Common mechanisms of DNA translocation motors in bacteria and viruses using one-way revolution mechanism without rotation

Abstract

Biomotors were once described into two categories: linear motor and rotation motor. Recently, a third type of biomotor with revolution mechanism without rotation has been discovered. By analogy, rotation resembles the Earth rotating on its axis in a complete cycle every 24h, while revolution resembles the Earth revolving around the Sun one circle per 365 days (see animations http://nanobio.uky.edu/movie.html). The action of revolution that enables a motor free of coiling and torque has solved many puzzles and debates that have occurred throughout the history of viral DNA packaging motor studies. It also settles the discrepancies concerning the structure, stoichiometry, and functioning of DNA translocation motors. This review uses bacteriophages Phi29, HK97, SPP1, P22, T4, and T7 as well as bacterial DNA translocase FtsK and SpoIIIE or the large eukaryotic dsDNA viruses such as mimivirus and vaccinia virus as examples to elucidate the puzzles. These motors use ATPase, some of which have been confirmed to be a hexamer, to revolve around the dsDNA sequentially. ATP binding induces conformational change and possibly an entropy alteration in ATPase to a high affinity toward dsDNA; but ATP hydrolysis triggers another entropic and conformational change in ATPase to a low affinity for DNA, by which dsDNA is pushed toward an adjacent ATPase subunit. The rotation and revolution mechanisms can be distinguished by the size of channel: the channels of rotation motors are equal to or smaller than 2 nm, that is the size of dsDNA, whereas channels of revolution motors are larger than 3 nm. Rotation motors use parallel threads to operate with a right-handed channel, while revolution motors use a left-handed channel to drive the right-handed DNA in an anti-chiral arrangement. Coordination of several vector factors in the same direction makes viral DNA-packaging motors unusually powerful and effective. Revolution mechanism that avoids DNA coiling in translocating the lengthy genomic dsDNA helix could be advantageous for cell replication such as bacterial binary fission and cell mitosis without the need for topoisomerase or helicase to consume additional energy.

Keywords: Binary fission; Bionanomotor; Bionanotechnology; Chromosome segregation; DNA packaging; DNA repair; Holliday junction; Homologous recombination; One-way traffic mechanism; Virus assembly.

Fig. 1

Depiction of the structure and function of phi29 DNA packaging motor. A. Schematic of hexameric pRNA. B. AFM images of loop-extended hexameric pRNA. C. Illustrations of the phi29 DNA packaging motor in side view (left) and bottom view (right) (adapted from (Schwartz et al.,2013a) with permission of Elsevier). D and E. Illustration of the rotation and revolution motions. (adapted from (De-Donatis et al.,2013)).

Fig. 2

Experiment of T4 and phi29 motor revealing that neither connector nor dsDNA rotation is required for active DNA packaging. A. Direct observation of DNA packaging horizontally using a dsDNA with its end linked to a cluster of magnetic beads for stretching the DNA. “a” and “b” are real-time sequential images of DNA-magnetic beads complexes (adapted from (Chang et al.,2008) with permission from the AIP publishing group). B. Schematic of the single molecule experimental geometry of phi29 DNA packaging motor to show no rotation of the connector during DNA packaging (adapted from (Hugel et al.,2007)). C. Experiment revealing that T4 motor connector does not rotate during packaging. The packaging activity is not inhibited with N-terminal of motor connector protein fused and tethered to its protease immune binding site on capsid (adapted from (Baumann et al.,2006) with permission from John Wiley and Sons).

Fig. 3

Single molecule photo-bleaching and confirmation of the presence of six copies of phi29 motor pRNA vial dual-view imaging of procapsids containing three copies of Cy3-pRNA and three copies of Cy5-pRNA. A. pRNA dimer design constructed with Cy3- and Cy5-pRNA. B. Typical fluorescence image of procapsids with dual-labeled pRNA dimers. C. Comparison of empirical photobleaching steps with theoretical prediction of Cy3-pRNA in procapsids bound with dual-labeled dimers. D. Photobleaching steps of procapsids reconstituted with the dimer. (adapted from (Shu et al.,2007) with permission from John Wiley and Sons).

