INTRODUCTION

We have shown in the accompanying articles that assembly of the X174-type primosome occurs in discrete steps (1), resulting in a preprimosomal complex on Escherichia coli single-stranded DNA-binding protein (SSB)¹-coated X174 single-stranded circular (ss(c)) DNA that contains five proteins: PriA, PriB, PriC, DnaT, and DnaB (2). The preprimosome, which is capable of both DNA translocation and DNA helicase (3) activities, forms the complete primosome with the association of DnaG as a result of a transient protein-protein interaction with DnaB (4).

Of the proteins present in the primosome, only three, PriA, DnaB, and DnaG, have independent biochemical activities that allow the assignment of a primosomal activity to an individual component. PriA provides the 3  5 DNA translocation (5) and DNA helicase activities (6, 7); DnaB provides the 5  3 DNA translocation and DNA helicase activities (8); and DnaG provides the primase activity (9). The roles of PriB, PriC, and DnaT in primosome function have remained obscure. We report here that our analyses of two mutant PriA proteins has allowed the definition of the role of PriB during primosome assembly.

PriA is a multifunctional protein. It is a primosome assembly site (PAS)-specific single-stranded DNA-dependent ATPase (10, 11) and a 3  5 DNA translocase and DNA helicase (4, 5, 6), and it serves as both the specific recognition factor and scaffold for primosome assembly. We have analyzed the interdependence of these activities by mutational analysis. ATPase mutant PriA proteins can no longer move along DNA but can still serve to assemble the primosome (12). Mutations in the extensive cysteine cluster in the center of the protein (13) uncouple the ATPase and DNA translocation activities (14). Based on our demonstration that priA disruption mutants are partially constitutively induced for the SOS response (15), we have selected temperature-sensitive mutant priA alleles.² Analyses of the activities of the purified mutant proteins revealed the role of PriB during primosome assembly.

Both PriA C439Y and PriA C445Y were defective in X174 ss(c)  replicative form (RF) DNA replication in vitro at 30 °C. We could attribute this defect to an inability of these proteins to form a complex at a PAS with PriB. Moreover, rescue of the activity of these proteins by high concentrations of DnaT correlated with the formation of PriA-DnaT complexes for both wild-type and mutant PriA proteins. This suggested that the major role of PriB during primosome assembly was to facilitate formation of a complex containing both PriA and DnaT.

MATERIALS AND METHODS

All proteins, DNAs, and reagents have been described in the accompanying articles (1, 2). Mutant PriA proteins were prepared as described by Zavitz and Marians (12). The selection procedure for isolating the mutant priA alleles and the characterization of the temperature sensitivity of the purified mutant proteins will be described elsewhere.^2,3

Standard reaction mixtures (15 µl) containing 5-[³²P]PAS304 DNA (1 nM), 50 mM Tris-HCl (pH 8.4 at 30 °C), 10 mM MgCl₂, 10 mM DTT, 50 µg/ml bovine serum albumin, and the indicated amounts of mutant or wild-type PriA proteins were incubated at 30 °C for 10 min. Washing buffer (1 ml of 25 mM Tris-HCl (pH 7.8 at 30 °C), 20 mM KCl, 5 mM MgCl₂, and 1 mM EDTA) was then added, and the mixture was passed at 1 ml/min through nitrocellulose filters (Millipore, HAWP) that had been soaked in 0.5 M NaOH for 45 min at room temperature and then in washing buffer for 45 min at room temperature just prior to use. The filters were then washed three times with washing buffer (1 ml each time) and dried, and the radioactivity retained was determined by liquid scintillation spectrometry. For challenge experiments, the reaction mixtures were first incubated for 5 min at 30 °C, cold competitor DNA was then added, and the incubation continued for an additional 5 min before filtering as above.

Gel Shift, Enhanced Chemiluminescence (ECL)-Western, and X174 ss(c)  RF Analyses

These were as described in the accompanying articles (1, 2) with the following minor changes. Gel electrophoresis to analyze primosomal protein complexes was through a 6.5% (80:1, acrylamide:bisacrylamide) gel at 6 V/cm for 6.5 h using 6 mM Tris-HCl (pH 7.8 at 23 °C), 4 mM MgOAc, 10 mM NaOAc, and 1 mM EDTA as the electrophoresis buffer. This maximized resolution of the PriA-PriB-PAS complexes from the PriA-PAS complexes. X174 ss(c)  RF replication assays were as described (16) and contained wild-type and mutant PriA proteins and DnaT as indicated in the figure legends.

