Chemistry Reference
In-Depth Information
Figure 11.3 (A) Repetitive shuttling of a Rep monomer revealed
by smFRET. (B) Physical mechanism of repetitive shuttling
involving transient loop formation via binding to the 3
0
end.
conformational change of the protein that enhances the af
nity on its secondary
binding site for a 3
0
end, forming a transient ssDNA loop before snapback and
repetition of translocation (Figure 11.3B). Supporting evidence includes: (1) periodic,
transient FRET increase when the two extremities of the ssDNA are labeled with the
FRET pair, (2) no repetitive shuttling when the 5
0
end is free, and (3) evidence of 2B
domain closing as the protein approaches the blockage . Repetitive shuttling was also
observed on ssDNA bounded by a stalled replication fork and an Okazaki fragment
analog, a DNA structure highly relevant to the function of Rep in vivo [94, 95], and it
was shown that Rep can interfere with RecA
filament formation on ssDNA. It is
possible that one of the in vivo functions of Rep is to shuttle repetitively on ssDNA
thereby keeping it free of unwanted proteins.
11.3.3.4 ssDNA Flexibility
In a separate study [13], we sought to understand the conformational properties of the
primary substrate of helicases by performing a systematic FRET study of ssDNA of
various lengths ((dT)
N
where N
10
-
70) over a wide range of NaCl concentrations
(0.025
-
2M). FRET increasedwith decreasing length andwith increasing salt. Using a
ΒΌ
