Cryptic Single-Stranded-DNA Binding Activities of the Phage P And

Proc. Natl. Acad. Sci. USA Vol. 94, pp. 1154–1159, February 1997 Biochemistry

Cryptic single-stranded-DNA binding activities of the phage ␭ P and Escherichia coli DnaC replication initiation proteins facilitate the transfer of E. coli DnaB helicase onto DNA (phage ␭ DNA replication͞E. coli DNA replication͞regulation of DNA helicase action)

BRIAN A. LEARN,SOO-JONG UM*, LI HUANG†, AND ROGER MCMACKEN‡

Department of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205

Communicated by Thomas Kelly, Johns Hopkins University, Baltimore, MD, December 5, 1996 (received for review October 2, 1996)

ABSTRACT The bacteriophage ␭ P and Escherichia coli tight complex with the hexameric DnaB helicase (16) and the DnaC proteins are known to recruit the bacterial DnaB repli- helicase is recruited to the viral origin through interactions of the cative helicase to initiator complexes assembled at the phage and P⅐DnaB complex with the O-some (7, 11, 12). This second-stage bacterial origins, respectively. These specialized nucleoprotein nucleoprotein structure is seemingly unreactive until it is partially assemblies facilitate the transfer of one or more molecules of disassembled by the action of the E. coli DnaJ, DnaK, and GrpE DnaB helicase onto the chromosome; the transferred DnaB, in molecular chaperone system (8–10, 17). This disassembly reac- turn, promotes establishment of a processive replication fork tion stimulates DnaB helicase action by freeing DnaB from its apparatus. To learn more about the mechanism of the DnaB strong association with the P protein, an interaction which is transfer reaction, we investigated the interaction of replication known to suppress the ATPase and helicase activities of DnaB initiation proteins with single-stranded DNA (ssDNA). These (16, 18). The DnaB helicase is believed to be loaded onto the studies indicate that both P and DnaC contain a cryptic ssDNA- DNA at the 40-bp AϩT-rich region of ori␭, since this DNA binding activity that is mobilized when each forms a complex segment acquires single-stranded character under the influence with the DnaB helicase. Concomitantly, the capacity of DnaB to of negative DNA supercoiling and O-mediated bending of the bind to ssDNA, as judged by UV-crosslinking analysis, is sup- four origin iterons (10, 11, 19, 20). pressed upon formation of a P⅐DnaB or a DnaB⅐DnaC complex. The initiation pathway at oriC, the E. coli chromosomal origin, This novel switch in ssDNA-binding activity evoked by complex shares many features with the ␭ reaction. The initial step involves formation suggests that interactions of P or DnaC with ssDNA the binding of multiple copies of the bacterial DnaA initiator may precede the transfer of DnaB onto DNA during initiation of protein to oriC to form a preinitiation complex, which in the DNA replication. Further, we find that the ␭ O replication presence of ATP and negative DNA supercoiling, destabilizes an initiator enhances interaction of the P⅐DnaB complex with AϩT-rich element near the left boundary of oriC (21–25). Next, ssDNA. Partial disassembly of a ssDNA:O⅐P⅐DnaB complex by DnaC, the bacterial analogue of ␭ P, forms a DnaB6⅐DnaC6 the DnaK͞DnaJ͞GrpE molecular chaperone system results in complex with the bacterial DnaB helicase (26, 27), which in turn the transfer in cis of DnaB to the ssDNA template. On the basis binds to the oriC:DnaA nucleoprotein structure to form a second- of these findings, we present a general model for the transfer of stage preinitiation complex. Transfer of DnaB onto DNA at oriC, DnaB onto ssDNA or onto chromosomal origins by replication presumably at the AϩT-rich element, ensues and bidirectional initiation proteins. DNA unwinding is initiated (13). The transfer step is believed to require ATP hydrolysis by DnaC (28) and is apparently accom- Biochemical studies of the initiation of Escherichia coli and panied by the release of DnaC (6). phage ␭ chromosomal DNA replication in reconstituted mul- We sought to learn more about the mechanisms involved in tienzyme systems have illuminated the molecular events that the transfer of DnaB helicase from nucleoprotein structures bring about these complex biosynthetic reactions (see refs. 1–3 onto DNA. Our examination of the single-stranded DNA for recent reviews). Both initiation pathways involve the (ssDNA)-binding properties of both ␭ and E. coli replication regulated assembly of large nucleoprotein structures at the initiation proteins indicates that the transfer of DnaB onto