Cre Recombinase
David Marcey
©2003
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, Cre recombinase is utilized to maintain the P1 genome in a
lysogenic state, but the Cre-lox system has been extensively employed in in
vivo and in in vitro genetic engineering applications in a variety
of organisms.
The mechanism of Cre-induced,
lox recombination is schematically represented in Figure
1.
Please reload the Cre molecule before proceeding to the
next sections.
II. Cre Monomer Structure
The protein at left is a Cre monomer.
The Cre protein 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).
reset molecule
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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 (Figure
1b).
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).
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, joining strands from opposite
DNA helices. This yields a Holliday junction intermediate (see Figure
1b-c).
The structure of the Holliday-type 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-type Intermediate will produce fully recombinant DNA helices
(Figure 1d-e).
The Cre subunits are assembled into a tetramer on the Holliday-type
junction DNA, each subunit bound to single lox half site.
reset molecule
(Cre-DNA Intermediate I)
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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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