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Chapter 3 : Nonhomologous Recombination

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Abstract:

This chapter focuses on the two principal and best-studied types of nonhomologous recombination event: site-specific recombination and transposition. In site-specific recombination, two alternative types of enzyme, the tyrosine (Y) and serine (S) recombinases, are involved. The enzymes involved in transposition reactions are known as transposases (Tpases). The majority are in a class generally called DDE transposases because of the presence of three key amino acids, two aspartate residues and a glutamate, as part of the active site. The chapter presents a short overview of nonhomologous prokaryotic recombination systems and of the mechanisms involved in their respective recombination reactions. It describes the protein and DNA components involved and considers the various levels of control in these systems. Accessory elements can play a crucial role in the recombination process. Although they are not directly involved in catalysis, they may influence all steps of the reaction. Transposable elements have developed numerous ways of controlling their activity since high transposition rates might be expected to be deleterious for the "host" cell. An extremely large number of genetic elements have now been detected which use the nonhomologous recombination mechanisms. Some of these are briefly described in the chapter. The importance of nonhomologous recombination in shaping bacterial genomes, in their evolution and their expression, is considerable.

Citation: Cornet F, Chandler M. 2004. Nonhomologous Recombination, p 36-66. In Miller R, Day M (ed), Microbial Evolution. ASM Press, Washington, DC. doi: 10.1128/9781555817749.ch3

Key Concept Ranking

Mobile Genetic Elements
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Gene Expression and Regulation
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Bacterial Proteins
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Genetic Elements
0.52113706
Chromosomal DNA
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0.77643263
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Figures

Image of FIGURE 1
FIGURE 1

Components of the horizontal gene pool.

Citation: Cornet F, Chandler M. 2004. Nonhomologous Recombination, p 36-66. In Miller R, Day M (ed), Microbial Evolution. ASM Press, Washington, DC. doi: 10.1128/9781555817749.ch3
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Image of FIGURE 2
FIGURE 2

Nuts and bolts of site-specific recombination. The core sequences and domain organization of the recombinases are shown for S recombinase-driven systems (A) and Y recombinase-driven systems (B). The core sequences are represented with their two inverted recombinase recognition sequences symbolized by the inverted arrows. The lengths of the central region separating these binding sites are indicated in base pairs. The major domain organization of the recombinases is symbolized for different members of the two families. These represent the major subclasses within each family. The names of the proteins and the assigned function of the domains are indicated. Catalytic domains are shown in gray with patches of residues important for catalysis indicated by white squares. The number of amino acid residues comprising each protein is also indicated. (A) For resolvase (Res) and invertase (Hin), the catalytic serine (S) that makes covalent bonds with DNA is shown together with additional important residues defining the two conserved patches of catalytic residues. These are highly conserved throughout the subfamilies. The position of the α-helix E that links the catalysis and the DNA-binding domain in the Tn/γδ resolvase is also indicated (see the text). For the less well described ϕC31 Int and IS Tpase, only the global domain organization is shown. (B) The two patches that contain the main catalytic residues are shown with the most important residues indicated. The catalytic tyrosine (Y) is shown in bold. The XerC and XerD recombinases have the same number of residues.

Citation: Cornet F, Chandler M. 2004. Nonhomologous Recombination, p 36-66. In Miller R, Day M (ed), Microbial Evolution. ASM Press, Washington, DC. doi: 10.1128/9781555817749.ch3
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Image of FIGURE 3
FIGURE 3

Accessory elements in model systems. Schematic representation of various recombination sites with the position and orientation of binding sites for the different accessory elements. The arrowheads represent recombinase-binding sites and their relative orientations. The core sequences are shown in gray and the accessory binding sites by open symbols. In the Tn, the sequence arrangement shown represents that earned by the excised circular element. I, II, and III are sites I, II, and III. See text for details.

Citation: Cornet F, Chandler M. 2004. Nonhomologous Recombination, p 36-66. In Miller R, Day M (ed), Microbial Evolution. ASM Press, Washington, DC. doi: 10.1128/9781555817749.ch3
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Image of FIGURE 4
FIGURE 4