RESULTS

PriA C439Y and C445Y Are Defective in X174 ss(c)  RF DNA Replication

Complementary strand X174 DNA replication can be reconstituted in vitro in the presence of SSB-coated X174 ss(c) DNA, PriA, PriB, PriC, DnaT, DnaB, DnaC, DnaG, and the DNA polymerase III holoenzyme. The mutant PriA proteins were defective in this assay (Fig. 1). At saturation, PriA C445Y and C439Y supported 70% and 40%, respectively, of the activity of the wild-type protein. Although the mutant proteins are temperature-sensitive in vitro³ (i.e., they can be heat-inactivated by short (about 3 min) incubations at 42 °C, conditions under which the wild-type protein is completely unaffected), this assay was at the permissive temperature of 30 °C. Similar defects for temperature-sensitive replication proteins have been noted at the permissive temperature in the past (17).

PriA C439Y and C445Y mutant proteins are defective in X174 ss(c)  RF DNA replication.

Such a biochemical phenotype could be reflective of a defect in any one of the many activities of PriA: PAS-DNA binding, 3  5 DNA translocation and DNA helicase activities, or the ability to form intermediate primosomal protein complexes during primosome assembly. Because PriA function is central to all subsequent primosomal functions, we sought the reason for the replication defect in the mutant proteins.

Equilibrium binding of PriA to PAS304 DNA was assessed by the nitrocellulose filter binding assay. Both the mutant and wild-type proteins bound the PAS304 DNA with nearly equivalent avidity (Fig. 2). The K_D for binding by the wild-type protein was 5 nM as determined according to Riggs et al. (18). This was similar to the value of 11.4 nM determined using the gel mobility shift assay as described in an accompanying article (1).

In the first article in this series (1) we reported that two distinct PriA-PAS304 DNA complexes, Pri-A-fast (PA-f) and Pri-A-slow (PA-s), could be resolved by gel mobility shift analysis. We examined the DNA binding activity of the mutant proteins further by assessing their ability to form these complexes (Fig. 3A). In this assay, the binding affinity of the mutant proteins was about 50% less than that of the wild-type (Fig. 3B). However, PriA C439Y and C445Y could form both PriA-PAS DNA complexes. At saturation, C445Y could form the same amounts of PA-f and PA-s complexes as the wild-type, whereas C439Y gave 67% of the wild-type level.

The mutant proteins were clearly more prone to aggregation than the wild-type. This manifested itself at high concentrations of the mutant proteins by the disappearance of the PA-f and PA-s complexes and the appearance of higher order complexes present as slowly moving smears in the gels, whereas there was very little aggregation evident for the wild-type protein.

The slight reduction in the activity of the mutant proteins compared with the wild-type protein in the formation of the PriA-PAS DNA complexes may be sufficient to account for the slight shift to higher saturation levels observed in the X174 ss(c)  RF replication assay; however, it is insufficient to account for the overall reduction in replication activity. We thus assessed the ability of the mutant proteins to participate in the next step in primosome assembly, the formation of a PriA-PriB-PAS DNA complex (1).

PriB adds to both the PA-F and PA-S complexes to form PriA-PriB-PAS304 DNA complexes termed PriA-PriB-fast (PA·PB-f) and PriA-PriB-slow (PA·PB-s) (1) (Fig. 4). The gel conditions used here were adjusted somewhat from those reported in the first article in this series (1) to maximize separation of the PA·PB complexes from the PA complexes. Thus, PriB addition is obvious both in the autoradiogram of the gel shift as a slight, but significant, retardation of the mobility of the PriA-PAS complexes (see, e.g. Fig. 4A, lanes 3-7) and in the Western blot probed with anti-PriB antisera and visualized by ECL (Fig. 4B, lanes 2-6).

Whereas PriA-PriB complex formation was evident in the presence of wild-type PriA (Fig. 4A, lanes 3-7 and Fig. 4B, lanes 2-6), PriB failed to form a complex with C439Y (Figs. 4, A, lanes 9-13, and B, lanes 8-12). PriB complex formation with C445Y was reduced to about one-tenth the level observed with the wild-type protein.

