The Ordered Assembly of the φX174-type Primosome

The φX-type primosome was discovered during the resolution and reconstitution in vitro of the complementary strand DNA replication step of the φX174 viral life cycle. This multienzyme bidirectional helicase-primase complex can provide the DNA unwinding and Okazaki fragment-priming functions at the replication fork and has been implicated in cellular DNA replication, repair, and recombination. We have used gel mobility shift assays and enhanced chemiluminescence Western analysis to isolate and identify the pathway of primosome assembly at a primosome assembly site (PAS) on a 300-nucleotide-long single-stranded DNA fragment. The first three steps do not require ATP and are as follows: (i) PriA recognition and binding to the PAS, (ii) stabilization of the PriA-PAS complex by the addition of PriB, and (iii) formation of a PriA-PriB-DnaT-PAS complex. Subsequent formation of the preprimosome involves the ATP-dependent transfer of DnaB from a DnaB-DnaC complex to the PriA-PriB-DnaT-PAS complex. The final preprimosomal complex contains PriA, PriB, DnaT, and DnaB but not DnaC. A transient interaction between the preprimosome and DnaG generates the five-protein primosome. As described in an accompanying article (Ng, J. Y., and Marians, K. J. (1996) J. Biol. Chem. 271, 15649-15655), when assembled on intact φX174 phage DNA, the primosome also contains PriC. The φX-type primosome was discovered during the resolution and reconstitution in vitro of the complementary strand DNA replication step of the φX174 viral life cycle. This multienzyme bidirectional helicase-primase complex can provide the DNA unwinding and Okazaki fragment-priming functions at the replication fork and has been implicated in cellular DNA replication, repair, and recombination. We have used gel mobility shift assays and enhanced chemiluminescence Western analysis to isolate and identify the pathway of primosome assembly at a primosome assembly site (PAS) on a 300-nucleotide-long single-stranded DNA fragment. The first three steps do not require ATP and are as follows: (i) PriA recognition and binding to the PAS, (ii) stabilization of the PriA-PAS complex by the addition of PriB, and (iii) formation of a PriA-PriB-DnaT-PAS complex. Subsequent formation of the preprimosome involves the ATP-dependent transfer of DnaB from a DnaB-DnaC complex to the PriA-PriB-DnaT-PAS complex. The final preprimosomal complex contains PriA, PriB, DnaT, and DnaB but not DnaC. A transient interaction between the preprimosome and DnaG generates the five-protein primosome. As described in an accompanying article (Ng, J. Y., and Marians, K. J. (1996) J. Biol. Chem. 271, 15649-15655), when assembled on intact φX174 phage DNA, the primosome also contains PriC. INTRODUCTIONThe φX-type primosome was identified initially as being required during the first stage of φX174 bacteriophage DNA replication, providing the priming activity for complementary strand DNA synthesis (1Wickner S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4120-4124Google Scholar, 2Weiner J. McMacken R. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 752-756Google Scholar). Subsequent studies showed that the primosome was (i) capable of translocation and primer synthesis along single-stranded DNA-binding protein (SSB) 1The abbreviations used are: SSBthe E. coli single-stranded DNA-binding proteinPASprimosome assembly sitePA-sthe PriA-slow complexPA-fthe PriA-fast complexPA·PB-sthe PriA-PriB-slow complexPA·PB-fthe PriA-PriB-fast complexPA·PB·Tthe PriA-PriB-DnaT complexBCthe DnaB-DnaC complexECLenhanced chemiluminescenceAMP-PNP5′-adenylyl-β,γ-imidodiphosphate. -coated single-stranded DNA (3Arai K. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 69-73Google Scholar, 4Arai K. Low R.L. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 707-711Google Scholar), (ii) a bidirectional DNA helicase (5Lee M.S. Marians K.J. J. Biol. Chem. 1989; 264: 14531-14542Google Scholar), and (iii) capable of providing the DNA helicase and Okazaki fragment-priming activities at replication forks reconstituted with purified proteins (6Mok M. Marians K.J. J. Biol. Chem. 1987; 262: 2304-2309Google Scholar, 7Wu C.A. Zechner E.L. Marians K.J. J. Biol. Chem. 1992; 267: 4030-4044Google Scholar). It has thus been suggested that the φX-type primosome is active at the Escherichia coli replication fork (7Wu C.A. Zechner E.L. Marians K.J. J. Biol. Chem. 1992; 267: 4030-4044Google Scholar). Additionally, recent genetic evidence has suggested its participation in DNA repair and recombination (8Sandler S.J. Samra H.S. Clark A.J. Genetics. 1996; 143: 5-13Google Scholar, 9Sandler S.J. Mol. Microbiol. 1996; 19: 871-880Google Scholar, 10Kogoma T. Cadwell G.W. Barnard K.G. Asai T. J. Bacteriol. 1996; 178: 1258-1264Google Scholar) as well as in stable DNA replication (11Lark C.A. Riazi J. Lark K.G. J. Bacteriol. 1978; 136: 1008-1017Google Scholar, 12Lark K.G. Lark C.A. Cold Spring Harbor Symp. Quant. Biol. 1978; 43: 537-549Google Scholar, 13Masai H. Bond M.W. Arai K. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1256-1260Google Scholar, 14Masai H. Arai K. J. Biol. Chem. 1988; 263: 15083-15093Google Scholar, 15Masai H. Asai T. Kubota Y. Arai K. Kogoma T. EMBO J. 1994; 13: 5338-5345Google Scholar).Eight E. coli proteins participate in the assembly of the primosome at a specific region of DNA known as a primosome assembly site (PAS) (16Marians K.J. CRC Crit. Rev. Biochem. 1984; 17: 153-215Google Scholar). They are PriA, PriB, PriC, DnaB, DnaC, DnaG, DnaT, and SSB (1Wickner S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4120-4124Google Scholar, 2Weiner J. McMacken R. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 752-756Google Scholar, 5Lee M.S. Marians K.J. J. Biol. Chem. 1989; 264: 14531-14542Google Scholar, 17Wickner S. Denhardt D.T. Dressler D. Ray D.S. The Single-stranded DNA Phages. Cold Spring Harbor Laboratory Press, New York1978: 255Google Scholar, 18Allen Jr., G.C. Kornberg A. J. Biol Chem. 1993; 268: 19204-19209Google Scholar, 19Arai K. Low R. Kobori J. Shlomai J. Kornberg A. J. Biol. Chem. 1981; 256: 5273-5280Google Scholar). On an SSB-coated φX174 phage DNA, the PAS, which has a hairpin-like structure (20Shlomai J. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 799-803Google Scholar), is exposed. A preprimosome is formed by the concerted action of PriA, PriB, PriC, DnaB, DnaC, and DnaT. The preprimosome is capable of translocating bidirectionally along single-stranded DNA (5Lee M.S. Marians K.J. J. Biol. Chem. 1989; 264: 14531-14542Google Scholar) displacing SSB in the process (19Arai K. Low R. Kobori J. Shlomai J. Kornberg A. J. Biol. Chem. 1981; 256: 5273-5280Google Scholar), as well as acting as a bidirectional DNA helicase (5Lee M.S. Marians K.J. J. Biol. Chem. 1989; 264: 14531-14542Google Scholar). An important feature of the preprimosome is its ability to attract DnaG, enabling it to synthesize primers for complementary strand synthesis, via a functional interaction with at least one primosomal protein, DnaB (21Tougu K. Peng H. Marians K.J. J. Biol. Chem. 1994; 269: 4675-4682Google Scholar).The roles of some of the primosomal proteins in primosome assembly and subsequent replication are more apparent than others. PriA specifically recognizes the PAS as evident from the hyperstimulation of its ATPase by that DNA sequence (20Shlomai J. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 799-803Google Scholar, 22Wickner S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3342-3346Google Scholar). It can be viewed as the recognition factor for the site of primosome assembly. In addition, its 3′→ 5′ helicase activity (23Lee M.S. Marians K.