replication origin that facilitate the transfer of the bacterial ssDNA is facilitated by a cryptic ssDNA-binding activity DnaB helicase (4) onto the chromosome (1, 5–12). The present in ␭ P and also in E. coli DnaC. Our findings suggest transfer of DnaB onto the DNA to initiate DNA unwinding is a general scheme for the transfer of DnaB helicase onto DNA a key step in the overall replication process because it inau- by replication initiation proteins. gurates the unregulated fork propagation phase of the reaction (4, 7, 10, 13–15). MATERIALS AND METHODS The molecular mechanisms responsible for the transfer of Reagents. Sources of reagents were as follows: adenosine DnaB helicase onto duplex DNA from preinitiation nucleopro- 5Ј-[␥-thio]triphosphate (ATP[␥S]) and 5Ј-adenylyl imido- tein structures at chromosomal origins remain ill defined. In the diphosphate (AMP-PNP), Boehringer Mannheim; [␥-32P]ATP case of ␭ DNA replication, it is known that a nucleoprotein (6000 Ci͞mmol; 1 Ci ϭ 37 GBq), Amersham; 14C-labeled complex at ori␭ that contains the phage O initiator protein, ␭ P protein molecular weight standards, Life Technologies; replication protein, and DnaB helicase is the progenitor complex required for DNA unwinding (7, 10). This ori␭:O⅐P⅐DnaB preini- ␭ Abbreviations: ssDNA, single-stranded DNA; ATP[␥S], adenosine tiation complex is assembled when multiple copies of P form a 5Ј-[␥-thio]triphosphate; AMP-PNP, 5Ј-adenylyl imidodiphosphate. *Present address: Institut National de la Sante´et de la Recherche The publication costs of this article were defrayed in part by page charge Me´dicaleUnite´184͞Centre National de la Recherche Scientifique payment. This article must therefore be hereby marked ‘‘advertisement’’ in Laboratorie de Ge´ne´tique Moleculaire des Eucaryotes, Institut de accordance with 18 U.S.C. §1734 solely to indicate this fact. Chimie Biologique, 11 Rue Humann, 67085 Strasbourg Cedex, France. †Present address: Institute of Microbiology, Chinese Academy of Copyright ᭧ 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA Sciences, P.O. Box 2714, Beijing 100080, People’s Republic of China. 0027-8424͞97͞941154-6$2.00͞0 ‡To whom reprint requests should be addressed. e-mail: rmcmacke@ PNAS is available online at http:͞͞www.pnas.org. phnet.sph.jhu.edu.

1154 Downloaded by guest on September 30, 2021 Biochemistry: Learn et al. Proc. Natl. Acad. Sci. USA 94 (1997) 1155

poly(dI-dC)⅐poly(dI-dC), Pharmacia Biotech; and T4 polynu- source, and irradiated (0.5 mJ͞sec at 254 nm) for 5 min. Control cleotide kinase, New England Biolabs. Synthetic oligonucleo- experiments indicated that no net increase in crosslinking of tides were synthesized in this department. protein to ssDNA occurred after 1 min of irradiation (data not Enzymes. All proteins were Ͼ95% homogeneous. The ␭ O (29) shown). Following irradiation, reaction mixtures were denatured and P (16) proteins and the E. coli DnaJ (30) and DnaK (30) for 5 min at 100ЊC and electrophoresed in SDS͞8% or 10% proteins were purified as described previously. The E. coli DnaB polyacrylamide gels as described (34). Gels were dried under and GrpE proteins were purified from overproducing strains by vacuum and autoradiographed. Quantitation of radioactivity modifications (B.A.L. and R.M, unpublished results; A. Mehl and associated with protein–DNA complexes was performed using a R.M., unpublished results) of previously described protocols (4, Fuji BAS 1000 Phosphor Image Plate Scanner fitted with Mac- 31). As judged by HPLC analysis of acid-denatured DnaB, Ͻ2% BAS computer software. The apparent molecular weight (Mr)of of the polypeptides in our preparation of purified DnaB con- nucleoprotein complexes was determined from the relative elec- tained bound ATP or ADP. Purified E. coli DnaA and DnaC trophoretic mobility of each complex in SDS͞PAGE as compared proteins were the generous gift of Kenneth Marians (Memorial with the mobilities of 14C-labeled protein molecular weight Sloan-Kettering Cancer Center, New York). Protein concentra- standards (35). The crosslinked polypeptide-ssDNA complex tions, with the exception of DnaA and DnaC, were determined migrates approximately at the position expected for a polypeptide spectrophotometrically under native conditions (32). whose Mr equals the sum of the Mrs of the individual components Oligonucleotides. The sequence of the synthetic DNA oli- of the complex (35). gonucleotide RM55 is 5Ј-TGACGAATAATCTTTTCTTTT- TTCTTTTGTAATAGTGTCTTTT. RM55 corresponds to RESULTS 43 nucleotides of the T-rich strand of the ori␭ AϩT-rich