Membrane-Embedded Channel of Bacteriophage Phi29 DNA-Packaging Motor for Translocation and Sensing of Double-Stranded DNA - Nanopores: Sensing and Fundamental Biological Interactions - page 101

During the dsDNA packaging process, the active motor was turned off by
adding a non-hydrolysable ATP analog, -S-ATP [ 51 ]. Both the completely and

partially packaged dsDNA remained in the procapsid and did not show a reverse
exit in the gradient under all centrifugation force tested (e.g., 149,000 g) [ 51 , 52 ].
The finding agrees with the phenomena observed previously that dsDNA did
not eject from the channel and remained within the channel of the DNA-packaging
intermediates [ 19 , 21 , 51 ]. DNA-packaging by the motor is an entropically unfa-
vorable process and chemical energy from ATP hydrolysis is required for DNA
translocation [ 19 , 53 ]. The pressure within viral procapsids gradually increases
when longer and longer dsDNA are packaged as the space within the cavity of
the procapsid becomes smaller and smaller [ 19 , 21 , 51 , 54 ].
To argue for the one-way traffic property of phi29 DNA-packaging channel in
a biologically active motor and to rule out the possibility that dsDNA remaining
inside the channel is due to the contributions of pRNA, gp16, and/or

g

-S-ATP,
sedimentation assays were performed. The fully packaged DNA and the DNA
packaging intermediates were treated individually and in combination with
DNase I and/or RNase A (Fig. 4.15 ). DNase treatment excludes the possibility
of any contact of motor components that could restrain the dsDNA from exiting,
while RNase A digestion specifically excludes the possibility of pRNA and gp16
from contacting and holding the DNA. Since pRNA serves as a bridge between
the motor and gp16 [ 20 ], RNase digestion will eliminate the fulcrum to prevent
the exercise of force,

g

if any, from pRNA and gp16 to dsDNA. When the

Fig. 4.15 Sedimentation assays for DNA packaging and retention within the procapsid with [3H]-
phi29 DNA via 5-20% sucrose gradient. DNA was packaged into the procapsid through the
channel of phi29 DNA packaging motor with the in vitro phi29 DNA packaging system [ 57 , 58 ]).
After DNA packaging, the samples were divided and digested with either DNase I or RNase A,
respectively. All samples were loaded onto the top of the gradient and sedimented from right to left
by ultracentrifugation followed by scintillation counting. Figures reproduced with permissions
from: Ref. [ 44 ], # American Chemical Society