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8345-8349Google Scholar, 24Lasken R.S. Kornberg A. J. Biol. Chem. 1988; 263: 5512-5518Google Scholar) probably accounts for the translocation of the preprimosome in the same direction (5Lee M.S. Marians K.J. J. Biol. Chem. 1989; 264: 14531-14542Google Scholar). DnaB, a 5′→ 3′ helicase (25LeBowitz J. McMacken R. J. Biol. Chem. 1986; 261: 4738-4748Google Scholar), is likely responsible for the 5′→ 3′ translocation activity of the preprimosome (5Lee M.S. Marians K.J. J. Biol. Chem. 1989; 264: 14531-14542Google Scholar) and the interaction with primase during primer synthesis (21Tougu K. Peng H. Marians K.J. J. Biol. Chem. 1994; 269: 4675-4682Google Scholar). DnaC forms a complex in solution with DnaB that facilitates binding of DnaB to DNA (26Wickner S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 921-925Google Scholar, 27Kobori J.A. Kornberg A. J. Biol. Chem. 1982; 257: 13770-13775Google Scholar, 28Wahle E. Lasken R.S. Kornberg A. J. Biol. Chem. 1989; 264: 2463-2468Google Scholar, 29Wahle E. Lasken R.S. Kornberg A. J. Biol. Chem. 1989; 264: 2469-2475Google Scholar). On the other hand, the lack of information on the enzymatic nature of PriB, PriC, and DnaT provides few clues to their functions in the assembly process. Although various efforts have been made to determine the composition of an active φX-type primosome, the evidence accrued has been mostly indirect, relying on detecting the biochemical activities of the proteins involved (18Allen Jr., G.C. Kornberg A. J. Biol Chem. 1993; 268: 19204-19209Google Scholar, 19Arai K. Low R. Kobori J. Shlomai J. Kornberg A. J. Biol. Chem. 1981; 256: 5273-5280Google Scholar, 20Shlomai J. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 799-803Google Scholar, 21Tougu K. Peng H. Marians K.J. J. Biol. Chem. 1994; 269: 4675-4682Google Scholar, 22Wickner S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3342-3346Google Scholar, 23Lee M.S. Marians K.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8345-8349Google Scholar, 24Lasken R.S. Kornberg A. J. Biol. Chem. 1988; 263: 5512-5518Google Scholar, 25LeBowitz J. McMacken R. J. Biol. Chem. 1986; 261: 4738-4748Google Scholar, 26Wickner S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 921-925Google Scholar, 27Kobori J.A. Kornberg A. J. Biol. Chem. 1982; 257: 13770-13775Google Scholar, 28Wahle E. Lasken R.S. Kornberg A. J. Biol. Chem. 1989; 264: 2463-2468Google Scholar, 29Wahle E. Lasken R.S. Kornberg A. J. Biol. Chem. 1989; 264: 2469-2475Google Scholar, 30Low R.L. Arai K. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 1436-1440Google Scholar). Questions remain as to (i) how and in what order the φX-type primosome is assembled, (ii) which primosomal proteins stably associate with the PAS during assembly, and (iii) whether the composition of the primosome changes as it makes the transition from assembly to complementary strand DNA synthesis.In order to resolve these issues, we examined the assembly process and subsequent complementary strand synthesis in a stepwise fashion. Ambiguity was minimized by physical detection of the primosomal proteins involved. In this report, the first two questions are addressed. We demonstrate the stepwise assembly of the primosome using gel mobility shift analysis to isolate intermediate complexes coupled with physical detection of the primosomal proteins present in these complexes. A model of ordered primosomal assembly is proposed. The accompanying articles describe studies on the composition of the primosome from assembly through replication (31Ng J.Y. Marians K.J. J. Biol. Chem. 1996; 271: 15649-15655Google Scholar), and the role of PriB during primosome assembly (32Liu J. Nurse P. Marians K.J. J. Biol. Chem. 1996; 271: 15656-15661Google Scholar).DISCUSSIONAlthough the φX-type primosome was first characterized during the analysis of the replication of an E. coli phage, its normal cellular functions are closely