The inability of the mutant PriA proteins to form a complex with PriB was consistent with the reduction in replication activity observed for them. If the PriA-PriB-PAS complex fails to form, all subsequent primosomal complexes would be ablated, leading to reduced primer synthesis and a reduction in observed DNA synthesis. Nevertheless, C439Y and C445Y were partially active in the replication assay. This suggested that an alternative pathway of primosome assembly might exist wherein the PriB requirement could be bypassed. A likely stage where this might occur was formation of the PriA-PriB-DnaT-PAS DNA complex (PA·PB·T). We reasoned that given the multiple equilibria that exist during the formation of this complex, excess DnaT might be able to achieve complex formation with PriA in the absence of PriB. As described in the remainder of ``Results,'' this proved to be the case.

The replication defects of PriA C439Y and C445Y were observed at a DnaT concentration of 35 nM, which is sufficient to saturate the X174 ss(c)  RF replication assay in the presence of wild-type PriA (Fig. 5D). Higher concentrations of DnaT increased the level of C445Y-supported replication to nearly wild-type levels and significantly stimulated the activity of C439Y (Fig. 5D).

Because X174 ss(c)  RF replication is known to convert to rolling circle DNA synthesis (19), measuring only the incorporation of labeled precursor into acid-insoluble product is not an accurate determination of the efficiency of replication. Therefore, the ³²P-labeled DNA products from the replication reactions were analyzed by agarose gel electrophoresis (Fig. 5, A-C). Rolling circle DNA product (multigenome-length double-stranded DNA) migrates very slowly in the gel. Some of this product is evident with wild-type PriA (Fig. 5A), but essentially all of the DNA product formed with either C445Y (Fig. 5B) or C439Y (Fig. 5C) was form II DNA. Thus, the observed rescue by DnaT resulted from an increase in the fraction of template utilized rather than from stimulation of rolling circle DNA synthesis on a small percentage of active templates.

Because large excesses of DnaT were added to these reactions, we tested whether rescue was the result of a PriA contamination in the DnaT. In all cases, irrespective of the DnaT concentration, maximal rates of DNA synthesis showed at least a 14-fold dependence on the presence of PriA (Table I). Thus, rescue was a function of the added DnaT. Similarly, increasing the concentration of the other primosomal proteins did not stimulate the replication activities of the mutant PriA proteins. An example of this is shown for PriB (Fig. 6). Whereas the ability of PriB to stimulate replication by the mutant proteins correlated with the ability to form PriA-PriB complexes, DNA synthesis saturated at essentially the same concentration of PriB for the wild-type and mutant proteins.

Table I. PriA dependence of the X174 ss(c)  RF DNA replication reaction at low and high concentrations of DnaT Standard X174 ss(c)  RF DNA replication reactions with the indicated concentrations of DnaT and in the presence and absence of either PriA or PriA C439Y were performed and analyzed as described under ``Materials and Methods.'' dAMP incorporated DnaT (37 nM) DnaT (700 nM) pmol/10 min No PriA 1.8 2.5 Wild-type PriAa 51.7 43.6 PriA C439Ya 12.7 35.7 a At 3.8 nM.

DnaT-mediated rescue of the replication defects of the mutant proteins, particularly in the case of C439Y, suggested that the principal reason for formation of PA·PB·T complex was to associate PriA and DnaT. PriB must contribute to this by providing a better target for DnaT binding. This could occur because PriB binding to PriA induces a conformational change in the latter protein that provides a preferred binding site for DnaT. Another possibility, because PriA is a helicase (6, 7) and will tend to move away from the PAS, is that PriB stabilizes the association of PriA with the PAS. This was tested using the nitrocellulose filter binding assay.

We assessed the stability of PriA on PAS304 DNA via a challenge protocol. PriA-PAS complexes were allowed to form and then were challenged with cold competitor PAS DNA. In the absence of PriB, binding by the wild-type protein was competed as expected for isotopic dilution. However, PriB effectively stabilized PriA on the DNA, making it resistant to competition (Fig. 7A). Interestingly, although C439Y appeared more stable on the DNA than the wild-type protein (because higher concentrations of competitor were required to destabilize it), there was no additional stabilization in the presence of PriB (Fig. 7B). Thus, whereas PriB clearly stabilizes PriA on the DNA, this cannot be the sole reason it facilitates binding of DnaT, and it is likely that PriB-induced conformational rearrangements contribute as well.

The data presented thus far suggest that PriB acts to facilitate entry of DnaT into a complex with PriA. If this were the case, then we should expect to be able to isolate a PriA-DnaT-PAS DNA complex for both the wild-type and mutant proteins at high concentrations of DnaT. This proved to be the case.

