16 S.D. Levene
complex is responsible for carrying out specific cleavage, strand exchange,
and ligation steps that result in the inversion, deletion, or fusion of DNA seg-
ments. This biologically essential mode of DNA recombination is involved
in gene amplification and copy-number control [32], viral and phage host
specificity [33], the generation of antibodies [34, 35], and the transposition
of drug-resistance genes [36, 37]. There are also many emerging DNA-delivery
applications of site-specific recombination systems in biotechnology such as
therapeutic gene targeting, generation of chromosomal translocations, large
deletions, and tissue-specific or conditional knockouts as well as site-specific
integration, and the precise removal of selectable markers [38, 39].
Flp is a eukaryotic site-specific recombinase from budding yeast
(Saccharomyces cerevisiae), which is believed to play a role in maintaining
the 2-μm circle, a yeast plasmid, at high levels independent of chromosomal
copy-number control. This enzyme is responsible for a reaction that causes in-
version of a DNA segment near the origin of replication on the 2-μm circle to
generate a quasirolling-circle replication intermediate. This intermediate form
can lead to the production of many tandem copies of the 2-μm genome, which
are subsequently split into individual monomeric circles via a deletion reaction
also carried out by the recombinase. Much of our understanding of the mecha-
nism of Flp recombination comes from in vitro studies employing naked DNA
and the purified protein. However, as a eukaryotic recombinase, Flp must con-
tend with the presence of nucleosomes and higher order chromatin structure
in vivo. Although as yet incompletely understood, some efforts to characterize
the in vivo behavior of this system have been reported (see below).
The Flp system is a member of the Int superfamily of site-specific recom-
binases, so called because of their mechanistic similarities to the integrative
recombination system of bacteriophageλ. The phageλsystem, which con-
sists of several proteins, is responsible for integration of the bacteriophage
genome at a specific location in theE. colichromosome and its subsequent
excision at the onset of the lytic stage of the phage life cycle. Some com-
mon features of the recombinases in the Int superfamily are the formation
of torus knots and catenanes and mechanisms that proceed via an obligate
four-stranded DNA structure during recombination, termeda Holliday junc-
tion(Fig. 2.9). Holliday junctions are key intermediates in a number of other
DNA-recombination events, including homologous or general recombination,
which, unlike site-specific recombination, can occur at essentially arbitrary
locations throughout a genome.
Torus knots and catenanes are so called because these particular forms can
be drawn on the surface of a torus. The fact that only products of this topology
are formed suggests that the juxtaposition of recombination sites takes place
through a “random-collision” mechanism that traps a variable number of su-
perhelical turns between the sites (Fig. 2.10). However, with relaxed circular
DNA a bias in the distribution of recombination-product topology indicates
that asymmetry exists within the synaptic complex, a feature that is likely to
be related to the structure of the Holliday-junction intermediate [40].