Fig. 4

Stoichiometric assays showing the formation of the hexamer of phi29 motor ATPase gp16. A. Native gel revealed six distinct bands characteristic of the six oligomeric states of the ATPase; the hexamer increases as the concentration of protein increased. B. Slab gel showing the binding of ATPase to dsDNA in a 6:1 ratio; imaged in GFP (upper) and Cy3 channels (lower) for ATPase and dsDNA, respectively. C. Quantification by varying the molar ratio of [ATPase]:[DNA]. The concentration of bound DNA plateaus at a molar ratio of 6:1 (adapted from (Schwartz et al.,2013a) with permission from the Elsevier)

Fig. 5

Experiment and the proposed mechanism revealing four steps of pauses for each circle during the packaging of phi29 dsDNA. A. Design (left) and results (right) of four sample packaging traces. (adapted from (Chistol et al.,2012) with permission of the Elsevier). B. The presence of four lysine residues of motor channel protein leads to the formation of four positively charged rings in different motors (adapted from (Fang et al.,2012) with permission from Elsevier). C. Diagram showing DNA revolution inside phi29 connector channel with four steps of pause due to the interaction of four positively charged lysine rings with the negatively charged dsDNA phosphate backbone. DNA revolution across the 12 connector channel subunits is shown. The uneven electrical static interaction resulting from the mis-gearing between 10.5-base pairs per DNA helical turn and 12 subunit per round of the channel is the cause leading to the observation of uneven steps (adapted with permission from (Zhao et al.,2013). Copyright (2013) American Chemical Society). (PDB IDs: Phi29-gp10, 1H5W. (Guasch et al.,2002); SPP1-gp6, 2JES. (Lebedev et al.,2007); P22-gp1, 3LJ5. (Olia et al.,2011)).

Fig. 6

Gel showing the binding of dsDNA with the ATPase under different conditions, revealing the different conformations and different dsDNA affinities of the ATPase upon ATP binding and hydrolysis (adapted from (Schwartz et al.,2013a) with the permission of Elsevier).

Fig. 7

Inhibition assays revealing stoichiometry of motor components and cooperativity of motor subunits. A. Binomial distribution assay reveals that ATPase gp16 possesses a 6-fold symmetry (highlighted) in the DNA packaging motor (adapted from (Schwartz et al.,2013a) with the permission of Elsevier). B. Binomial distribution assay demonstrating one inactive Walker B ATPase mutant subunit within the hexamer blocked motor activity. C. The inhibition ability of inactive Walker B mutants in the absence (C, E) and presence (D, F) of dsDNA reveals cooperativity of motor subunits, supporting the sequential action and revolution mechanism (adapted from (Schwartz et al.,2013b) with the permission of Elsevier).

Fig. 8

Schematic showing the mechanism of sequential revolution in translocating dsDNA. A. The binding of ATP to one ATPase subunit stimulates the ATPase gp16 to adopt a conformation with a higher affinity for dsDNA. ATP hydrolysis forces gp16 to assume a new conformation with a lower affinity for dsDNA, thus pushing dsDNA away from the subunit and transferring it to an adjacent subunit. Rotation of neither the hexameric ring nor the dsDNA is required since the dsDNA revolves around the diameter of the ATPase. In each transitional step, one ATP is hydrolyzed, and in one cycle, six ATPs are required to translocate dsDNA one helical turn of 360° (10.5 bp). An animation is available at http://nanobio.uky.edu/movie.htm. (adapted from (Schwartz et al.,2013b) with permission of the Elsevier). B. Diagram of CryoEM results showing offset of dsDNA in the channel of bacteriophage T7 DNA packaging motor. The dsDNA did not appear in the center of the channel, instead, the dsDNA tilted toward the wall of the motor channel (adapted from (Guo et al.,2013a) with the permission of the National Academy of Sciences). C. The revolution of dsDNA along the 12 subunits of the connector channel (adapted from (Schwartz et al.,2013b) with permission of the Elsevier).

