Regulation of site-specific recombination by the C-terminus of lambda integrase

Kazmierczak, Robert A.; Swalla, Brian M.; Burgin, Alex B.; Gumport, Richard I.; Gardner, Jeffrey F.

doi:10.1093/nar/gkf652

Cited by 34 publications

(42 citation statements)

References 34 publications

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“…One of the most useful mutants for studying the role of the C-terminal tail is a deletion of the seven most distal residues (W350ter), which results in a hyperactive topoisomerase (14,15,20,21). Previous results indicating that W350ter is similar to wild-type Int in its ability to cleave both half-and full-att suicide substrates (14) contrasts with the results from our laboratory.…”

Section: Discussioncontrasting

confidence: 66%

“…These structures, as well as a considerable amount of genetic and biochemical data, indicate that the C-terminal residues of each recombinase participate in trans (intermolecular) interactions that seem to have a regulatory function, albeit by different mechanisms in each system (8,(10)(11)(12)(13). Some of the experiments in this report are designed to reconcile and expand on the existing structural, genetic, and biochemical data pertaining to the role of the C-terminal tail of Int (6,7,(14)(15)(16).Despite many years of genetic and biochemical studies on the Int recombination system, it is still not known whether the Int recombinase is competent for DNA cleavage as a monomer. When Kikuchi and Nash first purified Int, they showed that at high salt it is a monomer in solution (17).…”

mentioning

confidence: 82%

“…This suggestion was initiated by the interesting finding by the Gardner and Gumport laboratories that an Int mutant (W350ter) lacking the C-terminal seven residues (C-terminal tail) retains topoisomerase activity but is unable to carry out recombination (15). Additional genetic and biochemical experiments led to the suggestion that the C-terminal tail is somehow involved in coordinating the multiple DNA cleavage͞ligation reactions of recombination (7,14,16).…”

Section: And In Materials and Methodsmentioning

confidence: 99%

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Two structural features of λ integrase that are critical for DNA cleavage by multimers but not by monomers

Lee

Aihara

Ellenberger

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Despite many years of genetic and biochemical studies on the integrase (Int) recombination system, it is still not known whether the Int protein is competent for DNA cleavage as a monomer. We have addressed this question, as part of a larger study of Int functions critical for the formation of higher-order complexes, by isolating ''multimer-specific'' mutants. We identify a pair of oppositely charged residues, E153 and R169, that comprise an intermolecular salt bridge within a functional Int multimer. Mutation of either of these residues significantly reduces both the cleavage of full-att sites and the resolution of Holliday junctions without compromising the cleavage of half-att site substrates. Allelespecific suppressor mutations were generated at these residues. Their interaction with wild-type Int on preformed Holliday junctions indicates that the mutated residues comprise an intermolecular salt bridge. We have also shown that the most C-terminal seven residues of Int, which comprise another previously identified subunit interface, inhibit DNA cleavage by monomeric but not multimeric Int. Taken together, our results lead us to conclude that Int can cleave DNA as a monomer. We also identify and discuss unique structural features of Int that act negatively to reduce its activity as a monomer and other features that act positively to enhance its activity as a multimer. T he integrase protein (Int) of Escherichia coli phage (1), which mediates the integration and excision of the viral genome in to and out of its host chromosome by recombination at specific loci (att sites) (2), belongs to the large Int family of recombinases (for review see refs. 3 and 4). They use transient covalent phospho-tyrosine intermediates to first generate and then resolve Holliday junction (HJ) recombination intermediates by means of two sequential pairs of strand exchanges. The locus of these reactions on each partner DNA duplex is a pair of 9-to 13-bp inverted binding sites for the recombinase (core-type sites) separated by a 6-to 8-bp overlap region (7 bp in the case of ) whose boundaries are defined by the staggered and precisely positioned DNA cleavage sites. The att sites of the pathway, and other ''heterobivalent'' recombinases have the additional complexity of nearby binding sites for accessory DNA bending proteins and binding sites for a second, N-terminal, recombinase domain that binds distant ''arm-type'' DNA sites.Mechanistic studies of the Int family have benefited enormously from the availability of crystal structures of several family members (reviewed in ref. 5), the most relevant to this work being the structures for the catalytic domain of Int (6), the covalent complex with DNA of the Int C75 domain (which is analogous to the monovalent recombinases Cre and Flp) (7), and the Cre recombinase complexed with a HJ and a recombination synapse (8, 9). The common motif that emerges from all of the -family structures is a C-terminal catalytic domain (containing the tyrosine nucleophile and active site pocket) appropriately po...

