Chemistry Reference
In-Depth Information
Monte Carlo simulation of the worm-like chainmodel we deduced that the persistent
length of poly-dT ranges from 1.5 nm in 2M NaCl to 3 nm in 25mM NaCl. We were
not able to observe any temporal changes in smFRETeven with 1ms time resolution
and therefore concluded that ssDNA conformation is rapidly averaged over a time
scale much faster than 1ms. Thus, smFRET detected during ssDNA translocation
and DNAunwindingmust report on the temporally averaged values over the range of
allowed ssDNA conformations.
11.4
SmFRET Studies of Holliday Junction
Homologous recombination is an essential process inmaintaining genomic stability
and its defects can lead to serious human diseases including cancer [96
-
99]. To cope
with DNA damage encountered during genome duplication, a four-way (Holliday)
junction (HJ) is formed by joining two nearly identical DNA molecules. Although
conventional ensemble tools such as gel electrophoresis,
uorescence resonance
energy transfer (FRET) [100
102], transient electric birefringence [103], chemical
-
probing [104
106], X-ray crystallography [107
111], atomic force microscopy [112
-
-
-
114], small-angle X-ray scattering [115] and NMR [116
121] have provided crucial
insights into the HJ lifecycle, many of its dynamic aspects remain poorly understood.
We have developed smFRET assays to study the structural dynamics of the HJ.
Many of these properties are dif
cult to address unambiguously using conventional
ensemble techniques. Our studies provide the
rmbaseline for future studies on the
various enzymes that participate in homologous recombination and how their
activities are modulated by the intrinsic properties of the HJ.
-
11.4.1
Structure and Function of HJ
The HJ is a four-way DNA junction in which four helices are connected by the
covalent continuity of the four single strands (see Figure 11.4 for conventions used
here). The HJ structure has been extensively studied using non-migratable (i.e.
sequence prohibits branch migration) HJs built from oligonucleotides [122
-
124].
In the absence of divalent ions such as Mg
2þ
, the HJ is unfolded and takes on a so-
called open structure, where each of the four helices points to a corner of a square
(Figure 11.4A) [101]. In physiologically relevant conditions, for instance with 1mM
Mg
2 þ
, the HJ folds into the antiparallel stacked-X structure (Figure 11.4B and
C) [100, 105]. A pair of helices is stacked against each other (that is, two helices form
an essentially continuous helix, much like a regular B-DNA). For instance, helix X in
Figure 11.4B can stack with helix R forming a super-helix XR while helices H and
B can stack to form a super-helix HB. These super-helices can be considered as
cylinders that are arranged in anX shape with an inter-helical angle of
60
[125
-
127].
An alternative stacking conformer is also possible where helix X stacks on helix R
and helix H stacks on helix B (Figure 11.4C).
