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Crystal Structure of the N-Terminal Domain of the Dnab Hexameric Helicase Deborah Fass1†, Cynthia E Bogden1 and James M Berger2*

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Crystal Structure of the N-Terminal Domain of the Dnab Hexameric Helicase Deborah Fass1†, Cynthia E Bogden1 and James M Berger2* View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Research Article 691 Crystal structure of the N-terminal domain of the DnaB hexameric helicase Deborah Fass1†, Cynthia E Bogden1 and James M Berger2* Background: The hexameric helicase DnaB unwinds the DNA duplex at the Addresses: 1Whitehead Institute, Cambridge, Escherichia coli chromosome replication fork. Although the mechanism by Massachusetts 02142, USA and 2Department of which DnaB both couples ATP hydrolysis to translocation along DNA and Molecular and Cell Biology, Stanley Hall, University of California, Berkeley, California 94720, USA. denatures the duplex is unknown, a change in the quaternary structure of the protein involving dimerization of the N-terminal domain has been observed and †Present Address: Department of Structural may occur during the enzymatic cycle. This N-terminal domain is required both Biology, Weizmann Institute of Science, Rehovot for interaction with other proteins in the primosome and for DnaB helicase 76100 Israel. activity. Knowledge of the structure of this domain may contribute to an *Corresponding author. understanding of its role in DnaB function. E-mail: [email protected] Results: We have determined the structure of the N-terminal domain of DnaB Key words: DnaB, DNA helicase, DNA replication, domain crystallographically. The structure is globular, highly helical and lacks a close structural relative in the database of known protein folds. Conserved residues Received: 1 February 1999 and sites of dominant-negative mutations have structurally significant roles. Revisions requested: 17 February 1999 Each asymmetric unit in the crystal contains two independent and identical Revisions received: 4 March 1999 Accepted: 11 March 1999 copies of a dimer of the DnaB N-terminal domain. Published: 1 June 1999 Conclusions: The large-scale domain or subunit reorientation that is seen in DnaB by electron microscopy might result from the formation of a true twofold Structure June 1999, 7:691–698 http://biomednet.com/elecref/0969212600700691 symmetric dimer of N-terminal domains, while maintaining a head-to-tail arrangement of C-terminal domains. The N-terminal domain of DnaB is the first © Elsevier Science Ltd ISSN 0969-2126 region of a hexameric DNA replicative helicase to be visualized at high resolution. Comparison of this structure to the analogous region of the Rho RNA/DNA helicase indicates that the N-terminal domains of these hexameric helicases are structurally dissimilar. Introduction 12 kDa called Fragment III [8]. Fragment II contains sig- The DnaB helicase is an essential protein that is involved nature sequences of nucleotide-binding proteins [9] and in both the initiation and elongation stages of Escherichia retains hexamerization, single-stranded DNA-binding and coli chromosome replication [1]. It is a hexameric ATPase ATPase activities [8,10]. The predicted secondary struc- that uses the hydrolysis of ATP to unwind double-stranded ture around the nucleotide-binding signature sequences DNA [2]. DnaB also contains binding sites for other pro- matches the consensus topography predicted for other teins making it a central component in assembly of the pri- hexameric helicase ATP-binding domains [11], and is mosome, movement of the replication apparatus and likely to comprise a Walker-type ATP-binding fold. Frag- termination of replication [1]. For example, DnaB interacts ment III has no known activity alone but this domain is with its DNA-loading factor DnaC [3,4], with the primase essential for DnaB helicase activity, general priming and DnaG [5] and with the replication termination protein, interaction with DnaC [8,10]. RTP [6]. Furthermore, translocation of DnaB is coordi- nated with the movement of the replication fork through On the basis of negative-stain electron microscopy direct contact to the τ-subunit of the pol III holoenzyme; studies, low-resolution structural models for the DnaB this complex couples the helicase and polymerase activities hexamer reveal six bi-lobed protomers arranged in a ring and appears to stimulate DnaB action as well [7]. around a channel [12,13]. Fragment II has been proposed to correspond to the larger, central lobe, because it is the The domain structure of DnaB has been examined to larger of the two protease-resistant regions and contains locate various functional regions (Figure 1a). Proteolysis of the primary multimerization activity [10,12,13]. The intact DnaB removes the N-terminal 14 residues to yield smaller domain farther from the sixfold axis was assumed the 50 kDa Fragment I [8]. Fragment