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Section: Discussioncontrasting

confidence: 66%

mentioning

confidence: 82%

Section: And In Materials and Methodsmentioning

confidence: 99%

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Two structural features of λ integrase that are critical for DNA cleavage by multimers but not by monomers

Lee

Aihara

Ellenberger

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…We believe that these aberrant hairpin products arise from uncoordinated DNA cleavages at "non-partner" Int binding sites, either during or after resolution. 14,16 These findings extend previous results 2, 3,5,6 indicating that while the C-terminal tail is not required for DNA cleavage by monomeric Int, it plays a critical role in the function and proper coordination of multimeric Int.…”

Section: C-terminal Tail Is Required For Efficient and Coordinated Hosupporting

confidence: 73%

Receipt of the C-terminal Tail from a Neighboring λ Int Protomer Allosterically Stimulates Holliday Junction Resolution

Hazelbaker¹,

Radman‐Livaja²,

Landy³

2005

Journal of Molecular Biology

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Bacteriophage λ integrase (Int) catalyzes the integration and excision of the phage λ chromosome into and out of the Esherichia coli host chromosome. The seven carboxy-terminal residues (Cterminal tail) of Int comprise a context-sensitive regulatory element that links catalytic function with protein multimerization and also coordinates Int functions within the multimeric recombinogenic complex. The experiments reported here show that the β5-strand of Int is not simply a placeholder for the C-terminal tail but rather exerts its own allosteric effects on Int function in response to the incoming tail. Using a mutant integrase in which the C-terminal tail has been deleted (W350ter), we demonstrate that the C-terminal tail is required for efficient and accurate resolution of Holliday junctions by tetrameric Int. Addition of a free heptameric peptide of the same sequence as the Cterminal tail partially reverses the W350ter defects by stimulating Holliday junction resolution. The peptide also stimulates the topoisomerase function of monomeric W350ter. Single residue alterations in the peptide sequence and a mutant of the β5 strand indicate that the observed stimulation arises from specific contacts with the β5 strand (residues 239-243). The peptide does not stimulate binding of W350ter to its cognate DNA sites and therefore appears to recapitulate the effects of the normal C-terminal tail intermolecular contacts in wild-type Int. Models for the allosteric stimulation of Int activity by β5 strand contacts are discussed. Keywords bacteriophage lambda; integrase; Holliday junction; site-specific recombination; peptide Bacteriophage lambda integrase (Int) is a tyrosine site-specific recombinase that functions as a higher-order tetrameric complex to catalyze the integration and excision of the viral chromosome into and out of the Escherichia coli chromosome by means of a highly coordinated sequence of cleavage and ligation reactions at specific loci (att sites, see Figure 1(a)). 1 Previous work has established that the seven carboxy-terminal residues (C-terminal tail) of Int are integral to regulatory mechanisms that link catalytic function with protein multi-merization and also coordinate Int functions within the multimeric recombinogenic complex. 2-6 Here we extend these results by demonstrating that the β5 strand of Int is not simply a placeholder for the multi-positional C-terminal tail but rather exerts its own allosteric effects on Int function in response to the incoming tail.Int is a member of the tyrosine recombinase family, whose hallmark is an ordered pair of transesterification reactions that first generate and then resolve a Holliday junction recombination intermediate (Figure 1(a)). 7-9 The transesterification reactions are mediated by transient covalent 3′ phospho-tyrosine bonds that are formed at each of the four "core-type"

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“…The phage-encoded integrase is a heterobivalent DNA-binding protein. It consists of a large C-terminal domain that binds to core-type DNA sequences and carries out the enzymatic steps of recombination (11,12). Additionally, Int contains a small N-terminal domain that binds to arm-type DNA sites that are distant from the sites of DNA strand exchange (13).…”

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confidence: 99%

Control and Regulation of KplE1 Prophage Site-specific Recombination

Panis¹,

Méjean

Ansaldi

2007

Journal of Biological Chemistry

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KplE1 is one of the 10 prophage regions of Escherichia coli K12, located at 2464 kb on the chromosome. KplE1 is defective for lysis, but it is fully competent for excisive recombination. In this study, we have mapped the binding sites of the recombination proteins, namely IntS, TorI, and IHF on attL and attR, and the organization of these sites suggests that the intasome is architecturally different from the canonical form. We also measured the relative contribution of these proteins to both excisive and integrative recombination by using a quantitative in vitro assay. These experiments show a requirement of the TorI excisionase for excisive recombination and of the IntS integrase for both integration and excision. Moreover, we observed a strong influence of the supercoiled state of the substrates. The KplE1 recombination module, composed of the integrase and excisionase genes together with the attL and attR DNA regions, is highly similar to that of several phages infecting various E. coli strains as well as Shigella flexneri and Shigella sonnei. The in vitro recombination data reveal that HK620 and KplE1 att sequences are exchangeable. This study thus defines a new sitespecific recombination module, and implications for the mechanism and regulation of recombination are discussed.Phage has long served as a model system for studies of regulated site-specific recombination (1). Indeed, bacteriophages such as may choose between the lytic and the lysogenic cycle for their propagation in the bacterial host (2). In conditions favorable for bacterial growth the phage genome is inserted into the host genome by an integrative recombination reaction, which takes place between DNA attachment sites called attP and attB, in the phage and the bacterial genome, respectively. As a result, the integrated DNA (or prophage DNA) is bounded by hybrid attachment sites, termed attL and attR. In response to a change in the physiological state of the bacteria, mainly in response to stress conditions such as DNA damage, phage DNA is excised from the host chromosome. This excisive reaction recombines attL and attR to restore the attP and attB sites on the circular and Escherichia coli

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Regulation of site-specific recombination by the C-terminus of lambda integrase

Cited by 34 publications

References 34 publications

Two structural features of λ integrase that are critical for DNA cleavage by multimers but not by monomers

Two structural features of λ integrase that are critical for DNA cleavage by multimers but not by monomers

Receipt of the C-terminal Tail from a Neighboring λ Int Protomer Allosterically Stimulates Holliday Junction Resolution

Control and Regulation of KplE1 Prophage Site-specific Recombination

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