DNA Formation of a ssDNA:O⅐P⅐DnaB Complex. A critical step in sequence (positions 39165–39123 of the ␭ sequence). RM55 the initiation of both E. coli chromosomal DNA replication and 32 was end-labeled at its 5Ј terminus using [␥- P]ATP [6000 coliphage ␭ DNA replication is the transfer of one or more Ci͞mmol] and T4 polynucleotide kinase and purified by poly- molecules of the E. coli DnaB helicase onto the genomic DNA at acrylamide gel electrophoresis under denaturing conditions. the respective bacterial and viral replication origins. The loading ssDNA-Binding Assays. Reaction mixtures (20 ␮l) were as- of DnaB at replication origins is facilitated by the action of sembled on ice and contained the following: 50 mM Tris⅐HCl, pH specific replication initiator proteins—e.g., E. coli DnaA and 7.6; 50 mM KCl; 10 mM magnesium acetate; 6 mM dithiothreitol; DnaC for oriC and ␭ O and P for ori␭. We have taken advantage 50 ␮g͞ml bovine serum albumin; 3 nM RM55 ssDNA oligonu- 32 of the capacity of O and P to load DnaB onto ssDNA in a cleotide (5Ј end-labeled with P); AMP-PNP, ATP[␥S], or ADP, sequence-independent manner (36, 37) to examine the roles of ␭ ␭ as indicated; and O (85 nM, as monomer), P (300 nM), DnaB these initiator proteins in the transfer reaction. Using gel- (100 nM, as monomer), DnaA (200 nM) and or DnaC (300 nM), ͞ retardation (band-shift) analysis, we investigated the interactions as indicated. After a 1-hr incubation at 30ЊC, each reaction of O, P, and DnaB, in various combinations, with a short mixture was supplemented with competitor DNA, either 2 ␮gof (43-base) ssDNA oligonucleotide derived from the T-rich strand poly(dI-dC)⅐poly(dI-dC) or 1 ␮g of unlabeled oligonucleotide, as of the ori␭ AϩT-rich region. Both O and DnaB individually indicated. Nucleoprotein complexes were analyzed either by interact with ssDNA (Fig. 1, lanes 2 and 5, respectively), albeit gel-retardation analysis or with a UV DNA-protein crosslinking weakly. As previously demonstrated (38, 39), the stable interac- assay (see below). tion of DnaB with ssDNA requires the continuous presence of Disassembly of nucleoprotein structures was initiated at 30ЊC, in the presence of excess unlabeled RM55 ssDNA ATP or an ATP analogue such as AMP-PNP (see also Fig. 4). competitor (1 ␮g), by the addition of ATP, DnaJ (to 60 nM), P protein alone does not detectably interact with ssDNA. DnaK (to 1.9 ␮M), and GrpE (to 400 nM). Immediately Unexpectedly, a stable and slowly migrating nucleoprotein following a 10-min (or 60-min) incubation, individual reaction structure was formed when a P⅐DnaB complex was incubated mixtures were irradiated with UV light as described below. In together with the labeled oligonucleotide (Fig. 1, lane 8). This certain experiments, disassembly reactions were modified to result was surprising, since we had earlier demonstrated that contain both AMP-PNP and ATP. In such instances, a mixture the P⅐DnaB protein complex does not interact stably with of DnaJ, DnaK, and GrpE was first preincubated with AMP- ssDNA, as assessed with a filter binding assay using phage M13 PNP for 40 min at 30ЊC to hydrolyze the small amount of ATP genomic ssDNA (16). The nucleoprotein structure formed on that contaminates commercial preparations of AMP-PNP. the oligonucleotide in the presence of P and DnaB apparently Preformed ssDNA:O⅐P⅐DnaB complexes were then mixed with is stabilized by the presence of AMP-PNP in the binding the molecular chaperone͞AMP-PNP mixture, so that the final reaction (lane 9). Unlike free DnaB, however, a stable nu- concentrations of ssDNA substrate and individual proteins cleoprotein complex is detected even when the nucleotide were as described above, and the final AMP-PNP concentration was 1 mM. Each combined mixture was equilibrated further for 15 min at 30ЊC, and disassembly of nucleoprotein complexes was initiated by the addition of unlabeled competitor ssDNA and ATP, as described above. Gel-Retardation Assays. Nucleoprotein complexes were formed, incubated for 5 min at 30ЊC in the presence of poly(dI-dC)⅐poly(dI-dC), and electrophoresed in nondenaturing 5% polyacrylamide gels equilibrated in 25 mM Tris͞190 mM glycine͞1 mM EDTA͞5 mM magnesium acetate, essen- tially as described (33). Where indicated, polyacrylamide gels and PAGE buffers were supplemented with 10 ␮MATP[␥S] FIG. 1. Formation of a ssDNA:O⅐P⅐DnaB nucleoprotein complex 32 or ADP. Following electrophoresis, the gels were dried under as assessed by band-shift analysis. P-labeled RM55 oligonucleotide vacuum and subjected to autoradiography. was