Fig. 9

Model of sequential mechanism of sequence action of phi29 DNA packaging motor. Binding of ATP to the conformationally disordered ATPase subunit stimulates an entropic and conformational change of the ATPase, thus fastening the ATPase at a less random configuration. This lower entropy conformation enables the ATPase subunit to bind dsDNA and prime ATP hydrolysis. ATP hydrolysis triggers the second entropic and conformational change, which renders the ATPase into a low affinity for dsDNA thus pushing the DNA to the next subunit that has already bound ATP. These sequential actions promote the movement and revolution of the dsDNA around the hexameric ATPase ring.

Fig. 10

Spooling of DNA within capsids of phages to support the revolution mechanism. The DNA Spooling inside the capsids are shown using example of A. phi29 bacteriophage (adapted from (Duda and Conway,2008b) with permission from Elsevier and (Tang et al.,2008) with permission from Elsevier), B. λ bacteriophage (adapted from (Petrov and Harvey,2011) with permission from Elsevier), C. ε 15 bacteriophage (adapted from (Jiang et al.,2006) with permission from Nature Publishing Group), D. T7 bacteriophage (adapted from (Cerritelli et al.,1997) with permission from Elsevier), and E. P22 bacteriophage (adapted and redrew from (Lander et al.,2006) and adapted from (Zhang et al.,2000) with permission from Elsevier).

Fig. 11

Quaternary structures showing the presence of the left-handed 30° tilting of the connector channel of different bacteriophages. A and B. The external view and the cross section view of the motor, showing the anti-parallel configuration between the left-handed connector subunits and the right-handed dsDNA helices. The 30° tilt of the helix (highlighted) relative to the vertical axis of the channel can be seen in a cross-section internal view of the connector channel and the view of its single subunit as shown in B (adapted from (Schwartz et al.,2013b) with permission from Elsevier, adapted from (Agirrezabala et al.,2005) with permission from Elsevier and adapted from (De-Donatis et al.,2013)). (PDB IDs: Phi29-gp10, 1H5W. (Guasch et al.,2002); HK97 family-portal protein, 3KDR; SPP1-gp6, 2JES. (Lebedev et al.,2007); P22-gp1, 3LJ5. (Olia et al.,2011), T7-gp8 EM ID: EMD-1231. (Agirrezabala et al.,2005)).

Fig. 12

The role of the flexible inner channel loop in DNA one-way traffic. A. Flexible loops within the phi29 (left) and SPP1 (right) connector channels function to interact with DNA, facilitating DNA to move forward but blocking reversal of DNA during DNA packaging. B. Demonstration of one-way traffic of dsDNA through wild type connectors using a ramping potential and by switching polarity (right). C. SsDNA is translocated via two-way traffic with a loop deleted connector (adapted from (Zhao et al.,2013) with permission from American Chemical Society).

Fig. 13

Bacterial hexameric DNA translocase FtsK may use the revolution mechanism. A. Diagram showing the contact of one strand of the dsDNA to the inner channel wall of the hexameric ATPase. The adjacent contact between DNA and ATPase will move around the inner surface of the channel without any rotation of the ATPase or DNA. B. Each of DNA contact points is expected to be separated by 60° along the inner surface of the ATPase hexameric channel. C. Sequential action of dsDNA translocation. DNA is shown as a line. T represents for ATP-bound and D for ADP-bound. (adapted from (Crozat and Grainge,2010) with permission from John Wiley and Sons).

