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The Serine Recombinases

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  • Author: W. Marshall Stark1
  • Editors: Phoebe Rice2, Nancy Craig3
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Institute of Molecular, Cell and Systems Biology, University of Glasgow, Bower Building, Glasgow G12 8QQ, Scotland, United Kingdom; 2: University of Chicago, Chicago, IL; 3: Johns Hopkins University, Baltimore, MD
  • Source: microbiolspec November 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0046-2014
  • Received 28 July 2014 Accepted 01 August 2014 Published 07 November 2014
  • Marshall Stark, marshall.stark@glasgow.ac.uk
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  • Abstract:

    In site-specific recombination, two short DNA sequences (‘sites’) are each cut at specific points in both strands, and the cut ends are rejoined to new partners. The enzymes that mediate recognition of the sites and the subsequent cutting and rejoining steps are called recombinases. Most recombinases fall into one of two families according to similarities of their protein sequences and mechanisms; these families are known as the tyrosine recombinases and the serine recombinases, the names referring to the conserved amino acid residue that attacks the DNA phosphodiester and becomes covalently linked to a DNA strand end during catalysis. This chapter gives an overview of our current understanding of the serine recombinases, their types, biological roles, structures, catalytic mechanisms, mechanisms of regulation, and applications.

  • Citation: Stark W. 2014. The Serine Recombinases. Microbiol Spectrum 2(6):MDNA3-0046-2014. doi:10.1128/microbiolspec.MDNA3-0046-2014.

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2014-11-07
2017-09-23

Abstract:

In site-specific recombination, two short DNA sequences (‘sites’) are each cut at specific points in both strands, and the cut ends are rejoined to new partners. The enzymes that mediate recognition of the sites and the subsequent cutting and rejoining steps are called recombinases. Most recombinases fall into one of two families according to similarities of their protein sequences and mechanisms; these families are known as the tyrosine recombinases and the serine recombinases, the names referring to the conserved amino acid residue that attacks the DNA phosphodiester and becomes covalently linked to a DNA strand end during catalysis. This chapter gives an overview of our current understanding of the serine recombinases, their types, biological roles, structures, catalytic mechanisms, mechanisms of regulation, and applications.

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FIGURE 1

Site-specific recombination. Two sites (pointed boxes) in double-helical DNA (shown as double lines) are recognized by a recombinase protein (not shown), and then cut and rejoined to form recombinants. doi:10.1128/microbiolspec.MDNA3-0046-2014.f1

Source: microbiolspec November 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0046-2014
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FIGURE 2

Site-specific recombination outcomes. (a) Recombination between two sites in separate linear DNA molecules results in two linear recombinant products. Usually, the sites have a polarity (indicated by the pointed boxes) such that the lower pathway (red arrow) is forbidden. (b) Recombination between two sites in separate DNA molecules, when one or both of the molecules is circular, results in a single product molecule containing two sites in direct repeat. This is called integration or fusion. The “reverse” reaction splits a molecule containing two sites into two product molecules, one or both of which are circular. This is called resolution, excision, or deletion (depending on the biological context). (c) Recombination between two sites in inverted repeat in a DNA molecule inverts the orientation of one segment of DNA relative to the other. This is called inversion. doi:10.1128/microbiolspec.MDNA3-0046-2014.f2

Source: microbiolspec November 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0046-2014
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FIGURE 3

Recombination sites. (a) A typical recombination site. The crossover site, where strand exchange takes place (at the position marked by the staggered red line), binds a recombinase dimer and typically has partial dyad symmetry (indicated by the blue arrows). “Accessory sites,” which may be adjacent on one side of the crossover site (as shown), on both sides or more distant, may bind additional recombinase subunits or other proteins, or may make looping interactions with recombinase bound at the crossover site. (b) Example of a real crossover site (, a site acted upon by Hin recombinase). The colors and symbols are as in part (a). Hin, like all serine recombinases characterized to date, cuts the DNA at the center of the crossover site with a 2 bp “stagger” as shown. doi:10.1128/microbiolspec.MDNA3-0046-2014.f3

Source: microbiolspec November 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0046-2014
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FIGURE 4

The serine recombinase strand-exchange mechanism. A synaptic complex of two crossover sites bridged by a recombinase tetramer (yellow ovals) is shown. The four subunits are spaced out, so that the catalytic steps can be seen clearly. The catalytic serine residues are indicated by S-OH. The scissile phosphodiesters are represented as circled Ps, and in the first and last panels the 2-bp overlap sequence is indicated by vertical lines. For further details, see text. doi:10.1128/microbiolspec.MDNA3-0046-2014.f4