At low concentrations, DnaT failed to form a complex that was stable during gel mobility shift analysis with either wild-type or C439Y PriA (Fig. 8, lanes 4 and 10). As expected, in the presence of PriB at this concentration of DnaT, a PA·PB·T complex was formed with wild-type but not C439Y PriA (Fig. 8, lanes 5 and 11). At 133-fold higher concentrations of DnaT, both wild-type and C439Y PriA formed complexes that moved more slowly than the corresponding PriA-PAS DNA complexes. (Fig. 8, lanes 6 and 12). At this high concentration of DnaT, no additional complex formation could be detected with C439Y in the presence of PriB, whereas complete PA·PB·T complex formation was observed for the wild-type protein (Fig. 8, lanes 7 and 13).

The data presented in Fig. 8 argue strongly that PriB facilitates complex formation between PriA and DnaT on a PAS. If this is the principal function of PriB during primosome assembly, we should expect to be able to demonstrate that X174 ss(c)  RF DNA replication at high concentrations of DnaT was independent of the presence of PriB for both wild-type and C439Y PriA proteins. This proved to be the case.

Replication supported by C439Y was stimulated only 2-fold by PriB at low concentrations of DnaT and not at all at high concentrations where rescue of the mutant protein was complete (Fig. 9). On the other hand, replication supported by wild-type PriA showed a greater than 10-fold dependence on PriB at low DnaT concentrations. As the DnaT concentration increased, this dependence disappeared so that at the highest concentrations of DnaT tested there was none (Fig. 10A). All replication observed resulted in the formation of only form II DNA product, either in the absence (Fig. 10B) or presence (Fig. 10C) of PriB. In addition, no PriB contamination could be detected by ECL-Western analysis in DnaT even at the highest amount used in the experiment (Fig. 10D), effectively ruling out the trivial explanation for the lack of PriB dependence at high DnaT concentrations.

PriA C439Y-supported X174 ss(c)  RF DNA replication is only stimulated minimally by PriB.

PriA-supported X174 ss(c)  RF DNA replication becomes PriB-independent at high concentrations of DnaT.

DISCUSSION

The ordered assembly of the X174-type primosome requires the participation of seven proteins to load a multienzyme, bidirectional helicase-primase onto an SSB-coated PAS. Previous studies have elucidated the role of a number of these proteins during and subsequent to assembly. We have described here the role of PriB during the assembly process.

Mutant PriA proteins were characterized that either failed to form, or were defective in the formation of, a complex with PriB. PriA-PriB complex formation is the second step in primosome assembly (1). Nevertheless, these mutant PriA proteins were partially active in X174 ss(c)  RF DNA replication, suggesting that PriB action was not essential for subsequent priming. This bypass was found to occur at the next step in primosome assembly, the formation of a PriA-PriB-DnaT complex (1).

High concentrations of DnaT could rescue the replication defects of the PriA mutants, and both wild-type and mutant PriA proteins were shown to be able to form a complex with DnaT in the absence of PriB. Thus, we concluded that PriB facilitates complex formation between PriA and DnaT. It appears to do so at least partially by stabilizing the complex of PriA with the PAS, presumably by preventing or inhibiting the PriA DNA translocation activity. We had noted previously that the presence of both PriB and DnaT inhibited the 3  5 DNA helicase activity of PriA (3). However, because the mutant PriA proteins themselves appeared to be more stable on the PAS (presumably because their DNA translocation activities are defective), it seems likely the PriA-PriB complex formation is also required to induce a conformational change in PriA that encourages DnaT binding.

The accompanying articles in this series have demonstrated that the multienzyme primosome complex on the DNA is composed of PriA, PriB, PriC, DnaT, DnaB, and DnaG. The function of only two of these proteins, DnaB, the 5  3 helicase, and DnaG, the primase, is obvious at a replication fork. Whether PriB has a role subsequent to primosome assembly remains to be determined. The proposed role of the primosome in establishing a replication fork during induced and constitutive stable DNA replication (20), the recombination defect found in strains carrying priA disruptions (21, 22), and the recent observation that mutations can be found in priA that suppress the lethality of recF overexpression⁴ suggest that the primosome may be required to interact directly with the homologous recombination machinery. Primosome assembly can only occur at a PAS in vitro. Assembly at a recombination intermediate in vivo may therefore require a protein-protein interaction between a recombination protein and a primosomal protein.