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Direct Physical Interaction Between Dnag Primase and Dnab Helicase

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Direct Physical Interaction Between Dnag Primase and Dnab Helicase Proc. Natl. Acad. Sci. USA Vol. 93, pp. 12902–12907, November 1996 Biochemistry Direct physical interaction between DnaG primase and DnaB helicase of Escherichia coli is necessary for optimal synthesis of primer RNA (protein–protein interactionylagging strand replication) YA-BIN LU,PILLARISETTY V. A. L. RATNAKAR,BIDYUT K. MOHANTY, AND DEEPAK BASTIA* Department of Microbiology, Duke University Medical Center, Durham, NC 27710 Communicated by Wolfgang K. Joklik, Duke University Medical Center, Durham, NC, September 3, 1996 (received for review July 2, 1996) ABSTRACT The primase DnaG of Escherichia coli re- To distinguish between the two alternative models of acti- quires the participation of the replicative helicase DnaB for vation of the primase andyor its loading on DNA, we have optimal synthesis of primer RNA for lagging strand replica- investigated the possible physical interaction between the tion. However, previous studies had not determined whether helicase and the primase of Escherichia coli. In this paper we the activation of the primase or its loading on the template was present evidence supporting direct protein–protein interaction accomplished by a helicase-mediated structural alteration of in vitro between the two enzymes. We have localized the the single-stranded DNA or by a direct physical interaction interacting surfaces on both enzymes and have isolated mu- between the DnaB and the DnaG proteins. In this paper we tations in DnaB that reduce the protein–protein interaction present evidence supporting direct interaction between the without causing a significant reduction in helicase activity. We two proteins. We have mapped the surfaces of interaction on show further that a mutant form of DnaB that is impaired in both DnaG and DnaB and show further that mutations that DnaB–DnaG interaction, also suffered from an '4- to 5-fold reduce the physical interaction also cause a significant re- reduction in promoting primer synthesis in vitro in comparison duction in primer synthesis. Thus, the physical interaction with wild-type (wt) DnaB. Conversely, we have shown that a reported here appears to be physiologically significant. mutant form of DnaG that is defective in DnaB-stimulated synthesis of primer RNA also showed reduced physical inter- The multiprotein complex that initiates, elongates, and termi- action with wt DnaB. Thus, direct physical interaction between nates DNA replication has been likened to a protein machine the primase and the helicase appears to be necessary for (1). The various component proteins of this machine have to optimal primer synthesis. work coordinately to successfully carry out the complex task of DNA replication. Thus, careful analyses of the interactions MATERIALS AND METHODS amongst the replisomal proteins are of critical importance for gaining further insights into the mechanism and control of the Plasmids and Bacterial Strains. The E. coli strains JM109 three steps of DNA replication. In both Escherichia coli and in (sup E44, rel A1, rec A, end A1, gyr A96, hsd R17D, D [lac, pro mammalian cells, discontinuous synthesis of Okazaki frag- AB], [F9tra D36, lacIq, D(lac Z)M15, pro A1B1),(rk2,mk1), ments is primed by a class of enzymes called primases (2) that and BL21[DE3] [F2omp T,hsd S, (rB2,mB2) gal] containing move along the length of the DNA template as a part of a the plasmid pLysS (9) were used for all cloning and protein multiprotein primosomal complex, synthesizing RNA primers expression, respectively. The proteins were expressed in the T7 at intermittent locations on the template DNA (3). Although promoter containing vector pET11b (Novagen) or the tac a great deal of information has been uncovered regarding the promoter containing pGEX vector (Pharmacia). The pET3c- enzymology of fork movement in prokaryotes and eukaryotes DnaG Q576A overproducer clone was a gift from Ken Marians (3, 4), a number of significant questions still remain unan- (Sloan–Kettering Cancer Center, New York). swered regarding the protein–protein interactions that control Site-Directed Mutagenesis. Site-directed mutagenesis was and drive fork movement. performed as described (10). The regions of DnaB having two Studies on priming of phage single-stranded DNA (ssDNA), or more contiguous charged residues were regarded as poten- which is not coated with ssDNA binding protein, have shown tial interaction surfaces for DnaG and these sites were re- that DnaB helicase (5) is needed along with DnaG primase for placed by alanine. Specifically, the following charged residues optimal primer synthesis (3). Two alternative mechanisms have of DnaB were replaced with alanine: mutant m1, D212A and been proposed to account for the need for DnaB in primer D213A; m2, K216A and K217A; m3, D253A and K254A. synthesis. One hypothesis