One-Way Traffic of a Viral Motor Channel for Double-Stranded DNA Translocation

Nanobiomedical Center, College of Engineering and College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267, USA.
Nano Letters (Impact Factor: 13.59). 09/2010; 10(9):3620-7. DOI: 10.1021/nl101939e
Source: PubMed


Linear double-stranded DNA (dsDNA) viruses package their genome into a procapsid using an ATP-driven nanomotor. Here we report that bacteriophage phi29 DNA packaging motor exercises a one-way traffic property for dsDNA translocation from N-terminal entrance to C-terminal exit with a valve mechanism in DNA packaging, as demonstrated by voltage ramping, electrode polarity switching, and sedimentation force assessment. Without the use of gating control as found in other biological channels, the observed single direction dsDNA transportation provides a novel system with a natural valve to control dsDNA loading and gene delivery in bioreactors, liposomes, or high throughput DNA sequencing apparatus.

Download full-text


Available from: Carlo Montemagno,
37 Reads
  • Source
    • "As for all other members of the superfamily, the packaging machine of the phi29 phage is an AAA+ ATPase. The extensive researches by Guo and co-workers have unambiguously demonstrated that the packaging motor is hexameric protein complex that functions in a one-way revolution model to push the viral DNA into the procapsid [49-53]. The mechanism involves the four electropositive lysine rings of the connector channel that facilitate the DNA revolution, and this occurs without DNA rotation inside the channel during the packaging process [42,54]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Nanobiomotors perform various important functions in the cell, and they also emerge as potential vehicle for drug delivery. These proteins employ conserved ATPase domains to convert chemical energy to mechanical work and motion. Several archaeal nucleic acid nanobiomotors, such as DNA helicases that unwind double-stranded DNA molecules during DNA damage repair, have been characterized in details. XPB, XPD and Hjm are SF2 family helicases, each of which employs two ATPase domains for ATP binding and hydrolysis to drive DNA unwinding. They also carry additional specific domains for substrate binding and regulation. Another helicase, HerA, forms a hexameric ring that may act as a DNA-pumping enzyme at the end processing of double-stranded DNA breaks. Common for all these nanobiomotors is that they contain ATPase domain that adopts RecA fold structure. This structure is characteristic for RecA/RadA family proteins and has been studied in great details. Here we review the structural analyses of these archaeal nucleic acid biomotors and the molecular mechanisms of how ATP binding and hydrolysis promote the conformation change that drives mechanical motion. The application potential of archaeal nanobiomotors in drug delivery has been discussed.
    Cell and Bioscience 06/2014; 4(1):32. DOI:10.1186/2045-3701-4-32 · 3.63 Impact Factor
  • Source
    • "These evidences have brought up a puzzle concerning how packaging can involve a rotation motor without any rotating components. In 2010, another question was raised regarding the inverse orientations of the Phi29 motor channel and dsDNA helices [52], which further questioned the involvement of rotational motion, since the rotation mechanism of dsDNA as a bolt threading onto a motor channel as a nut requires that the threads of the bolt and the nut have the same directionality. Recently, we have discovered that bacteriophage Phi29 dsDNA packaging motor uses a revolution mechanism without rotation, coiling, or torque forces (Figure 1) [9,50,53,54]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background Double-stranded DNA translocation is ubiquitous in living systems. Cell mitosis, bacterial binary fission, DNA replication or repair, homologous recombination, Holliday junction resolution, viral genome packaging and cell entry all involve biomotor-driven dsDNA translocation. Previously, biomotors have been primarily classified into linear and rotational motors. We recently discovered a third class of dsDNA translocation motors in Phi29 utilizing revolution mechanism without rotation. Analogically, the Earth rotates around its own axis every 24 hours, but revolves around the Sun every 365 days. Results Single-channel DNA translocation conductance assay combined with structure inspections of motor channels on bacteriophages P22, SPP1, HK97, T7, T4, Phi29, and other dsDNA translocation motors such as bacterial FtsK and eukaryotic mimiviruses or vaccinia viruses showed that revolution motor is widespread. The force generation mechanism for revolution motors is elucidated. Revolution motors can be differentiated from rotation motors by their channel size and chirality. Crystal structure inspection revealed that revolution motors commonly exhibit channel diameters larger than 3 nm, while rotation motors that rotate around one of the two separated DNA strands feature a diameter smaller than 2 nm. Phi29 revolution motor translocated double- and tetra-stranded DNA that occupied 32% and 64% of the narrowest channel cross-section, respectively, evidencing that revolution motors exhibit channel diameters significantly wider than the dsDNA. Left-handed oriented channels found in revolution motors drive the right-handed dsDNA via anti-chiral interaction, while right-handed channels observed in rotation motors drive the right-handed dsDNA via parallel threads. Tethering both the motor and the dsDNA distal-end of the revolution motor does not block DNA packaging, indicating that no rotation is required for motors of dsDNA phages, while a small-angle left-handed twist of dsDNA that is aligned with the channel could occur due to the conformational change of the phage motor channels from a left-handed configuration for DNA entry to a right-handed configuration for DNA ejection for host cell infection. Conclusions The revolution motor is widespread among biological systems, and can be distinguished from rotation motors by channel size and chirality. The revolution mechanism renders dsDNA void of coiling and torque during translocation of the lengthy helical chromosome, thus resulting in more efficient motor energy conversion.
    Cell and Bioscience 06/2014; 4(1):30. DOI:10.1186/2045-3701-4-30 · 3.63 Impact Factor
  • Source
    • "If the motor components do not rotate, then what is the mechanism of a motor that translocates a dsDNA helix? Unidirectional movement by a " push through a one-way valve " model was recently proposed (Schwartz et al., 2012; Fang et al., 2012; Zhao et al., 2013; Zhang et al., 2012; Jing et al., 2010) and solves this puzzle. Pushing DNA through the connector channel by the ATPase is entropically unfavorable, requiring the connector to function like a valve to prevent DNA from slipping out during DNA packaging (Black, 1989; Casjens, 2011; Guo and Lee, 2007). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Biomotors have been classified into linear and rotational motors. For 35 years, it has been popularly believed that viral dsDNA-packaging apparatuses are pentameric rotation motors. Recently, a third class of hexameric motor has been found in bacteriophage phi29 that utilizes a mechanism of revolution without rotation, friction, coiling, or torque. This review addresses how packaging motors control dsDNA one-way traffic; how four electropositive layers in the channel interact with the electronegative phosphate backbone to generate four steps in translocating one dsDNA helix; how motors resolve the mismatch between 10.5 bases and 12 connector subunits per cycle of revolution; and how ATP regulates sequential action of motor ATPase. Since motors with all number of subunits can utilize the revolution mechanism, this finding helps resolve puzzles and debates concerning the oligomeric nature of packaging motors in many phage systems. This revolution mechanism helps to solve the undesirable dsDNA supercoiling issue involved in rotation.
    Virology 11/2013; 446(1-2):133-43. DOI:10.1016/j.virol.2013.07.025 · 3.32 Impact Factor
Show more