tied to the well being and survival of the bacterium. Mutational analysis has lent insight to the significance of the primosomal proteins. Conditional-lethal mutations in dnaB, dnaC, dnaG, and dnaT that cease DNA replication at the nonpermissive temperature have been identified, a clear sign of their indispensable involvement in the chromosomal replication machinery. priA disruption mutants were viable but manifested partial induction of the SOS response, UV sensitivity, and inefficiencies in recombination, plasmid replication, cell growth, and cell division (41Nurse P. Zavitz K.H. Marians K.J. J. Bacteriol. 1991; 173: 6686-6693Google Scholar, 42Lee E.H. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3029-3032Google Scholar). This has led to several models proposing that the φX-type primosome provides the helicase and primase activities at the E. coli replication fork (41Nurse P. Zavitz K.H. Marians K.J. J. Bacteriol. 1991; 173: 6686-6693Google Scholar, 43Zavitz K.H. Marians K.J. Mol. Microbiol. 1991; 5: 2869-2873Google Scholar, 44Allen G.C. Dixon N.E. Kornberg A. Cell. 1993; 74: 713-722Google Scholar).The phenotypes of the priA disruption mutants were also informative in providing clues to other possible functions of PriA and the φX-type primosome in E. coli. Recent studies have shown that the primosome assembly function of PriA was essential for DNA repair and recombination (8Sandler S.J. Samra H.S. Clark A.J. Genetics. 1996; 143: 5-13Google Scholar, 9Sandler S.J. Mol. Microbiol. 1996; 19: 871-880Google Scholar, 10Kogoma T. Cadwell G.W. Barnard K.G. Asai T. J. Bacteriol. 1996; 178: 1258-1264Google Scholar). Compelling genetic data pointed to the participation of PriA, DnaC, and DnaT in inducible, as well as constitutive stable DNA replication (10Kogoma T. Cadwell G.W. Barnard K.G. Asai T. J. Bacteriol. 1996; 178: 1258-1264Google Scholar, 11Lark C.A. Riazi J. Lark K.G. J. Bacteriol. 1978; 136: 1008-1017Google Scholar, 12Lark K.G. Lark C.A. Cold Spring Harbor Symp. Quant. Biol. 1978; 43: 537-549Google Scholar, 13Masai H. Bond M.W. Arai K. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1256-1260Google Scholar, 14Masai H. Arai K. J. Biol. Chem. 1988; 263: 15083-15093Google Scholar, 15Masai H. Asai T. Kubota Y. Arai K. Kogoma T. EMBO J. 1994; 13: 5338-5345Google Scholar), which are alternative pathways of DNA replication activated during stress. It has also been suggested that φX-type primosome activity was required for resolution of recombination intermediates that arise during double-strand break repair (45Asai T. Kogoma T. J. Bacteriol. 1994; 176: 1807-1812Google Scholar). We therefore set out to develop a clear picture of the mechanism of primosome assembly. A gel mobility shift protocol was designed and optimized to isolate and resolve intermediates in primosome assembly. The identity of primosomal proteins present in each complex was then determined using specific antibodies and a highly sensitive immunoblotting technique. Our interpretation of the results is summarized in a model of ordered φX primosome assembly and is primosomal proteins were for their ability to to PriA showed for the DnaB also a complex with in the of DnaC and this the ability of DnaB to single-stranded DNA K. Kornberg A. J. Biol. Chem. 1981; 256: Scholar). In of a complex be when the gel shift assays were with SSB-coated not it likely that PriA is the recognition factor in φX primosome assembly step the role of PriA is not to and binding to the The PriA-PAS interaction the binding of other primosomal at least one of PriB, in the PriA-PAS As described in the accompanying by (32Liu J. Nurse P. Marians K.J. J. Biol. Chem. 1996; 271: 15656-15661Google Scholar), the interaction results in a for PriA from the the complex is likely the of the PriA-PriB-DnaT complex step is in an accompanying article (32Liu J. Nurse P. Marians K.J. J. Biol. Chem. 1996; 271: 