was that DnaB interacted with Purification of Proteins and Enzymes. Wt and mutant forms ssDNA and created a conformation that allowed DnaG to load of DnaG and DnaB were purified as described before (7, 11). on to the DNA. This idea was based on the observation that The reading frame of full-length DnaG or the N-terminal DnaB binding induced a change in the secondary structure of (bases 1–1265) and the C-terminal domains were fused in ssDNA (5). The alternative hypothesis was that DnaB inter- frame with the DNA encoding glutathione S-transferase acted directly with DnaG and with ssDNA, thus facilitating the (GST) present in the pGEX vector and the fusion proteins loading of the primase on the template andyor the activation were purified by affinity chromatography on glutathione- of the enzyme (3). Although functional interaction between agarose. The C-terminal bases 1266–1740 were fused in frame the C terminus of DnaG with DnaB was reported by Marians with the His-6 tag present in the vector pET30a (Novagen) and and coworkers (7, 8), critical evidence showing physical inter- the fusion protein was purified on Ni-nitrilotriacetic acid- action between the two protein has been elusive. agarose columns. The publication costs of this article were defrayed in part by page charge Abbreviations: wt, wild type; GST, glutathione S-transferase, tr.BC3, payment. This article must therefore be hereby marked ‘‘advertisement’’ in truncated BC3; ssDNA, single-stranded DNA. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 12902 Downloaded by guest on September 27, 2021 Biochemistry: Lu et al. Proc. Natl. Acad. Sci. USA 93 (1996) 12903 ELISA. The procedure used was similar to the one published RESULTS in ref. 12. One member of interacting pair of proteins was DnaB and DnaG Physically Interact in Vitro with Each dissolved in 100 ml of coating buffer (0.1 M NaHCO , pH 9.2) 3 Other. Because evidence supporting direct interaction be- at a concentration of 100 mg ml and adsorbed to each well of y tween the two proteins had been elusive, we assumed that such a 96-well plastic microtiter plate (Corning) by incubating for 1 interaction, if it occurred, would probably be weak. We hr at 208C. The wells were rinsed with 200 ml of wash buffer therefore endeavored to modify and develop procedures to (20 mM Tris, pH 7.4y0.15 M NaCly0.5% Tween 20). Blocking detect this potentially weak interactions. We modified ELISA buffer (200 mlof5%BSAy0.5% Tween 20 in wash buffer) was to detect the possible interaction between the helicase and the added to each well and incubated for 1 hr at 208C. The primase as described in a previous section. The modifications adsorbed protein in each well was challenged with 10–500 ng were in the washing procedure to reduce the background of the second protein dissolved in 100 ml of the blocking buffer nonspecific binding. Different amounts of DnaG were ad- and incubated at 208C for 1 hr. Each well was washed five times sorbed onto the wells of plastic microtiter plates and blocked with 200 ml of wash buffer. Rabbit antiserum raised against the the remainder of the free surface with BSA. We challenged the second protein was diluted 2000-fold in blocking buffer, and immobilized DnaG with various amounts of purified DnaB. 100 ml of the diluted antibody was added to the designated DnaA protein, which is known to interact with DnaB, was also wells and incubated for 1 hr at 208C. After five washes with the immobilized on plastic and used as a positive control, whereas washing buffer, a 1:40,000-fold dilution of a goat anti-rabbit helicase II was used as a negative control. The binding of DnaB IgG antiserum that was conjugated with alkaline phosphatase to DnaG, DnaA, and helicase II was measured as described in (Sigma) was added to each of the wells and incubated further a preceding section. The results showed that DnaB readily for1hrat208C. Following five washes with the wash buffer, bound to the immobilized DnaG. DnaB also bound to DnaA 100 ml of the chromogenic substrate, p-nitrophenyl disodium in agreement with previously published data (14). Helicase II phosphate hexahydrate was added to each well and incubated did not show significant levels of interaction with DnaB (Fig. at 208C for 5–20 min. The absorbance was read at 405 nm in 1A). We performed reciprocal binding experiments in which an ELISA plate reader. purified DnaB, DnaC, and BSA were immobilized on plastic Coupled in Vitro Transcription-Translation. DNAs encod- surface and challenged with increasing amounts of DnaG. The ing full-length wt DnaB, mutant forms of DnaB, or peptides of results showed that DnaG bound to immobilized DnaB but no DnaB were cloned downstream from a T7 promoter and significant binding to immobilized DnaC and BSA was detect- able (Fig. 1B). transcribed and translated in vitro in the presence of 35S- We wished to confirm the observed DnaB–DnaG interac- methionine (10 mCiyml;1Ci537 GBq), 10 mM amino acid tion by an independent technique.
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