I can be further to be Fragment III [12,13]. DnaB can display either cleaved into two species: a 33 kDa C-terminal region threefold or sixfold symmetry [13] and dimerization of termed Fragment II and a smaller, N-terminal domain of the Fragment III domain is associated with the switch 692 Structure 1999, Vol 7 No 6 Figure 1 the Fragment III domains in DnaB alone do not appear to protrude radially but instead may lie closer to the oligomer (a) Fragment III Fragment II axis stacked above the Fragment II domains [16]. This Regulatory ssDNA binding, ATPase arrangement is consistent with fluorescence energy transfer T 12 kDa T T 33 kDa experiments that suggest that the Fragment III domains N C 2º oligomerization 'Hinge' 1º oligomerization face the 5′ end of the replication fork arm [17]. Positioning of the N-terminal domains atop the ATPase regions is also (b) α1 α2 PPHSIEAEQSVLGGLMLDNERWDDVA consistent with the known low-resolution structures of ••• 30 40 50 phage T7 hexameric helicase/primase protein [18] and the 3 1 10 α3 RuvB branch migration protein [19]; these enzymes also ERVVADDFYTRPHRHIFTEMARLQES •• 60 70 have a pronounced polarity, with both domains of the bi- α4 α5 lobed protomers circling the hexamer axis. GSPIDLITLAESLERQGQLDSVGGFA ••• 80 90 100 α6 α7 The similarities between DnaB and other hexameric heli- YLAELSKNTPSAANISAYADIVRER case assemblies suggest that these enzymes may share •• 110 120 Structure certain mechanistic features [20]. Hexameric ATPases with Walker-type nucleotide-binding regions, however, Organization of DnaB. (a) The primary domain structure of DnaB. The have been found to play diverse roles in numerous cellular polypeptide chain has been divided into distinct regions by proteolytic events, such as ATP synthesis via a proton gradient [21], mapping [8]. Nomenclature, sizes and positions of these domain boundaries are indicated. (b) Secondary structure of DnaB-III. The termination of transcription [22] and facilitation of vesicle sequence of the DnaB N-terminal domain is shown with helices boxed fusion [23,24]. The molecular motors responsible for each and secondary structure labels assigned. These secondary structural of these functions must be adapted to their particular tasks assignments agree well with previous assignments from NMR [41]. through structural differences between regions outside the nucleotide-binding motifs. A high-resolution view of DnaB, in particular of regions that distinguish it from [12,13]. A similar distribution of symmetry states has other hexameric ATPases, is ultimately necessary to recently been observed for the papillomavirus E1 hexam- understand the details of its reaction mechanism. The eric helicase [14]. N-terminal domain (Fragment III) is critical to the func- tion of DnaB, regulating the conformational consequences Fragments II and III of DnaB are connected by a of ATP binding and hydrolysis for both priming of replica- 45 amino acid linker, which may facilitate the quaternary tion and unwinding of DNA. To begin to address how the structural changes of the enzyme. The rate of protease hexameric ATPase DnaB is customized for its cellular role cleavage at this linker decreases dramatically in the pres- in DNA replication, we have determined the structure of ence of a non-hydrolyzable ATP analog, as compared with Fragment III, which we will refer to as DnaB-III, using ATP or ADP [8], indicating that ATP hydrolysis results in X-ray crystallography. conformational changes affecting accessibility of the hinge. Interestingly, protease treatment to uncouple Frag- Results ments II and III leads to a fivefold increase in the ATPase Structure of the DnaB N-terminal domain activity of Fragment II without affecting ATP binding The DnaB N-terminal domain structure was solved by ([8], but also see [10]), suggesting that the presence of the multiple isomorphous replacement and has been refined to N-terminal domain of DnaB can regulate ATP hydrolysis. 2.3 Å resolution (Figure 2; Table 1). The working R factor Despite having enhanced ATPase activity, the hexameric is 26.3% and the free R factor is 29.3% (Table 2). The Fragment II shows no helicase activity [10], which further overall dimensions of the DnaB-III domain are 25 Å × 25 Å indicates that the Fragment III domain is critical for cou- × 35 Å, which is roughly consistent with the size of the pling ATPase activity to enzyme function. Other globules at the vertices in the electron microscope (EM) members of the replication machinery that require the reconstruction of intact DnaB after correction for typical DnaB N-terminal domain for binding may either bind this losses in measured volume because of artifacts of negative domain directly or may recognize a quaternary structure of staining [12]. The DnaB-III structure is largely helical the enzyme that is assumed only when the Fragment III (Figures 2b,c) as previously reported
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