15656-15661Google Scholar) that DnaT can associate with PriA on the DNA, the of DnaT required is by a factor of in the of PriB also the preprimosomal complexes by of its interaction with This has been suggested R.L. Shlomai J. Kornberg A. J. Biol. Chem. 1982; 257: Scholar) and is in the accompanying by and Marians (31Ng J.Y. Marians K.J. J. Biol. Chem. 1996; 271: 15649-15655Google Scholar).The final step in preprimosome assembly is the addition of DnaB from a DnaB-DnaC complex in solution to the PriA-PriB-DnaT-PAS complex step This step ATP as suggested by and Kornberg E. Lasken R.S. Kornberg A. J. Biol. Chem. 1989; 264: 2463-2468Google Scholar, 29Wahle E. Lasken R.S. Kornberg A. J. Biol. Chem. 1989; 264: 2469-2475Google Scholar), it is DnaC that the ATP and not This be with the DnaC is not in of the primosomal that DnaC is acting as a protein during primosome assembly, the interaction of DnaB with at least one of the proteins in the PriA-PriB-DnaT the described a preprimosomal complex is of PriA, PriB, DnaT, and PriA out of this DNA, it clear that formation of the PriA-PriB-DnaT-PAS complex specific interaction of PriB with PriA and interaction of DnaT with PriA of DnaB with this complex be a of an specific interaction a stabilization of DnaB to single-stranded DNA in to the primosomal the studies on primosomal complex formation and not be in of the complexes. in vitro of the complementary strand synthesis step of the φX174 viral life is in using proteins are known not to be with on purified from the replication by This that be a factor role was more during the of the of primosome when the primosomal proteins were and This is with the in the accompanying article (31Ng J.Y. Marians K.J. J. Biol. Chem. 1996; 271: 15649-15655Google Scholar), that is present in the preprimosomal complex when it on bacteriophage DNA, is a of DNA present in the assembly well with on the assembly steps required to a bidirectional helicase complex in the of the primosomal proteins (5Lee M.S. Marians K.J. J. Biol. Chem. 1989; 264: 14531-14542Google Scholar). Our from on detection of proteins by biochemical assays (18Allen Jr., G.C. Kornberg A. J. Biol Chem. 1993; 268: 19204-19209Google Scholar, K. McMacken R. S. Kornberg A. J. Biol. Chem. 1981; 256: Scholar) in that we demonstrate the of a complex and a PriA-PriB-DnaT complex and that DnaT with the with (1Wickner S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4120-4124Google Scholar, 2Weiner J. McMacken R. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 752-756Google Scholar) and with the transient nature of its interaction with DnaB (21Tougu K. Peng H. Marians K.J. J. Biol. Chem. 1994; 269: 4675-4682Google Scholar), we not isolate a stable primosome complex the accompanying we the detection described to the composition of the primosome from assembly through DNA and replication and the activities of some PriA mutants to the role of PriB during primosome assembly. INTRODUCTIONThe φX-type primosome was identified initially as being required during the first stage of φX174 bacteriophage DNA replication, providing the priming activity for complementary strand DNA synthesis (1Wickner S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4120-4124Google Scholar, 2Weiner J. McMacken R. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 752-756Google Scholar). Subsequent studies showed that the primosome was (i) capable of translocation and primer synthesis along single-stranded DNA-binding protein (SSB) 1The abbreviations used are: SSBthe E. coli single-stranded DNA-binding proteinPASprimosome assembly sitePA-sthe PriA-slow complexPA-fthe PriA-fast complexPA·PB-sthe PriA-PriB-slow complexPA·PB-fthe PriA-PriB-fast complexPA·PB·Tthe PriA-PriB-DnaT complexBCthe DnaB-DnaC complexECLenhanced chemiluminescenceAMP-PNP5′-adenylyl-β,γ-imidodiphosphate. -coated single-stranded DNA (3Arai K. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 69-73Google Scholar, 4Arai K. Low R.L. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 707-711Google Scholar), (ii) a bidirectional DNA helicase (5Lee M.S. Marians K.J. J. Biol. Chem. 1989; 264: 14531-14542Google Scholar), and (iii) capable of providing the DNA helicase and Okazaki fragment-priming activities at replication forks reconstituted with purified proteins (6Mok M. Marians K.J. J. Biol. Chem. 1987; 262: 2304-2309Google Scholar, 7Wu C.A. Zechner E.L. Marians K.J. J. Biol. Chem. 1992; 267: 4030-4044Google Scholar). It has thus been suggested that the φX-type primosome is active at the Escherichia coli replication fork (7Wu C.A. Zechner E.L. Marians K.J. J. Biol. Chem. 1992; 267: 4030-4044Google Scholar). Additionally, recent genetic evidence has suggested its participation in DNA repair and recombination (8Sandler S.J. Samra H.S. Clark A.J. Genetics. 1996; 143: 5-13Google Scholar, 9Sandler S.J. Mol. Microbiol. 1996; 19: 871-880Google Scholar, 10Kogoma T. Cadwell G.W. Barnard K.G. Asai T. J. Bacteriol. 1996; 178: 1258-1264Google Scholar) as well as in stable DNA replication (11Lark C.A. Riazi J. Lark K.G. J. Bacteriol. 1978; 136: 1008-1017Google Scholar, 12Lark K.G. Lark C.A. Cold Spring Harbor Symp. Quant. Biol. 1978; 43: 537-549Google Scholar, 13Masai H. Bond M.W. Arai K. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1256-1260Google Scholar, 14Masai H. Arai K. J. Biol. Chem. 1988; 263: 15083-15093Google Scholar, 15Masai H. Asai T. Kubota Y. Arai K. Kogoma T. EMBO J. 1994; 13: 5338-5345Google Scholar).Eight E. coli proteins participate in the assembly of the primosome at a specific region of DNA known as a primosome assembly site (PAS) (16Marians K.J. CRC Crit. Rev. Biochem. 1984; 17: 153-215Google Scholar). They are PriA, PriB, PriC, DnaB, DnaC, DnaG, DnaT, and SSB (1Wickner S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4120-4124Google Scholar, 2Weiner J. McMacken R. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 752-756Google Scholar, 5Lee M.S. Marians K.J. J. Biol. Chem. 1989; 264: 14531-14542Google Scholar, 17Wickner S. Denhardt D.T. Dressler D. Ray D.S. The Single-stranded DNA Phages. Cold Spring Harbor Laboratory Press, New York1978: 255Google Scholar, 18Allen Jr., G.C. Kornberg A. J. Biol Chem. 1993; 268: 19204-19209Google Scholar, 19Arai K. Low R. Kobori J. Shlomai J. Kornberg A. J. Biol. Chem. 1981; 256: 5273-5280Google Scholar). On an SSB-coated φX174 phage DNA, the PAS, which has a hairpin-like structure (20Shlomai J. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 799-803Google Scholar), is exposed. A preprimosome is formed by the concerted action of PriA, PriB, PriC, DnaB, DnaC, and DnaT. The preprimosome is capable of translocating bidirectionally along single-stranded DNA (5Lee M.S. Marians K.J. J. Biol. Chem. 1989; 264: 14531-14542Google Scholar) displacing SSB in the process (19Arai K. Low R. Kobori J. Shlomai J. Kornberg A. J. Biol. Chem. 1981; 256: 5273-5280Google Scholar), as well as acting as a bidirectional DNA helicase (5Lee M.S. Marians K.J. J. Biol. Chem. 1989; 264: 14531-14542Google Scholar). An important feature of the preprimosome is its ability to attract DnaG, enabling it to synthesize primers for complementary strand synthesis, via a functional interaction with at least one primosomal protein, DnaB (21Tougu K. Peng H. Marians K.J. J. Biol. Chem. 1994; 269: 4675-4682Google Scholar).The roles of some of the primosomal proteins in primosome assembly and subsequent replication are more apparent than others. PriA specifically recognizes the PAS as evident from the hyperstimulation of its ATPase by that DNA sequence (20Shlomai J. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 799-803Google Scholar, 22Wickner S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3342-3346Google Scholar). It can be viewed as the recognition factor for the site of primosome assembly. In addition, its 3′→ 5′ helicase activity (23Lee M.S. Marians K.