNA polymerase II, as well as DNA4 polymerases III and III*, is able to catalyze the conversion of id DNA SS ----* RF in crude extracts.
The reason for the discrepancy between this study and that previously reported (23) is not clear. It rnay reflect the difference in the method of in activation of the temperature-sensitive dnaE gene product (temperature inactivation employed in this study versus a freeze-thaw procedure (23)).
It has been reported (31,36) that the DNAbinding (unwinding) protein is required for DNA synthesis on hI13 (fd) DNA in partially fractionated extracts of E. coli. We also believe that the binding protein is essential for DNA polymerase II-catalyzed synthesis of fd DNA RF, as DNA polymerase II itself cannot synthesize a DNA molecule 5000 nucleotides in length.
Given our results with the use of crude cell-free extracts as well as purified enzymes, we believe that DNA polymerase II, as well as DNA polymerase III, is able to catalyze fd DNA RF formation. Furthermore, our results with the use of purified components suggest t'hat, in addition to the RNA polymerase requirement, at least two separate pathways for fd DNA RF formation are possible: (a) DNA polymerase II and DNA-binding protein and (b) DNA polymerase III (with or without binding protein) and other factors (23,31,36).
Even though DNA polymerase II catalyzes RF formation in vitro, it is not clear that it does so in viva. The amount of DNA polymerase II required to achieve normal rates of RF formation in vitro is at least lo-fold higher than its physiological concentration. Furthermore, fd replicative form catalyzed by DNA polymerase II in crude extracts is devoid of interspersed ribonucleotides.
Either the mixed polymer is a normal product from which the ribonucleotides are excised and replaced by deoxyribonucleotides or there exists a mechanism whereby their inprimed DNA synthesis are to reduce DNA polymerase I-cata-6 I. J. Molineux, unpublished observation. corporation is prevented. However, we have shown with purified components that the binding protein is required for the synthesis of DNA polymerase II-catalyzed unit length material. To date, no physiological role for the E. coli binding protein nor for DNA polymerase II is known. 30 mutants which ma) help to elucidate a funct,ion have been isolated.
The protein has been isolated in both normal amounts and activity from recAamber mutants.
It has been shown (37) that the bacteriophage T4-gene 32 protein plays a basic structural role in the production of mature phage and that the bacteriophage fd-gene 5 protein is necessary in noncatalytic amounts for progeny singlestranded DNA synthesis (38). It has been suggested (2) that single-stranded DNA-binding protein might function in viva to unwind the double helix.
However, DNA ploymerase 11 appears dispensable for E. coli DNA replication (17,39), and a major in vitro property of the binding protein is its stimulation of that enzyme.
Thus, the physiological significance of the protein interactions reported here remain to be elucidated.