and new DNA molecules are formed by breakage and joining of the homo-
logous segments. This process is facilitated by the protein RecA and is used
by cells to repair damaged DNA. The process has been seen by use of
electron microscopy of DNA undergoing homologous recombination.
In genetic recombination, a DNA molecule is formed with a sequence
derived from two strands. In 1964 Robert Holliday proposed a model
that involved the formation of an intermediate state that is now termed
a Holliday junction. The first step is the alignment of two homologous
duplexes (Figure 14.21). A strand from one duplex and the corresponding
strand are nicked, leading to a cross-over in which strands from differ-
ent duplexes are joined. The junction point can move along the strands
through a process called branch migration. The strands are then nicked
again, resulting in mixed strands. The structure of the Holliday junction
has been determined primarily using NMR, although some X-ray three-
dimensional structures are available.
To study the properties of the intermediate states, the DNA is first immo-
bilized by a 5′biotin tag to a streptavidin-coated glass slide (Churchill 2003;
McKinney et al. 2003). The motions of the DNA strands are observed by
placing fluorescent markers at the ends of the DNA (Figure 14.22). For
this particular set of experiments, a dye is attached at the end of one strand
and serves as the energy donor and a different dye is attached to the end of
another strand and serves as the energy acceptor. The Holliday junction
CHAPTER 14 OPTICAL SPECTROSCOPY 313
(b)Figure 14.21Model for a Holiday junction from two homologous duplexes. (a) The proposed
pathway involving alignment, cleavage, invasion, sealing, and migration followed by different
views of the cleavage process. (b) Three-dimensional view showing alignment of duplexes and
minor and major groves. Modified from Berg et al. (2007).
(a)