Catalysis by serine recombinases. (A) The essential features of recombinase binding to a single core sequence (site I in Fig. 3B ). The catalytic domain is shown as a square including the catalytic serine, S, and is connected to the DNA-binding domain that contacts the DNA on the opposite side of the helix (oval), by α-helix E. Open circles indicate the sessile phosphates: one above and the other (dotted line) below the DNA. (B) The catalytic mechanism of S recombinase. (i) The synapse. The two core sequences are shown in a parallel configuration. A tetramer of recombinase is bound at the core sequences. Open circles indicate the scissile phosphates and vertical lines indicate the 2 base pairs of the central region. S indicates the catalytic serine that attacks the scissile phosphodiester bonds (symbolized by the curved arrows), (ii) Cleaved intermediate before rotation. Cleavage generates four covalent DNA-recombinase 5′-phosphoserine bonds and liberates four 3′-OH ends. Strand exchange involves a 180° rotation of the two left-half sites with respect to the two right-half sites (symbolized by the large circular arrow), (iii) Cleaved intermediate after rotation. After the rotation step, the four 5′-OH ends perform a nucleophilic attack (symbolized by the curved black arrows) on the four 3′-phosphoscrine bonds, thereby closing the four DNA nicks and liberating the recombinase monomers from their covalent attachment to the DNA substrate. (iv) Recombination products.

Citation: Cornet F, Chandler M. 2004. Nonhomologous Recombination, p 36-66. In Miller R, Day M (ed), Microbial Evolution. ASM Press, Washington, DC. doi: 10.1128/9781555817749.ch3
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Image of FIGURE 5
FIGURE 5

Structure of the synaptic complex and catalytic mechanism of Y recombinases. (i) Synapse. The two core sequences are in an antiparallel configuration bound with a recombinase tetramer. The small open circles on the DNA represent the scissile phosphates that border the central region. The large circles in front of the DNA represent the N-terminal DNA-binding domain (not shown in the rest of the figure). A flexible linker connects this domain to the catalytic C-terminal domain (the large ellipse with the catalytic tyrosine residue shown) to form a C-shaped clamp around the DNA. The main interaction domains between the recombinases (the hairpin) extend from the C-terminal domains to dock into specific recipient pockets into the adjacent monomer. The main DNA deformation (the pronounced kink at the uncleaved edge of the central region) is shown. The gray recombinases are in an active conformation for cleavage and perform a nucleophilic attack on the scissile phosphodiester bonds (symbolized by the small curved arrows). (ii) Transfer of the first pair of strands. Cleavage generates two DNA-recombinase 3′-phosphotyrosine bonds (symbolized by the link between the DNA and the catalytic tyrosines of the gray monomers) and liberates two 5′-OH ends. The 5′-OH ends then attack the phosphotyrosine bond of the partner core sequence, thereby liberating the gray recombinase monomers from their covalent bond with the DNA and creating a four-way Holliday junction intermediate, (iii) The Holliday junction intermediate before isomerization. In the four-way junction, generated by exchange of the first pair of strands, the angles formed between the four DNA arms are not equivalent. The interactions between adjacent recombinase monomers are also not equivalent (here two C-terminal extensions are represented kinked and the two others are straight). This Holliday junction intermediate must isomerize to allow exchange of the second pair of strands, (iv) The Holliday junction intermediate after isomerization. Isomerization inverts the small and large angles between the DNA arms. Similarly, the short (here kinked) C-terminal interaction domains extend and the previously extended (here straight) domains contract. This leads to catalytic activation of the previously inactive (white) pair of recombinase monomers and inactivation of the previously active (gray) monomers and allows exchange of the second pair of strands by a mechanism similar to that involved in exchange of the first pair of strands.

Citation: Cornet F, Chandler M. 2004. Nonhomologous Recombination, p 36-66. In Miller R, Day M (ed), Microbial Evolution. ASM Press, Washington, DC. doi: 10.1128/9781555817749.ch3
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Image of FIGURE 6
FIGURE 6

Topological filters in S-recombinase-driven systems. A linear representation of the recombination sites is shown (top panels) with the arrows representing the binding sites for the recombinases. The core sequences are shown in gray. The enhancer sequence of the Hin system is shown as an open bar edged with the two open squares that represent the binding sites for the Fis protein. The drawing at the bottom of each section represents the organization of the respective synaptic complexes as postulated by recent models (see text). The open curved bars symbolize the recombination sites, and the enclosed small black arrows indicate their relative orientation (in direct repetition in the sites and inverted in the sites)′. The black lines represent the rest of the DNA of the closed, negatively supercoiled, circular DNA molecules that carry the recombination sites. The cubes symbolize the recombinases (12 monomers in the Res system and 4 monomers in the Hin system). (A) Synapse between two directly repeated Tn sites. The 12 monomers are thought to form a filament-like structure around which the two sites interwrap. The three subsites, each carrying two inversely repeated binding sites for Res, are indicated (sites I are the core sequences). Interwrapping of the two sites around the Res filament constrains three crossings of negative supercoiling (indicated by the stars). (B) Synapse between two inversely repeated sites. The synapse is thought to be preferentially formed at a branch of negative supercoiling and involves an interaction between the Fis-bound enhancer sequence and the Hin-bound sites. Two monomers of Fis (the gray ellipses) bind at each edge of the enhancer.

