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Chapter 12 : Theme and Variation in Tyrosine Recombinases: Structure of a Flp-DNA Complex

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Abstract:

The recently determined structure of a Flp-DNA complex confirms that its catalytic domain closely resembles that of other family members, although the active site itself is assembled differently. The reaction mechanism as drawn is the distillation of years of work by many laboratories. In addition to the structure of the Flp-DNA complex, which is the focus of this chapter, crystal structures of four members of this family have been determined: the core domains of the λ () and HP1 (23) phage integrases, intact XerD (52), and intact bacteriophage P1 Cre. The active site of Flp has been known for some time to differ from that of its relatives in one very important aspect: in contrast to other tyrosine recombinases, including the λ and HP1 phage integrases, Cre (20), and XerC and D (5, 7), the active sites within Flp complexes are composed of amino acid residues from two different monomers. The chapter briefly describes the structure of a Flp-Holliday junction complex and its biochemical implications, and explores its similarities to and differences from other tyrosine recombinases and the type IB topoisomerases.

Citation: Rice P. 2002. Theme and Variation in Tyrosine Recombinases: Structure of a Flp-DNA Complex, p 219-229. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch12

Key Concept Ranking

Eukaryotic Topoisomerase I
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Type IB Topoisomerase
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Spacer DNA
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Figures

Image of Figure 1
Figure 1

Proposed mechanism of site-specific recombination catalyzed by the tyrosine recombinases. Each recombinase monomer is represented by a single Y. Circles indicate the two active tyrosines, which form transient 3′ phosphotyrosine linkages with the DNA. The central isomerization step (right) activates the alternate pair of active sites, allowing for exchange of the second pair of DNA strands.

Citation: Rice P. 2002. Theme and Variation in Tyrosine Recombinases: Structure of a Flp-DNA Complex, p 219-229. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch12
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Image of Figure 2
Figure 2

DNA substrate in the Flp crystal structure. (a) The symmetrized Flp binding site used in crystallization, numbered from center. Two identical half-sites (plain and bold text) anneal to form the duplex shown, with nicks at the junction. Flp cleaves DNA between nucleotides −4 and −5, one base away from the nick. (b) Pathway for formation of the Holliday junction present in the crystals. Two duplexes are synapsed by a tetramer of Flp protein. Only the sequence at the center is shown, and small circles represent the scissile phosphates. Due to the nicks, T-4 is lost into solution upon attack of the tyrosine. Since the spacer sequence used allows slippage of the base pairing, T-3 of the opposite duplex can fit into the pocket vacated by T-4, and its 5′ hydroxyl can attack the phosphotyrosine intermediate. After isomerization, the same sequence of events can occur at the other pair of active sites, resulting in a Holliday junction with a central unpaired A. All four scissile bonds were hydrolyzed at some point before data collection. This probably occurred because the Holliday junction was in equilibrium with the phosphotyrosine intermediate, which is subject to slow hydrolysis ( ).

Citation: Rice P. 2002. Theme and Variation in Tyrosine Recombinases: Structure of a Flp-DNA Complex, p 219-229. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch12
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Image of Figure 3
Figure 3

Synaptic complexes of Flp and Cre. (a) Tube-and-arrow representation of the Flp complex. The N-terminal domain is above the DNA, and the catalytic domain, below. Inactive monomers are shaded. The trans-donated active site tyrosine is shown in black. The tyrosine donated by one of these monomers is disordered. Dotted lines represent connections not visible in the electron density. (b) Similar representation of the Cre complex ( ). The nucleophilic tyrosine is in dark gray but is largely occluded in this view. (c) and (d) The Flp and Cre complexes, rotated such that the catalytic domains are at the bottom of the picture. (e) and (f) Cartoons of the Flp and Cre complexes, as seen from the underside of panels a and b (catalytic domains above the DNA). HelicesMand N are shown explicitly. Note that in both cases, the helix M-N segment that traverses a type I interface contains the active tyrosine, regardless of the provenance of that tyrosine.

Citation: Rice P. 2002. Theme and Variation in Tyrosine Recombinases: Structure of a Flp-DNA Complex, p 219-229. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch12
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Image of Figure 4
Figure 4

Proposed Holliday junction isomerization step for Flp-mediated recombination. Yapos;s represent the nucleophilic tyrosines and the circles denote the active pair. The two half-FRT sites under the attack of Y343 are below the plane of the figure. The conformational changes that must occur during isomerization can be deconvoluted into a translation of each monomer by ∼1.5 Å along the DNA axis, followed by 3 rotations pivoting about the scissile phosphate: a scissoring of the DNA arms by ∼7°, then a rotation of ∼15° that moves the DNA arms into or below the plane of the junction, followed by a rotation of ∼15° around the DNA axis itself. This isomerization changes a type I interface to a type II, and vice versa.

Citation: Rice P. 2002. Theme and Variation in Tyrosine Recombinases: Structure of a Flp-DNA Complex, p 219-229. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch12
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Image of Figure 5
Figure 5

Proposed advantage for trans cleavage by Flp. (a) Cartoon of the Futcher model for amplification of the 2μm plasmid copy number by Flp recombination ( ). If Flp inverts the segment of DNA between the two FRT (Flp recombination target) sites after the replication fork has passed one, multiple copies of the plasmid are eventually made from a single firing of the origin. (b) Since Flp must act during replication, there is a danger of the replication machinery colliding with Flp in its covalent intermediate form. Although the original monomer may become unfolded or degraded after this encounter (“corpse”), a second monomer could catalyze the religation of the nick and release of the covalently bound damaged protein.

Citation: Rice P. 2002. Theme and Variation in Tyrosine Recombinases: Structure of a Flp-DNA Complex, p 219-229. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch12
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