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Oligomeric States of Bacteriophage T7 Gene 4 Primase/Helicase

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Oligomeric States of Bacteriophage T7 Gene 4 Primase/Helicase doi:10.1016/j.jmb.2006.05.037 J. Mol. Biol. (2006) 360, 667–677 Oligomeric States of Bacteriophage T7 Gene 4 Primase/Helicase Donald J. Crampton1⁎, Melanie Ohi2, Udi Qimron1, Thomas Walz2 and Charles C. Richardson1 1Department of Biological Electron microscopic and crystallographic data have shown that the gene 4 Chemistry and Molecular primase/helicase encoded by bacteriophage T7 can form both hexamers Pharmacology, Harvard and heptamers. After cross-linking with glutaraldehyde to stabilize the Medical School, Boston oligomeric protein, hexamers and heptamers can be distinguished either by MA 02115 USA negativestainelectronmicroscopyor electrophoretic analysis using polyacrylamide gels. We find that hexamers predominate in the presence 2Department of Cell Biology, of either dTTP or β,γ-methylene dTTP whereas the ratio between hexamers Harvard Medical School, and heptamers is nearly the converse in the presence of dTDP. When Boston, MA 02115, USA formed, heptamers are unable to efficiently bind either single-stranded DNA or double-stranded DNA. We postulate that a switch between heptamer to hexamer may provide a ring-opening mechanism for the single-stranded DNA binding pathway. Accordingly, we observe that in the presence of both nucleoside di- and triphosphates the gene 4 protein exists as a hexamer when bound to single-stranded DNA and as a mixture of heptamer and hexamer when not bound to single-stranded DNA. Furthermore, altering regions of the gene 4 protein postulated to be conformational switches for dTTP-dependent helicase activity leads to modulation of the heptamer to hexamer ratio. © 2006 Elsevier Ltd. All rights reserved. *Corresponding author Keywords: helicase; DNA binding; heptamer; hexamer; phosphate sensor Introduction At the replication fork, hexameric helicases translocate along the lagging strand and unwind Helicases are motor proteins that translocate duplex DNA to expose single-stranded DNA along single-stranded nucleic acids using energy template for the leading strand DNA polymerase. derived from nucleotide hydrolysis.1–3 This activity Synthetic duplex DNA substrates used to measure ′ facilitates many nucleic acid metabolic processes the unwinding activity require a 5 -single-stranded 10 including those that require the separation of tail onto which the hexameric helicase can bind. A ′ double-stranded nucleic acids into their component 3 -single-stranded tail is also required in order to single strands. The gene 4 protein of bacteriophage assure that this strand remains outside the central T7 contains within its C-terminal half the six channel of the helicase through which the other 11 conserved amino acid sequence motifs associated strand passes. Kinetic data suggest that DNA 12,13 with the superfamily 4 of helicases.4 Helicases of this binding is a multiple step process, but the exact family, which include the replicative Escherichia coli mechanism by which the gene 4 hexamer binds to DnaB helicase, assemble as ring-shaped hexamers DNA is unknown. that encircle single-stranded DNA.5–7 In the pres- Unlike many other hexameric helicases, such as ence of dTTP, the gene 4 protein translocates DnaB from E. coli and the gene 41 protein of bac- unidirectionally 5′ to 3′ by using the energy of teriophage T4, gene 4 protein loads onto single- dTTP hydrolysis.8,9 The movement of the protein stranded DNA without the aid of a helicase loader 14 can be inhibited by a non-hydrolyzable analog such protein. In order for the gene 4 protein to load onto as β,γ-methylene dTTP. single-stranded DNA it must either disassemble or the ring must open, since the protein loads onto circular DNA as efficiently as it does on linear DNA.15 Biochemical studies argue against the dis- E-mail address of the corresponding author: assembly and reassembly of the oligomeric complex [email protected] as a mechanism for ssDNA binding.12,13 Rather, it 0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved. 668 Oligomeric States of T7 Gene 4 Primase/Helicase has been suggested that the gene 4 protein adopts a Oligomerization of the gene 4 protein is nucleotide ‘‘lock-washer’’ conformation as has been observed in dependent.22 However, the residues involved in the the crystal structure of transcription termination fac- conformational changes required for the assembly of tor Rho16 and in electron micrographs of bacterio- free subunits into a ring-shaped and active gene 4 phage T4 gene 41 protein,17 and in so doing allows protein oligomer are largely unidentified. The ssDNA access to the central channel.12,13,18 Another crystal structure of the helicase fragment showed mechanism for binding to DNA is suggested from histidine 465 contacting the γ-phosphate of the studies with the RuvB protein from Thermus thermo- bound nucleotide in a manner consistent with this philus in which the loss of one subunit from a residue acting as a conformational