Fig. 14

Sequential rotation mechanism of the hexameric DNA helicase DnaB. One strand of the dsDNA displaces within the hexameric channel and the other strand is outside the ATPase channel(adapted from (Itsathitphaisarn et al.,2012) with permission from Elsevier).

Fig. 15

Comparison of the size of channels between biomotors using rotation mechanism (left panel) and biomotors using revolution mechanism (right panel). The dsDNA biomotors with a channel size of twice the width of dsDNA argues against the a bolt and nut treading mechanism, supporting the conclusion of revolution rather than rotation (adapted from (Agirrezabala et al.,2005) with permission from Elsevier, adapted from (Sun et al.,2008) with permission from Elsevier and adapted from (De-Donatis et al.,2013)). (PDB IDs: RepA, 1G8Y. (Niedenzu et al.,2001); TrwB: 1E9R. (Gomis-Ruth et al.,2001); ssoMCM, 2VL6. (Liu et al.,2008); Rho, 3ICE. (Thomsen and Berger,2009); E1, 2GXA. (Enemark and Joshua-Tor,2006); T7-gp4D, 1E0J. (Singleton et al.,2000); FtsK, 2IUU. (Massey et al.,2006); Phi29-gp10, 1H5W. (Guasch et al.,2002); HK97 family-portal protein, 3KDR; SPP1-gp6, 2JES. (Lebedev et al.,2007); P22-gp1, 3LJ5. (Olia et al.,2011). T7-gp8 EM ID: EMD-1231. (Agirrezabala et al.,2005)).

Fig. 16

The use of channel chirality to distinguish revolution motors from rotation motors. A. In revolution motors, the right-handed DNA revolves within a left-handed channel (Guasch et al.,2002; Olia et al.,2011; Zhao et al.,2013). B. In rotation motors, the right-handed DNA rotates through a right-handed channel via the parallel thread, with RecA (Xing and Bell,2004a) and DnaB (Itsathitphaisarn et al.,2012) shown as examples (adapted from (De-Donatis et al.,2013)). (PDB IDs: RecA, 1XMS. (Xing and Bell,2004b); P22-gp1, 3LJ5. (Olia et al.,2011))

Fig. 17

Effect of DNA chemistry and structure on its packaging. A. Demonstration of blockage of dsDNA packaging by single-stranded gaps. When a single-stranded gap is present, only the left-end fragment of phi29 genomic DNA is packaged (adapted from (Moll and Guo,2005) with permission from Elsevier). B. T4 DNA packaging assays reveals that single stranded extensions with less than 12 bases at the DNA end do not inhibit translocation, whereas the ones with more bases do have a significant effect on the packaging (adapted from (Oram et al.,2008) with permission from Elsevier). C. Chemical modification of the negatively charged phosphate backbone on DNA packaging. Modification on the 3′→5′ strand does not block dsDNA packaging, but alternation on the other direction seriously affects DNA packaging. The results support the finding of the revolution mechanism showing that only one strand of the dsDNA interacts with the motor channel during revolution (adapted from (Aathavan et al.,2009) with permission from Nature Publishing Group).

Fig. 18

Elucidation of how the motor transports closed circular dsDNA without breaking any covalent-bonds or changing the topology of the DNA. A. Direct observation of ATPase complexes queuing and moving along dsDNA. Cy3 conjugated gp16 were incubated with (a, b, e) and without (d) dsDNA tethered between two polylysine beads. The zoomed in images are shown in (c) and (f). (g, h) The motion of the Cy3-gp16 spot was analyzed and a kymograph was produced to characterize the ATPase walking. (adapted from (Schwartz et al.,2013b) with permission from Elsevier). B. A model showing that gp16 hexamer acts as an open spiral filament similar to RecA filament with a different chirality rather than as a closed ring. (adapted from (De-Donatis et al.,2013)). C. Illustration of the assembly of the motor subunit on circular dsDNA. Subunits gather around the dsDNA chain first and then form a hexamer complex surrounding the DNA.