incubated with ␭ O (O), ␭ P (P), and E. coli DnaB (B) and͞or UV ssDNA-Protein Crosslinking Assays. DnaA (A) proteins, as indicated, in the presence or absence of Assembled nucleo- AMP-PNP. Assembled nucleoprotein complexes were challenged with protein complexes were irradiated, using a hand-held UV source poly(dI-dC)⅐poly(dI-dC) and electrophoresed through a nondenatur- (model UVG-11, Ultraviolet Products, San Gabriel, CA), as ing 5% polyacrylamide gel in the presence of 5 mM magnesium follows: reaction mixtures were spotted onto plastic Petri dishes acetate. Arrow, free ssDNA. The slowest moving species in lane 9 is positioned in an ice bucket approximately 8 cm from the UV believed to represent two P⅐DnaB complexes bound to one DNA chain. Downloaded by guest on September 30, 2021 1156 Biochemistry: Learn et al. Proc. Natl. Acad. Sci. USA 94 (1997)

cofactor is absent. The nucleotide independence of the DNA complex or in a ssDNA:O⅐P⅐DnaB nucleoprotein complex interaction raises the possibility that DnaB polypeptides in the (Fig. 2, lanes 8–11) is also consistent with this conclusion. On P⅐DnaB complex do not directly interact with DNA. the other hand, crosslinks to P and O were readily observed When the ␭ O and P initiators are both present together with with the latter complex (lanes 10 and 11). We infer, therefore, DnaB and oligonucleotide, a very stable nucleoprotein com- that interactions of these two ␭ replication proteins with plex is formed that does not enter the gel (Fig. 1, lanes 10 and ssDNA contribute prominently to the stability of the presump- 11). UV-crosslinking analysis of this complex (see below) tive ssDNA:O⅐P⅐DnaB complex. indicates that it is extremely stable, even in the absence of For each nucleoprotein complex, single polypeptides of O, ATP. We infer that specific protein–protein interactions be- P, or DnaB were the primary species crosslinked to the tween O and the P⅐DnaB complex are responsible for the radiolabeled ssDNA by UV irradiation. However, for the greatly enhanced stability of this putative ssDNA:O⅐P⅐DnaB presumptive ssDNA:P⅐DnaB and ssDNA:O⅐P⅐DnaB nucleo- nucleoprotein structure. The specificity of O in this interaction protein complexes, significant levels of higher-order is bolstered by the finding that the E. coli DnaA initiator fails crosslinked species were also detected (Fig. 2, lanes 8–11). One to interact with the P⅐DnaB complex on ssDNA (compare such species, labeled with an asterisk, has an electrophoretic lanes 11 and 12 in Fig. 1). mobility consistent with that expected for a complex contain- ␭ P Protein Contains a Cryptic ssDNA-Binding Activity. To ing two polypeptides of P crosslinked to the labeled oligonu- identify which proteins were in intimate contact with ssDNA in cleotide. Additional experimentation indicated that multiple the various nucleoprotein complexes that are formed in this study, subunits of P can bind the same oligonucleotide within a single we made use of a UV-crosslinking approach. Nucleoprotein P⅐DnaB complex, a complex that is apparently composed of six structures were assembled on a radiolabeled ssDNA oligonucle- subunits of DnaB and three to six subunits of P (16). otide and irradiated with 254-nm light. Only proteins that directly Interaction of DnaB Helicase with ssDNA During Disas- contact DNA become crosslinked to the 32P-labeled oligonucle- sembly of the ssDNA:O⅐P⅐DnaB Complex by Molecular Chap- otide. The identity of the crosslinked proteins can be established erones. Previous studies of ␭ DNA replication indicated that from the apparent molecular weights of the polypeptide–ssDNA partial disassembly of preinitiation complexes by the DnaJ͞ complexes, as determined from their relative mobilities under DnaK͞GrpE molecular chaperone system is required to com- denaturing conditions during SDS͞PAGE (35). plete the transfer of DnaB onto DNA (8–10, 12, 17, 36, 40). As expected from the results presented in Fig. 1, control Taking these findings together with our present results, we experiments indicated that ␭ O (33.9 kDa) is readily considered it likely that molecular chaperone action will be crosslinked to the ssDNA substrate (Fig. 2, lanes 2, 6, and 7) required to permit DnaB polypeptides present in an and that DnaB (52.4 kDa) is crosslinked to oligonucleotide in oligonucleotide:O⅐P⅐DnaB complex to interact stably with the the presence of AMP-PNP, but not in the absence of a ssDNA. To test this idea, nucleoprotein complexes containing nucleotide cofactor (Fig. 2, lanes 4 and 5). The crosslinking O, P, and DnaB were formed on a 