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(iii) Unwinding of a 40-bp duplex often paused, resulting in either DNA rezipping
(FRETrecovery) or the restart of unwinding; the latter required free proteins in
solution. We interpreted the results as unwinding stalls when a monomer
dissociates from the functional dimer. DNA rezips if the remaining monomer
also dissociates. If instead another monomer comes to the rescue, they can
form a functional dimer and continue to unwind (Figure 11.2).
11.3.3.2 Protein
DNA FRET
In a subsequent study [37], we carried out site-directed mutagenesis to create single-
cysteine Rep mutants that were fully functional both in vivo,testedthroughthephage
replication assay [93], and in vitro, tested by bothmultiple turnover and single turnover
unwindingwithandwithout dye label.We showedthat thePEGsurfaceeliminatednon-
speci
c binding of labeled protein to the surface and we could detect the moment of
singleRepproteinbindingtoanimmobilized3
0
-tailedDNAasthesuddenappearanceof
a
fluorescence signal. By working with sub-nanomolar protein concentrations (K
D
is
about 5
-
10 nM [48]) and with over 90% labeling ef
ciency, single protein binding
events were predominant and could be easily distinguished from raremultiple protein
binding events which showed higher
fluorescence signal and multi-step binding and
dissociation (or photobleaching). We measured FRET from a donor on the protein to
an acceptor on the DNA junction for each of the eight single cysteine mutants in the
absence of ATP. Importantly, we obtained highly similar FRET ef
ciencies when the
donor and the acceptor locations were swapped or when different donors with similar
spectral properties were used. Our data con
rmed the crystallographic binding orien-
tation of ssDNA relative toRep and showed that the proteinobtains primarily the closed
conformation when it is bound to a 3
0
-tailed DNA in solution, consistent with the PcrA
structure bound to a 3
0
-tailed DNA [76]. This study validated the smFRET approach
using the labeled protein and laid a
-
firm foundation for functional studies such as
DNA translocation [38] and unwinding powered by ATP hydrolysis.
11.3.3.3 Repetitive Shuttling
In a recent article [38], we showed that the 3
0
to 5
0
ssDNA translocation of a Rep
monomer can be detected as gradual change in FRET between the protein and
the DNA. ssDNA is highly
flexible so FRET here is averaged over conformations of
the intervening ssDNA. Surprisingly, when the protein encountered an insurmount-
able blockade, either the dsDNA that it cannot unwind as amonomer or a streptavidin
attached to a 5
0
-biotin, it snapped back to near the 3
0
end abruptly within a time
resolution of 15ms and repeated the gradual translocation followed by another
snapback and so on. This was observed in the data as the sawtooth pattern of the
smFRET time traces (Figure 11.3A). We termed this novel behavior repetitive
shuttling. Several lines of evidence strongly indicated that a single monomer was
doing the repetitive shuttling, not multiple monomers in succession [38], and
repetitive shuttling was observed over a wide range of salt concentrations and
temperature, and also on DNA with mixed sequences. The repetition period was
proportional to the tail length, insensitive to the DNA sequence, and was longer at
lower ATP concentrations. We proposed its physical mechanismas blockage-induced
