Strand and nucleotide-dependent ATPase activity of gp16 of bacterial virus phi29 DNA packaging motor

Department of Biomedical Engineering, College of Engineering and College of Medicine, University of Cincinnati, Cincinnati, OH 45267, USA.
Virology (Impact Factor: 3.32). 09/2008; 380(1):69-74. DOI: 10.1016/j.virol.2008.07.003
Source: PubMed


Similar to the assembly of other dsDNA viruses, bacterial virus phi29 uses a motor to translocate its DNA into a procapsid, with the aid of protein gp16 that binds to pRNA 5'/3' helical region. To investigate the mechanism of the motor action, the kinetics of the ATPase activity of gp16 was evaluated as a function of DNA structure (ss- or ds-stranded) or chemistry (purine or pyrimidine). The k(cat) and K(m) in the absence of DNA was 0.016 s(-1) and 351.0 microM, respectively, suggesting that gp16 itself is a slow-ATPase with a low affinity for substrate. The affinity of gp16 for ATP was greatly boosted by the presence of DNA or pRNA, but the ATPase rate was strongly affected by DNA structure and chemistry. The order of ATPase stimulation is poly d(pyrimidine)>dsDNA>poly d(purine), which agreed with the order of the DNA binding to gp16, as revealed by single molecule fluorescence microscopy. Interestingly, the stimulation degree by phi29 pRNA was similar to that of poly d(pyrimidine). The results suggest that pRNA accelerates gp16 ATPase activity more significantly than genomic dsDNA, albeit both pRNA and genomic DNA are involved in the contact with gp16 during DNA packaging.

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    • "The motor undergoes a cycle of conformational changes between two distinct states during its interaction with ATP; the initial step is the binding of ATP that results in a reduction of entropy in the ATPase by a conformational change (Guo et al., 1987c; Ibarra et al., 2001; Lee et al., 2008). The entropy lost is compensated by a subsequent step of ATP hydrolysis resulting in entropy increase with another conformational change. "
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    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
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    • "Enzymatic activity via fluorescent labeling was described previously (Lee et al., 2008). Briefly, a phosphate binding protein conjugated to a fluorescent probe that senses the binding of phosphate was used to assay ATP hydrolysis. "
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    ABSTRACT: The AAA+ superfamily of proteins is a class of motor ATPases performing a wide range of functions that typically exist as hexamers. The ATPase of phi29 DNA packaging motor has long been a subject of debate in terms of stoichiometry and mechanism of action. Here, we confirmed the stoichiometry of phi29 motor ATPase to be a hexamer and provide data suggesting that the phi29 motor ATPase is a member of the classical hexameric AAA+ superfamily. Native PAGE, EMSA, capillary electrophoresis, ATP titration, and binomial distribution assay show that the ATPase is a hexamer. Mutations in the known Walker motifs of the ATPase validated our previous assumptions that the protein exists as another member of this AAA+ superfamily. Our data also supports the finding that the phi29 DNA packaging motor uses a revolution mechanism without rotation or coiling (Schwartz et al., this issue).
    Virology 05/2013; 443(1). DOI:10.1016/j.virol.2013.04.004 · 3.32 Impact Factor
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    • "In f29, gp16 itself was found to be a slow ATPase with low affinity to ATP, while it exhibited much higher activity in ATP hydrolysis when ssDNA, dsDNA, or RNA were bound (Guo et al., 1987d; Shu et al., 2003a). Interestingly, the strength of ATPase stimulation is dependent on the structure and chemistry of the nucleic acids, with an order of pRNA poly d(pyrimidine) > dsDNA > poly d(purine)(Lee et al., 2008). Similar DNA-dependent ATPase activities were also found in other viral systems, including l (Hwang et al., 1996) and T3 (Morita et al., 1993), and in many AAA þ helicases where ATP hydrolysis is stimulated from 10-to 100-folds. "
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    ABSTRACT: Double-stranded (ds)DNA viruses package their genomic DNA into a procapsid using a force-generating nanomotor powered by ATP hydrolysis. Viral DNA packaging motors are mainly composed of the connector channel and two DNA packaging enzymes. In 1998, it was proposed that viral DNA packaging motors exercise a mechanism similar to the action of AAA+ ATPases that assemble into ring-shaped oligomers, often hexamers, with a central channel (Guo et al. Molecular Cell, 2:149). This chapter focuses on the most recent findings in the bacteriophage ϕ29 DNA packaging nanomotor to address this intriguing notion. Almost all dsDNA viruses are composed entirely of protein, but in the unique case of ϕ29, packaging RNA (pRNA) plays an intermediate role in the packaging process. Evidence revealed that DNA packaging is accomplished via a "push through one-way valve" mechanism. The ATPase gp16 pushes dsDNA through the connector channel section by section into the procapsid. The dodecameric connector channel functions as a one-way valve that only allows dsDNA to enter but not exit the procapsid during DNA packaging. Although the roles of the ATPase gp16 and the motor connector channel are separate and independent, pRNA bridges these two components to ensure the coordination of an integrated motor. ATP induces a conformational change in gp16, leading to its stronger binding to dsDNA. Furthermore, ATP hydrolysis led to the departure of dsDNA from the ATPase/dsDNA complex, an action used to push dsDNA through the connector channel. It was found unexpectedly that by mutating the basic lysine rings of the connector channel or by changing the pH did not measurably impair DNA translocation or affect the one-way traffic property of the channel, suggesting that the positive charges in the lysine ring are not essential in gearing the dsDNA. The motor channel exercises three discrete, reversible, and controllable steps of gating, with each step altering the channel size by 31% to control the direction of translocation of dsDNA. Many DNA packaging models have been contingent upon the number of base pairs packaged per ATP relative to helical turns for B-type DNA. Both 2 and 2.5 bp per ATP have been used to argue for four, five, or six discrete steps of DNA translocation. The "push through one-way valve" mechanism renews the perception of dsDNA packaging energy calculations and provides insight into the discrepancy between 2 and 2.5 bp per ATP. Application of the DNA packaging motor in nanotechnology and nanomedicine is also addressed. Comparison with nine other DNA packaging models revealed that the "push through one-way valve" is the most agreeable mechanism to interpret most of the findings that led to historical models. The application of viral DNA packaging motors is also discussed.
    Advances in Virus Research 07/2012; 83:415-65. DOI:10.1016/B978-0-12-394438-2.00009-8 · 4.57 Impact Factor
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