32P-labeled oligonucleotide, analysis of the P⅐DnaB complex on ssDNA, however, yielded challenged with a 1000-fold excess of unlabeled oligonucleo- an unanticipated result. The primary covalent polypeptide- tide, and incubated for 10 min in the presence of DnaJ, DnaK, ssDNA species obtained migrated in SDS͞PAGE with a Mr Ϸ and GrpE and 1 mM ATP prior to UV irradiation. Following 41,000, the size expected for a complex composed of a single the action of the molecular chaperones, neither O, P, nor DnaB molecule each of ␭ P and of the 43-base oligonucleotide (Fig. was crosslinked to the labeled ssDNA (Fig. 3, lane 4), sug- 2, lanes 8 and 9). This finding suggests that the ␭ P replication gesting that few nucleoprotein complexes survived the chap- protein contains a cryptic ssDNA-binding activity that is erone treatment. Control experiments indicated that the mobilized as a consequence of its interaction with the DnaB ssDNA:O⅐P⅐DnaB structures were stable to ssDNA challenge helicase. Moreover, the ATP-independence of the binding of for periods up to several hours in the absence of added the P⅐DnaB complex to ssDNA (Figs. 1 and 2) is a strong chaperones (Fig. 3, lanes 1–3) or ATP (lane 5). indicator that DnaB does not directly participate in the DNA- The failure to detect the transfer of DnaB onto the oligo- binding event (4, 39). Our inability to detect crosslinking to nucleotide in the presence of the chaperones and ATP was ssDNA of DnaB when it is present in a ssDNA:P⅐DnaB predictable. We anticipated that the interaction of DnaB with an oligonucleotide might prove to be fleeting, because of the capacity of DnaB, when ATP is present, to directly dissociate from the DNA or translocate off the ends of short ssDNA fragments. We surmised that a lower concentration of ATP might slow the movement of DnaB helicase along a ssDNA fragment (4), yet still be adequate to support the chaperone-mediated nucleoprotein disassembly reactions required for the transfer of DnaB from the ssDNA:O⅐P⅐DnaB complex. The results of an ATP titration made in the presence of 1 mM AMP-PNP [a nucleotide cofactor that supports DNA binding but not DNA translocation by DnaB (4)] are depicted in Fig. 3 (lanes 5–13). Indeed, a band that comigrates with the oligonucleotide-DnaB standard (lane 14) is observed (lanes 7 and 8) when the nucleoprotein disassembly reaction is carried out in the presence of 10–25 ␮M ATP. This presumptive DnaB:ssDNA species is not detected at ATP concentrations above 100 ␮M, perhaps because FIG. 2. UV-crosslinking analysis of ssDNA:O⅐P⅐DnaB nucleopro- the DnaB helicase molecules under these conditions no longer tein complexes. Radiolabeled RM55 oligonucleotide was incubated in remain bound to ␭ P protein and, thus, are capable of rapid the presence of AMP-PNP, ␭ O (O), ␭ P (P), and͞or E. coli DnaB (B) translocation or dissociation off the ssDNA oligonucleotide. We proteins as indicated. Assembled nucleoprotein complexes were mixed conclude that the ‘‘transfer’’ of DnaB to the ssDNA occurs in cis with unlabeled competitor, irradiated with UV light, and resolved by from an ssDNA:O⅐P⅐DnaB complex, since the transfer reaction to electrophoresis through an SDS͞10% polyacrylamide gel. The migration positions of covalent protein-DNA complexes containing 1 mol- labeled ssDNA is Ϸ80% efficient (relative to DnaB alone, lane ecule of ssDNA and 1 monomer of either O (␭ O1),P(␭P1), or DnaB 14) despite the presence of a 1000-fold excess of unlabeled specific (DnaB1) proteins are indicated. M, 14C-labeled protein molecular competitor DNA. Furthermore, we also conclude from the data weight standards (ϫ 10Ϫ3). presented in Fig. 3 that ATP concentrations that yield maximal Downloaded by guest on September 30, 2021 Biochemistry: Learn et al. Proc. Natl. Acad. Sci. USA 94 (1997) 1157

FIG. 4. Formation of ssDNA:DnaA⅐DnaB⅐DnaC nucleoprotein complexes. 32P-labeled RM55 oligonucleotide was incubated in the presence of 100 ␮MATP[␥S] (lanes 1–8) or 100 ␮M ADP (lanes 9–16) and DnaA (A), DnaB (B), and͞or DnaC (C) proteins, as indicated. Assembled nucleoprotein complexes were challenged with poly(dI- dC)⅐poly(dI-dC) and electrophoresed in a nondenaturing 5% polyacrylamide gel in the presence of 5 mM magnesium acetate and either 10 ␮MATP[␥S] or 10 ␮M ADP. Arrow, free ssDNA. FIG. 3. Disassembly of the ssDNA:O⅐P⅐DnaB nucleoprotein complex by molecular chaperones transfers DnaB to ssDNA. Nucleopro- not stably interact with this oligonucleotide either in the tein ssDNA:O⅐P⅐DnaB complexes were assembled on 32P-labeled presence (Fig. 4, lane 11) or