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8345-8349Google Scholar, 24Lasken R.S. Kornberg A. J. Biol. Chem. 1988; 263: 5512-5518Google Scholar) probably accounts for the translocation of the preprimosome in the same direction (5Lee M.S. Marians K.J. J. Biol. Chem. 1989; 264: 14531-14542Google Scholar). DnaB, a 5′→ 3′ helicase (25LeBowitz J. McMacken R. J. Biol. Chem. 1986; 261: 4738-4748Google Scholar), is likely responsible for the 5′→ 3′ translocation activity of the preprimosome (5Lee M.S. Marians K.J. J. Biol. Chem. 1989; 264: 14531-14542Google Scholar) and the interaction with primase during primer synthesis (21Tougu K. Peng H. Marians K.J. J. Biol. Chem. 1994; 269: 4675-4682Google Scholar). DnaC forms a complex in solution with DnaB that facilitates binding of DnaB to DNA (26Wickner S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 921-925Google Scholar, 27Kobori J.A. Kornberg A. J. Biol. Chem. 1982; 257: 13770-13775Google Scholar, 28Wahle E. Lasken R.S. Kornberg A. J. Biol. Chem. 1989; 264: 2463-2468Google Scholar, 29Wahle E. Lasken R.S. Kornberg A. J. Biol. Chem. 1989; 264: 2469-2475Google Scholar). On the other hand, the lack of information on the enzymatic nature of PriB, PriC, and DnaT provides few clues to their functions in the assembly process. Although various efforts have been made to determine the composition of an active φX-type primosome, the evidence accrued has been mostly indirect, relying on detecting the biochemical activities of the proteins involved (18Allen Jr., G.C. Kornberg A. J. Biol Chem. 1993; 268: 19204-19209Google Scholar, 19Arai K. Low R. Kobori J. Shlomai J. Kornberg A. J. Biol. Chem. 1981; 256: 5273-5280Google Scholar, 20Shlomai J. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 799-803Google Scholar, 21Tougu K. Peng H. Marians K.J. J. Biol. Chem. 1994; 269: 4675-4682Google Scholar, 22Wickner S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3342-3346Google Scholar, 23Lee M.S. Marians K.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8345-8349Google Scholar, 24Lasken R.S. Kornberg A. J. Biol. Chem. 1988; 263: 5512-5518Google Scholar, 25LeBowitz J. McMacken R. J. Biol. Chem. 1986; 261: 4738-4748Google Scholar, 26Wickner S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 921-925Google Scholar, 27Kobori J.A. Kornberg A. J. Biol. Chem. 1982; 257: 13770-13775Google Scholar, 28Wahle E. Lasken R.S. Kornberg A. J. Biol. Chem. 1989; 264: 2463-2468Google Scholar, 29Wahle E. Lasken R.S. Kornberg A. J. Biol. Chem. 1989; 264: 2469-2475Google Scholar, 30Low R.L. Arai K. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 1436-1440Google Scholar). Questions remain as to (i) how and in what order the φX-type primosome is assembled, (ii) which primosomal proteins stably associate with the PAS during assembly, and (iii) whether the composition of the primosome changes as it makes the transition from assembly to complementary strand DNA synthesis.In order to resolve these issues, we examined the assembly process and subsequent complementary strand synthesis in a stepwise fashion. Ambiguity was minimized by physical detection of the primosomal proteins involved. In this report, the first two questions are addressed. We demonstrate the stepwise assembly of the primosome using gel mobility shift analysis to isolate intermediate complexes coupled with physical detection of the primosomal proteins present in these complexes. A model of ordered primosomal assembly is proposed. The accompanying articles describe studies on the composition of the primosome from assembly through replication (31Ng J.Y. Marians K.J. J. Biol. Chem. 1996; 271: 15649-15655Google Scholar), and the role of PriB during primosome assembly (32Liu J. Nurse P. Marians K.J. J. Biol. Chem. 1996; 271: 15656-15661Google Scholar).