Citation: Cornet F, Chandler M. 2004. Nonhomologous Recombination, p 36-66. In Miller R, Day M (ed), Microbial Evolution. ASM Press, Washington, DC. doi: 10.1128/9781555817749.ch3
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Image of FIGURE 7
FIGURE 7

Transposase organization. The positions of protease-sensitive sites are delimited by the open boxes. This information is not yet available for IS and IS. Potential or real helix-turn-helix (HTH) motifs are shown in grey boxes. Potential HTH motifs are indicated by “?”. The catalytic core is indicated by grey and carries the DDE motif. These residues, together with others referred to in the text, are indicated in upper-case letters above each transposase molecule. LZ indicates the leucine zipper motif with the four repeating heptads observed in the IS transposase. A second region involved in multimerization is also shown slightly downstream. In those cases investigated, the catalytic core is also capable of promoting multimerization. Known functions of the different transposase regions are indicated below. Transposase alignments are centered on the second aspartate residue. The length of each protein in amino acids is indicated at the right. The function of each region, where known, is indicated under the respective proteins.

Citation: Cornet F, Chandler M. 2004. Nonhomologous Recombination, p 36-66. In Miller R, Day M (ed), Microbial Evolution. ASM Press, Washington, DC. doi: 10.1128/9781555817749.ch3
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Image of FIGURE 8
FIGURE 8

Organization of transposon ends. (A) General organization of the ends of different prokaryote transposable elements. Terminal inverted repeats (IRs) are indicated in black. Transposase-binding sites are indicated in grey or white, and their relative orientation is indicated by a small arrow within each box. (B) Functional organization of the ends of a typical insertion sequence. This figure shows an IR that can be divided into a domain required for catalysis and a domain required for transposase recognition. The 1 and 30 represent base pairs to indicate approximately the general length of IS terminal IRs.

Citation: Cornet F, Chandler M. 2004. Nonhomologous Recombination, p 36-66. In Miller R, Day M (ed), Microbial Evolution. ASM Press, Washington, DC. doi: 10.1128/9781555817749.ch3
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Image of FIGURE 9
FIGURE 9

Cointegrate formation. From top to bottom, this figure shows synapsis of the transposon ends (dark gray); initial cleavage to generate a free 3′-OH at the ends of the transferred strand and a staggered attack on the target DNA; the formation of a branched molecule with a suitable 3′-OH capable of being used as a primer for replication of the element; and the result of replication and repair, which generated two copies of the element, leaving each attached both to donor DNA at one end and target DNA at the other.

Citation: Cornet F, Chandler M. 2004. Nonhomologous Recombination, p 36-66. In Miller R, Day M (ed), Microbial Evolution. ASM Press, Washington, DC. doi: 10.1128/9781555817749.ch3
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Image of FIGURE 10
FIGURE 10

Variant pathways in DDE transposase-catalyzed transposition. This figure demonstrates alternative catalytic pathways observed for various transposons encoding DDE transposases. The basic initial enzyme-catalyzed hydrolysis to generate a 3′-OH end on the DNA strand that will be transferred into the target molecule and the final strand transfer step in which the 3′-OH are used as nucleophiles to attack the target phosphodiester bonds are chemically identical. The major differences arise from the manner in which the second nontransferred strand is processed. (Left panels) Initial hydrolysis of the strand to be transferred occurs at both ends to liberate a 3′-OH. This then attacks the opposite strand to generate an intermediate carrying a hairpin structure at each end and separates the transposon from the donor backbone DNA. A second round of hydrolysis, chemically identical to the first, generates an intermediate with two 3′-OH residues at its ends capable of inserting into the target DNA molecule. (Center panels) Initial hydrolysis of the strand to be transferred occurs at both ends to liberate a 3′-OH. The other strand is cleaved by a second non-DDE endonuclease (in the Tn7, this resembles a restriction endonuclease). (Right panels) Initial hydrolysis of the strand to be transferred occurs at only one end to liberate a single 3′-OH. This then attacks the opposite end to generate a single-strand bridge between the ends. In a further step, this form gives rise to a double-stranded covalently closed transposon circle, which undergoes subsequent cleavage at each end to regenerate a 3′-OH at each end.