in the absence of ADP (data not RM55 ssDNA in the presence of 1 mM ATP (lanes 1–4) or 1 mM shown). Since the presence of a rNTP is not required for stable AMP-PNP (lanes 5–13) as described in the text. Subsequently, reaction DNA binding of the DnaB⅐DnaC complex, it is an indication mixtures were supplemented with the following: (i)1␮g of unlabeled that DnaC, rather than DnaB, may be responsible for the RM55 ssDNA (lanes 2–13); (ii)2␮g of poly(dI-dC)⅐poly(dI-dC) (lanes interaction of the complex with ssDNA. Further analysis 1 and 14); (iii) DnaJ, DnaK, and GrpE proteins (lanes 4–13); and (iv) ATP, at the indicated final concentration in micromolar (lanes 6–13). indicated that DnaA is capable of interacting with the After an additional incubation for 10 min at 30ЊC (60 min for the DnaB⅐DnaC complex to form a highly stable nucleoprotein reaction mixture applied to lane 3), each reaction mixture was UV complex that apparently contains all three E. coli replication irradiated and subjected to SDS͞PAGE as described in the legend of proteins (Fig. 4, lane 8). The formation of this presumptive Fig. 2. For the lane 14 sample, purified DnaB alone was incubated in ssDNA:DnaA⅐DnaB⅐DnaC complex is strictly dependent upon the presence of AMP-PNP with 32P-labeled RM55 ssDNA substrate the presence of ATP[␥S]; it is notable that ADP does not serve and crosslinked by UV irradiation. The migration positions of certain as an effective nucleotide cofactor for formation of this crosslinked nucleoprotein complexes and of protein molecular weight higher-order nucleoprotein structure (Fig. 4, lane 16). standards ( 10Ϫ3) are indicated as described for Fig. 2. ϫ To determine which proteins directly interact with ssDNA in levels of crosslinked DnaB support substantial disassembly of O these nucleoprotein structures, we used UV-crosslinking anal- and P from the starting ssDNA:O⅐P⅐DnaB complex, as judged by ysis as described earlier for the studies with O, P, and DnaB. the significant reduction in those DNA species that contain a Consistent with the results depicted in Fig. 4, DnaB, but single crosslinked O or P polypeptide in such reaction mixtures. neither DnaA nor DnaC, was crosslinked to a labeled oligo- DnaC Contains a Cryptic ssDNA-Binding Activity That Is nucleotide when present alone with ATP[␥S] (Fig. 5). Never- theless, in the DnaB⅐DnaC nucleoprotein complex, our results Elicited upon Interaction with DnaB Helicase. The discovery indicate that it is DnaC, not DnaB, that is crosslinked to the that the ␭ P replication protein contains a cryptic ssDNA- DNA under these conditions (Fig. 5, lane 7). Complexes of binding activity that is revealed when it interacts with a oligonucleotide covalently coupled to one or to two DnaC molecule of DnaB helicase caused us to consider the possibility polypeptides were readily detected. We conclude that DnaC that the functionally homologous DnaC protein of E. coli acquires the capacity to interact with ssDNA when it is bound contains a similar activity. To test this idea, various combina- to DnaB. Concomitantly, the DnaB subunits present in the tions of DnaC, DnaB, and DnaA were incubated with a labeled DnaB⅐DnaC nucleoprotein structure are apparently rendered RM55 oligonucleotide, and the resulting nucleoprotein com- incapable of interacting with the ssDNA chain, even when plexes were electrophoresed through nondenaturing polyacryl- ATP[␥S] is present. Interestingly, in the presence of ADP, a amide gels in the presence of ATP[␥S] (Fig. 4, lanes 1–8) or small fraction of the DnaB subunits in the DnaB⅐DnaC ADP (Fig. 4, lanes 9–16). Of these proteins, when present nucleoprotein structure become crosslinked to ssDNA (Fig. 5, alone, only DnaB formed a stable nucleoprotein complex with lane 10). Finally, the presence of the DnaA initiator in the RM55, and then only when ATP[␥S] (or AMP-PNP; data not nucleoprotein complex had no distinctive effect on the UV- shown) was present in both the binding reaction mixture and crosslinking pattern of either DnaC or DnaB regardless of the the electrophoresis running buffer (Fig. 4, lane 3). The two nucleotide cofactor, although in ATP[␥S] the efficiency of retarded bands produced by the interaction of DnaB with DnaC crosslinking was reproducibly higher when DnaA was RM55 apparently represent oligonucleotides containing one present (Fig. 5). These crosslinking results were obtained with and two bound hexamers of DnaB helicase. Consistent with an oligonucleotide derived from ori␭, but identical results were this interpretation, just a single retarded