Cre
Recombinase
David Marcey
© 2006
I.
Introduction
II. Cre Monomer Structure
III. Cre-DNA Recombination Intermediates
IV. References
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I.
Introduction
The integrase
family of DNA recombinases includes over sixty members identified
by sequence similarity. Cre (shown at left) is a bacteriophage P1
member of the integrase family, catalyzing site-specific recombination
between two, 34-base pair lox DNA sequences. In vivo, the function
of Cre recombinase is to circularize the P1 genome during infection
and maintain the genome in the monomeric state for cell division.
Cre-lox recombination has also been extensively employed in in
vivo genetic engineering applications in a variety of organisms.
Before
examining structural features of Cre Recombinase, it may be helpful
to review the mechanism of Cre-induced, lox recombination as shown
in Figure 1, below.
Figure
1. The site specific recombination reaction between two, lox DNA molecules
(red and blue),
catalyzed by Cre recombinase.
(a) two Cre monomers bind to each lox site. A conserved, active
site tyrosine (tyr324) from one of
the monomers on each lox DNA molecule cleaves the DNA backbone, forming
a covalent, 3' phosphotyrosine bond, leaving a free, 5' hydroxyl (OH)
on one strand of each DNA double helix. (b) The 5' OH's perform
a nucleophillic attack on the phophotyrosines from the partner DNA
substrates, yielding a Holliday junction intermediate (c).
(c)-(d) A second round of tyr324-catalyzed breakage,
followed by strand joining reactions (nucleophillic attack of free
OH's on phosphotyrosines) resolves the Holliday junction into recombinant
products (e). Note that Cre monomers in the active conformation
(green) and inactive conformation (purple) change sequentially in
the process. Adapted from Figure 11-8, Watson, et al., 2003,
Molecular Biology of the Gene.
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to Cre-DNA Recombination Intermediates, below
II. Cre Monomer
Structure
The
monomeric Cre protein shown at left contains amino
terminal, linker, and carboxy
terminal domains.
The
amino terminal domain comprises 5 alpha
helices. Helices A
and E are involved in Cre recombinase
tetramer formation (see below). Helices
B and D contact the lox DNA major
groove.
The
carboxy terminal domain contains 9 alpha
helices and 3 beta strands. Helix
J, as well as several prominent
loops, are involved
in DNA interactions. Helix
N protrudes from the domain and plays a role in intersubunit
contacts (see below).
The
carboxy terminal domain harbors the Cre recombinase active
site, consisting of arg173,
his289,
arg292,
trp315,
and the phosphate-attacking tyr324
(see Figure 1).
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III. Cre-DNA
Recombination Intermediates
Let's now explore the structure of intermediates
in the Cre recombinase reaction. At left are shown two Cre monomers,
each bound to a single lox half site of one DNA
molecule. This corresponds to the top half of the schematic
representation of Cre-DNA Intermediate I in Figure
1b. Note that the two monomers are in different conformations,
only one of which is the active
conformation. These conformations are switched as the reaction
progresses (see below and Figure 1).
Several of the helices
mentioned in the previous section as being important in inter-Cre
subunit interaction are positioned to promote association of the two
monomers.
Helix N from one subunit has one face
buried in a hydrophobic pocket of the other monomer. Helices A
and E from different monomers interact
in a helix-helix interface. Other helices
are positioned to interact with the Cre dimer bound to the partner
lox DNA (not shown).
Note
that the B, D, and J helices from each
monomer are inserted into the major grooves of the lox half sites.
It can be seen that tyr324
from the active site of one monomer is
covalently linked to the DNA through
a 3' phosphate, a result of tyrosine-induced
cleavage of the DNA backbone (see Figure
1b).
The cleavage leaves a free
5' OH on one strand of DNA.
The 5' OH will act as a nucleophile,
attacking the phosphotyrosine from the partner DNA substrate (not
shown), joining strands from opposite DNA helices. This yields a Holliday
junction intermediate (see Figure 1b-c).
The structure of the Holliday
junction intermediate in the Cre recombinase reaction is shown at
left (compare to Figure 1c). The
DNA of the the formerly separate lox sites is now joined through one
recombinant strand. Cleavage and joining of the non-recombinant strands
of the Holliday junction will produce fully recombinant DNA helices
(Figure 1d-e).
The Cre subunits are assembled
into a tetramer on the Holliday junction DNA (Figure
1c), with each subunit bound to single lox half site. As
noted above, the two subunits bound to each DNA molecule are in different
conformations, with only one monomer on each DNA partner being active
at one time. The pair of subunits that are active
switches in the sequential “one strand at a time” mechanism of exchange
(Figure 1).
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IV.
References
Guo, F., Gopaul,
D. N., van Duyne, G. D.: Structure of Cre recombinase complexed with
DNA in a site-specific recombination synapse. Nature 389:
40-46 (1997).
Gopaul, D. N.,
Guo, F., Van Duyne, G. D.: Structure of the Holliday junction intermediate
in Cre-loxP site-specific recombination. EMBO J 17:
4175-4187 (1998).
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