Cre Recombinase
David Marcey
2003

I. Introduction
II. Cre Monomer Structure
III.
Cre-DNA Recombination Intermediates
IV. References


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.

reset Cre recombinase Please reload the Cre molecule before proceeding to the next sections.



II. Cre Monomer Structure

reset Cre recombinase 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 Cre recombinase 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.


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(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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