protein-DNA species obtained with an oligonucleotide derived from the oriC AϩT- is formed on a shorter, (dT)20, oligonucleotide over a broad rich region. This suggests that the DNA-binding interactions range of DnaB concentrations (1.2–600 nM monomer). documented here are relatively sequence independent. DnaC is capable of interacting with DnaB to form a new nucleoprotein structure on ssDNA (Fig. 4, lane 7). This DISCUSSION complex migrates slightly behind the slower of the two DnaB- Our investigation of the ssDNA-binding properties of the ␭ P and oligonucleotide species under native conditions, even though E. coli DnaC proteins has revealed that each of these replication this species apparently represents just a single DnaB6⅐DnaC6 initiation proteins harbors a cryptic ssDNA-binding activity. For complex (27) bound to the oligonucleotide. Surprisingly, a both P and DnaC, this DNA-binding activity is mobilized only similar DnaB⅐DnaC complex is formed on RM55 in the when each protein forms a complex with the bacterial DnaB presence of ADP (Fig. 4, lane 15), whereas DnaB alone does helicase. The discovery of this new DNA-binding activity in the Downloaded by guest on September 30, 2021 1158 Biochemistry: Learn et al. Proc. Natl. Acad. Sci. USA 94 (1997)

in results to be published elsewhere, that the second-stage ori␭:O⅐P⅐DnaB nucleoprotein structure (7, 9), assembled on supercoiled ori␭ templates during the initiation of ␭ DNA replication, has the unique capacity to form a stable pre-open complex that can trap the energy of DNA supercoiling. (L.H., B.A.L., D. S. Sampath, and R.M., unpublished results). It appears probable that interactions with ori␭ DNA sequences of one or more of the P polypeptides that are present in this nucleoprotein assembly must play a critical role in the formation of this key replication intermediate. The foregoing results, when taken together with the findings presented in this report, suggest that P and DnaC carry out multiple functions during the initiation of ␭ and E. coli DNA replication, respectively. In addition to their known role as FIG. 5. UV-crosslinking analysis of ssDNA:DnaA⅐DnaB⅐DnaC and ssDNA:DnaB⅐DnaC nucleoprotein complexes. Nucleoprotein molecular matchmakers in recruiting DnaB helicase to an complexes were assembled on 32P-labeled RM55 ssDNA substrate, in origin preinitiation nucleoprotein complex, P and DnaC ap- the presence of 100 ␮MATP[␥S] (lanes 1–8) or 100 ␮M ADP (lanes parently also actively participate in the transfer of DnaB from 9–11), and DnaA (A), DnaB (B), and͞or DnaC (C) as indicated. the complex onto template DNA. In Fig. 6, we depict one Following UV irradiation, nucleoprotein complexes were resolved in pathway for the O- and P-mediated transfer of DnaB onto an SDS͞8% polyacrylamide gel. The relative electrophoretic mobili- ssDNA that is consistent with our results. In step a, O binds ties of covalent protein-DNA complexes containing 1 molecule of 1 1 weakly and nonspecifically to the DNA fragment. After bind- ssDNA and 1 monomer of DnaB (B ) or DnaC (C ) proteins are ing of P to DnaB in solution to form what is presumably a indicated. M, 14C-labeled protein molecular weight standards. P6⅐DnaB6 complex (16), the P⅐DnaB complex interacts with the P⅐DnaB and DnaB⅐DnaC complexes was initially surprising, since bound O protein (step b) to form a stable ssDNA:O⅐P⅐DnaB free DnaB itself was known to have an intrinsic ATP-dependent complex. The driving force for this assembly reaction is a ssDNA-binding activity (4, 38, 41, 42). This DNA-binding capac- specific interaction between O and P (7, 46–48). With ssDNA ity of DnaB was generally assumed to be both necessary and templates, it is possible that the first two steps occur in reverse sufficient for the loading of DnaB onto DNA at replication order; the P⅐DnaB complex may initially bind weakly to ssDNA origins. UV-crosslinking analysis of P⅐DnaB or DnaC⅐DnaB and then be stabilized by protein–protein interactions with O. complexes bound to ssDNA fragments, however, identified Regardless, it is clear from the UV-crosslinking data that both the O and P polypeptides present in the ssDNA:O⅐P⅐DnaB crosslinks between DNA and P or between DNA and DnaC, complex interact directly with the ssDNA at this stage. Trans- respectively, but failed to detect any interaction of DnaB subunits fer of DnaB onto the DNA requires the partial disassembly of with DNA, whether or not ATP was present. The absence of the nucleoprotein structure by the DnaJ, DnaK, and GrpE DnaB crosslinking is consistent with the ATP independence