Citation: Cornet F, Chandler M. 2004. Nonhomologous Recombination, p 36-66. In Miller R, Day M (ed), Microbial Evolution. ASM Press, Washington, DC. doi: 10.1128/9781555817749.ch3
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Image of FIGURE 11
FIGURE 11

Systems using site-specific recombination. Black arrowheads symbolize the recombination sites. See text for a description of the different systems. (A and B) Gene-shuffling systems. Arrows represent the various genes with their names indicated below. The genes coding for the respective recombinases are shown in gray and may contain the recombination enhancer sequence (the enclosed white bar in the and genes). P is the main promoters with the arrows above indicating the sense of transcription. (C and D) Resolution systems. The open circles symbolize replication origins. The open bars represent the ends of a replicative transposon. (C) Different copies of a circular replicon (in this case, two monomers of a plasmid, left drawing) may be fused by recombination (in this case, a dimer is formed, central drawing). Site-specific recombination resolves these multimeric forms to monomers (right). (D) Replicative transposition (symbolized by the dashed arrow; see also Fig. 9 ) fuses the DNA molecules carrying the transposon (the black line) and the transposition target site (the dotted circle), respectively (in this case, two plasmids, left drawing). This generates a cointegrate that consists of the fused DNA molecules separated by two directly repeated copies of the transposon (central drawing). Site-specific recombination resolves this cointegrate. This generates a copy of the donor molecule and a target molecule carrying the transposon at the target site. (E) Integration/excision systems. The elements are inserted into a DNA molecule of their host (here the chromosome, left drawing). Excision by site-specific recombination generates a DNA circle that carries one recombination site. The circular element is eventually transferred to another cell. Phage genomes are transferred by infection following encapsidation. These may be injected into the recipient cell as a linear molecule and are subsequently circularized. Conjugative transposons and other conjugative elements are transferred by conjugation (see text). These may be injected into the recipient cell as single-strand DNA molecules and are subsequently replicated. Integration may occur at a specific site (phages and site-specific integrative elements) or at a variety of different sequences whose specificity depends on the system considered (conjugative transposons).

Citation: Cornet F, Chandler M. 2004. Nonhomologous Recombination, p 36-66. In Miller R, Day M (ed), Microbial Evolution. ASM Press, Washington, DC. doi: 10.1128/9781555817749.ch3
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Image of FIGURE 12
FIGURE 12

Diversity in transposable elements. A collection of transposable elements is presented to demonstrate their diversity. Reactive ends are indicated as dark-grey triangles, genes involved in recombinational mobility are shown in white with arrows indicating the direction of transcription/translation. Additional genes such as antibiotic resistance genes are shown as white bars (Tn) A conjugative transposon is an example of a transposon using a Y transposase () and excisase (). Also shown is the tetracycline resistance gene TetM and various genes involved in conjugal transfer. (IS and IS) Two insertion sequences, with one and two transposase ORFs, respectively. (Tn) A composite transposon based on the IS element showing the resistance and genes flanked by two IS copies. Note that the left IS copy has reduced activity and depends on the right copy for its activity. (Tn) A member of the unit transposons showing the DDE Tpase gene the S recombinase involved in resolution of Tpase-generated cointegrates (), and the site on which it acts (). The β-lactamase gene () carried by Tn is also indicated. (Tn) A second member of the “Tn” unit transposons showing an alternative arrangement of , , and together with the genes specifying resistance to mercury salts. (Tn) Found as part of multiple antibiotic resistance plasmids, this member of the Tn family illustrates the high complexity of certain transposable elements. The basic module is one similar to Tn in organization. In addition, Tn also carries an integron landing platform with its own Y recombinase (integrase; ) and attachment site () together with several integron cassettes (, δ1, ). This is inserted into a second structure bordered by short inverted repeats (IRi) and additional mobility functions (, ). Finally, two insertion sequences, an IS family member (IS) with its transposase genes ( and ) and an IS family member, IS, are also present. Here, IS is inserted into IS. (Bacteriophage Mu) This scheme shows the organization of the genes involved in transposition of this 37-kb bacteriophage. From left to right, it shows the repressor gene c, involved in regulating early gene expression; the internal activating sequence (IAS) involved in assembly of the transpososome; an accessory gene, also involved in regulation of early gene expression; the transposase, MuA; and the accessory protein Mu. The late genes are not shown in detail. (Tn) From left to right, the integrase gene and three integron resistance cassettes; the five genes involved in transposition, , the transposase, , a second endonuclease involved in processing the nontransferred strand, and involved in the recognition of the specific chromosomal target site, and , which, in conjunction with , permits integration at many different target sites.

Citation: Cornet F, Chandler M. 2004. Nonhomologous Recombination, p 36-66. In Miller R, Day M (ed), Microbial Evolution. ASM Press, Washington, DC. doi: 10.1128/9781555817749.ch3
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References

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