Proc. Natl. Acad. Sci. USA Vol. 94, pp. 1154–1159, February 1997 Biochemistry Cryptic single-stranded-DNA binding activities of the phage l P and Escherichia coli DnaC replication initiation proteins facilitate the transfer of E. coli DnaB helicase onto DNA (phage l DNA replicationyE. coli DNA replicationyregulation 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 l 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 PzDnaB 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 A1T-rich region of oril, 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 PzDnaB or a DnaBzDnaC 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 l 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 l O replication presence of ATP and negative DNA supercoiling, destabilizes an initiator enhances interaction of the PzDnaB complex with A1T-rich element near the left boundary of oriC (21–25). Next, ssDNA. Partial disassembly of a ssDNA:OzPzDnaB complex by DnaC, the bacterial analogue of l P, forms a DnaB6zDnaC6 the DnaKyDnaJyGrpE 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 A1T-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 l 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 l 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 l 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- 59-[g-thio]triphosphate (ATP[gS]) and 59-adenylyl imido- tein structures at chromosomal origins remain ill defined. In the diphosphate (AMP-PNP), Boehringer Mannheim; [g-32P]ATP case of l DNA replication, it is known that a nucleoprotein (6000 Ciymmol; 1 Ci 5 37 GBq), Amersham; 14C-labeled complex at oril that contains the phage O initiator protein, l P protein molecular weight standards, Life Technologies; replication protein, and DnaB helicase is the progenitor complex required for DNA unwinding (7, 10). This oril:OzPzDnaB preini- l Abbreviations: ssDNA, single-stranded DNA; ATP[gS], adenosine tiation complex is assembled when multiple copies of P form a 59-[g-thio]triphosphate; AMP-PNP, 59-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´184yCentre 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 q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA Sciences, P.O. Box 2714, Beijing 100080, People’s Republic of China. 0027-8424y97y941154-6$2.00y0 ‡To whom reprint requests should be addressed. e-mail: rmcmacke@ PNAS is available online at http:yywww.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)zpoly(dI-dC), Pharmacia Biotech; and T4 polynu- source, and irradiated (0.5 mJysec 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 .95% homogeneous. The l 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 1008C and electrophoresed in SDSy8% 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, ,2% 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 SDSyPAGE 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 59-TGACGAATAATCTTTTCTTTT- TTCTTTTGTAATAGTGTCTTTT. RM55 corresponds to RESULTS 43 nucleotides of the T-rich strand of the oril A1T-rich DNA Formation of a ssDNA:OzPzDnaB Complex. A critical step in sequence (positions 39165–39123 of the l sequence). RM55 the initiation of both E. coli chromosomal DNA replication and 32 was end-labeled at its 59 terminus using [g- P]ATP [6000 coliphage l DNA replication is the transfer of one or more Ciymmol] and T4 polynucleotide kinase and purified by poly- molecules of the E. coli DnaB helicase onto the genomic DNA at acrylamide gel electrophoresis