of molecular chaperone system of E. coli (Fig. 6, steps c and d). the binding reaction as judged by band-shift analysis of the We surmise that this reaction is mechanistically similar to the P⅐DnaB and DnaB⅐DnaC complexes (Fig. 2 and data not shown). closely related nucleoprotein disassembly reaction required to Nevertheless, we cannot absolutely exclude the possibility that establish DnaB as an active helicase at ori␭ (8, 9, 12). there are interactions between DnaB and ssDNA that cannot be Although the precise molecular details of the DnaB transfer detected by UV crosslinking. reaction remain to be determined, the UV-crosslinking data These results suggest that the same P⅐DnaB and DnaB⅐DnaC provide some clues. First, it is apparent (Fig. 2 and unpublished protein–protein interactions responsible for mobilizing the data) that multiple subunits of P (at least two and perhaps as DNA-binding activity of P or of DnaC also suppress the many as three) in each ssDNA:P⅐DnaB complex and each capacity of DnaB to bind to ssDNA. Because DnaB acts as a ssDNA:O⅐P⅐DnaB complex interact with a single ssDNA frag- highly processive helicase in chromosomal DNA replication (4, ment. This being the case, chaperone-mediated partial disas- 43), and because it has the capacity to drive priming and sembly of the ssDNA:O⅐P⅐DnaB complex in the presence of establishment of the replication fork apparatus once loaded ATP (Fig. 6, step c) may initially involve stepwise removal of onto duplex DNA, it presumably is critical to cellular viability a subset of the P subunits bound to DnaB. This would yield an that there be molecular mechanisms in place that preclude the intermediate in which some of the subunits of the presumptive loading of DnaB nonspecifically onto the chromosome. In vivo, DnaB hexamer are liberated from the inhibitory action of P it is likely that cytoplasmic DnaB exists primarily in a complex protein and are now free to bind ssDNA in an ATP-dependent with DnaC (26, 44), or in a P⅐DnaB complex following binding reaction. The ssDNA template would be expected to infection by phage ␭ (16, 18, 45). Our data are consistent with be present in high local concentration, since other subunits of the idea that P and DnaC each serve to modulate the activity the same DnaB hexamer would still be indirectly tethered to of DnaB by restricting its capacity to bind to ssDNA. DNA by their interaction with DNA-bound P subunits. This Although the P⅐DnaB and DnaB⅐DnaC complexes are also model may explain why DnaB is apparently transferred in cis capable of binding to ssDNA, this interaction is relatively weak. to a ssDNA fragment (Fig. 3) even in the presence of a In fact, the interaction of the P⅐DnaB complex with long 1000-fold excess of competitor ssDNA. Ultimately, the chap- ssDNA chains, such as M13 viral DNA, cannot be detected by erone-mediated disassembly reaction completes the transfer of using standard filter binding assays (16). Stable interaction of a hexamer of free DnaB helicase onto DNA. The mode of the P⅐DnaB complex with ssDNA requires the presence of the binding of ssDNA to DnaB is uncertain, but the high proces- ␭ O chromosomal initiator protein. Preliminary experiments sivity of DnaB action once transferred to DNA may indicate indicate that a P⅐DnaB complex bound to oligonucleotide that the ssDNA template passes through the center of the RM55 has a half-life of only 3 min in the presence or absence DnaB ring as depicted in Fig. 6 (49, 50). of nucleotide cofactor, whereas when the ␭ O initiator is We envision that the transfer of DnaB onto negatively present, the half-life of the P⅐DnaB interaction with ssDNA is supercoiled oriC DNA templates by DnaA and DnaC during increased to several hours. Thus, in vivo it is likely that stable the initiation of E. coli chromosomal DNA replication shares interactions of the P⅐DnaB complex with ssDNA are restricted some of the mechanistic features described in Fig. 6. A to ori␭ sequences and occur only in the context of initiation of DnaB6⅐DnaC6 complex interacts with an assembly of DnaA ␭ DNA replication. In this regard, we have recently discovered, molecules bound to oriC to form an oriC:DnaA⅐DnaB⅐DnaC Downloaded by guest on September 30, 2021 Biochemistry: Learn et al. Proc. Natl. Acad. Sci. USA 94 (1997) 1159

FIG. 6. Proposed model for the transfer of DnaB onto DNA from a ssDNA:O⅐P⅐DnaB preinitiation complex. See text for details. For simplicity, all proteins are depicted as single shapes: ␭ O (O), ␭ P (P), and DnaB (B).

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