switch by heptamer accompanies binding to double-stranded ‘‘sensing’’ the presence or absence of the γ-phos- DNA. The loss of a subunit provides a mechanism phate. Additionally, helicase motif 4 of the hexame- for opening the oligomeric ring providing access to ric helicases has also been postulated to be a region the central channel for double-stranded DNA bind- with an important role in modulating the confor- ing to the hexameric form.19 mational changes required for dTTPase-dependent In the presence of dTDP, dTTP, or β,γ-methylene helicase activity.23,24 dTTP, the gene 4 protein assembles into oligomeric In the present study, we have investigated the role complexes that bind to single-stranded DNA.20 of the heptameric form of the gene 4 protein. After Although hexamers are the predominant form of cross-linking of the gene 4 protein to stabilize the oligomerized gene 4 protein detected by electron oligomeric form,25 the heptamer and hexamer forms microscopy,5 heptamers of gene 4 protein have also can be observed by negative staining electron been observed.18 Furthermore, a gene 4 protein microscopy or by migration on a polyacrylamide fragment lacking the primase domain crystallized as gel. Significantly, no DNA binding by the heptamer a hexamer21 whereas a nearly full length gene 4 is observed. Rather, the heptamer is disfavored protein crystallized as a heptamer18 (Figure 1). The under conditions that promote the binding of heptamer revealed an expanded ring with dimen- DNA. The ratio of heptamer to hexamer can be sions suitable for accommodating double-stranded manipulated by the presence of nucleotide di- or DNA in the central channel. Thus, it was proposed triphosphates, mixing nucleotides in the presence or that the heptameric form of the gene 4 protein could absence of single-stranded DNA, or altering func- translocate along double-stranded DNA. Such a tionally important residues thought to be switches model is of biological relevance, since the gene 4 for conformational change. Our data are compatible protein has been proposed to translocate along with a model in which the loss of a subunit from the double-stranded DNA when it remodels Holiday heptamer provides a ring-opening mechanism for structures.7 the hexamer to bind single-stranded DNA. Figure 1. Crystal structures of the gene 4 protein helicase region: (a) 4D fragment (residues 241–566)21 and (b) 56 kDa 4B helicase.18 In each case, residues 261–549 are shown with individual subunits uniquely colored. The 4D helicase fragment crystallized as a hexamer with 2-fold symmetry while the altered 4B protein crystallized as a heptamer. Oligomeric States of T7 Gene 4 Primase/Helicase 669 Results Table 1. Effect of nucleotide on the ratio of heptamer to hexamer Oligomeric structure of the gene 4 protein is Nucleotide Particles Heptamer (%) Hexamer (%) nucleotide dependent dTDP 3172 79 21 dTTP 3175 29 71 β γ A representative electron microscopic image of , -Methylene dTTP 2714 17 83 negatively stained bacteriophage T7 gene 4 protein The number of hexamers and heptamers was determined using is shown in Figure 2(a). As previously described for the display program WEB.41 thegene4protein,5,18 the electron micrograph shows many ring-shaped particles. After incuba- Oligomerization of the T7 gene 4 protein is tion of gene 4 protein with nucleotide followed routinely determined by polyacrylamide gel electro- by cross-linking with glutaraldehyde to stabilize phoresis under non-denaturing conditions.22,23 Fre- the ring structure, both heptameric and hexameric quently, multiple bands appear at sizes greater than forms are observed in electron microscopic ima- 300 kDa (Figure 3, lane 1), but their significance has ges of negatively stained samples (Figure 2(b) and not been evaluated other than to verify oligomeri- (c)). Glutaraldehyde is a bi-functional compound zation. The ability of the gene 4 protein to form both that links covalently the amine groups of lysine heptamers and hexamers as revealed by the crystal in protein molecules creating a structure more structure of the 56 kDa gene 4 protein 18 suggests stable than that attained by aggregation or that the two discrete but closely associated protein oligomerization. bands seen in the non-denaturing gel in Figure 3 The ratio of heptameric to hexameric rings is represent the heptameric and hexameric forms of the dependent upon whether the protein is incubated enzyme. with thymidine diphosphate (dTDP) or triphos- To explore potential roles of the heptamer, poly- phate (dTTP) (Table 1). In the presence of dTDP, acrylamide gel electrophoresis was used in an at- electron microscopy shows that approximately four tempt to observe both oligomeric states under out of five ring structures formed by the gene 4 various conditions. Again, glutaraldehyde was protein are heptameric. Conversely, the ratios of used as a cross-linking agent so that oligomerized heptameric to hexameric rings are reversed when gene 4 protein would remain stable during electro- the gene 4 protein is incubated with either dTTP or phoresis .
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