Source: microbiolspec November 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0046-2014
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FIGURE 5

Domain structures of serine recombinases ( 26 ). The SR (catalytic) domain (shown in pink; typically ∼150 amino acids) is common to all serine recombinases and contains the active site (red star). Small serine recombinases (including resolvases and invertases) have a helix–turn–helix DNA-binding domain (blue; ∼40 amino acids) at the C terminus. Some related recombinases such as ISXc5 resolvase have additional C-terminal domains (orange) of unknown function. Serine transposases have a similar helix–turn–helix domain at the N terminus. Large serine recombinases have multiple domains at the C terminus of the SR domain ( 27 ). doi:10.1128/microbiolspec.MDNA3-0046-2014.f5

Source: microbiolspec November 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0046-2014
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FIGURE 6

Crystal structures of γδ resolvase–NA complexes. (a) Wild-type γδ resolvase dimer bound to crossover-site DNA (PDB 1GDT; 40). The subunits are in cartoon representation (green and orange). The active site serine residues (α carbons) are indicated by magenta spheres. (b) Activated mutant γδ resolvase tetramer in a cleaved-DNA synaptic intermediate (PDB 1ZR4; 41). The resolvase is rendered as in (a). The active-site serines are covalently linked to DNA ends (see Fig. 4 ); only two are visible. This view emphasizes the flat interface (marked by a dashed red line) between “rotating pairs” of resolvase subunits. The red arrows indicate positions of double-strand breaks in the DNA. The structure corresponds to the intermediates cartooned in the two central panels of Fig. 4 . doi:10.1128/microbiolspec.MDNA3-0046-2014.f6

Source: microbiolspec November 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0046-2014
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FIGURE 7

Topologically selective recombination by Tn/γδ resolvase. (a) The reaction pathway of resolvase (lower row) is contrasted with that of a non-selective recombinase (upper row). Random collision of sites results in products with a variety of topologies (a 6-noded catenane is shown as an example here). Selective synapsis by resolvase results in a product with a specific topology (2-noded catenane). (b) Architecture of the synapse. The Tn/γδ site is diagrammed on the left. On the right, the arrangement of DNA in the synapse is shown. The catalytic tetramer bound to the crossover sites (the “catalytic module”) is represented as an orange oval, and the eight resolvase subunits bound at the accessory sites (the “regulatory module”) are collectively represented by the pink oval. Chapter [X], this volume, gives more details on the structures of this and other synaptic complexes. doi:10.1128/microbiolspec.MDNA3-0046-2014.f7

Source: microbiolspec November 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0046-2014
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FIGURE 8

Subunit rotation mechanism of resolvase. (a) Topologies of first round and “iteration products” observed by Cozzarelli's group ( 58 , 59 , 60 ). The upper part shows the products predicted by a rotation mechanism in a resolvase synapse with topology as shown in Fig. 7 . The lower panels show the simplified topologies of these products. “Mismatched” substrates (see text) form only the nonrecombinant knot products, starting with the 4-noded knot. (b). Cartoon illustrating the proposed subunit rotation mechanism. DNA is represented as ribbons and recombinase subunits as ovals. The crystal structure of a proposed intermediate in subunit rotation is shown in Fig. 6b . doi:10.1128/microbiolspec.MDNA3-0046-2014.f8

Source: microbiolspec November 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0046-2014
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FIGURE 9

Cartoon of proposed inversion synaptic complex. An invertase tetramer bridging the two crossover sites is shown as an orange oval, and contacts the enhancer DNA (brown) and a FIS dimer bound there (pink). The DNA in the complex is intertwined as shown. Supercoiled loops of DNA outside the complex are shown as dashed lines. doi:10.1128/microbiolspec.MDNA3-0046-2014.f9

Source: microbiolspec November 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0046-2014
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FIGURE 10

Recombination by serine integrases. In nature, integrase promotes recombination between a crossover site, , in the circular bacteriophage DNA and a different crossover site, , in the host bacterial genome (indicated by a squiggly line). Integrase alone does not promote any reaction between the product sites and . However, the presence of a bacteriophage-encoded recombination directionality factor (RDF) protein alters the properties of integrase so that it preferentially promotes × recombination (red arrow). See text for further details. doi:10.1128/microbiolspec.MDNA3-0046-2014.f10

Source: microbiolspec November 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0046-2014
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Tables

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TABLE 1

Structural data for serine recombinases

Source: microbiolspec November 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0046-2014

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