Site-specific recombination intermediates trapped with suicide substrates

Nunes-Düby, Simone E.; Matsumoto, Lloyd; Landy, Arthur

doi:10.1016/0092-8674(87)90336-9

Cited by 229 publications

(205 citation statements)

References 29 publications

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“…In both the integrative and excisive recombination reactions, the top strands of the core sites are exchanged first to form the HJ intermediate (32,33). The source of this strong bias is evident from our models (Figs.…”

Section: Discussionmentioning

confidence: 78%

Nucleoprotein architectures regulating the directionality of viral integration and excision

Seah

Warren

Tong

et al. 2014

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

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The virally encoded site-specific recombinase Int collaborates with its accessory DNA bending proteins IHF, Xis, and Fis to assemble two distinct, very large, nucleoprotein complexes that carry out either integrative or excisive recombination along regulated and essentially unidirectional pathways. The core of each complex consists of a tetramer of Integrase protein (Int), which is a heterobivalent DNA binding protein that binds and bridges a core-type DNA site (where strand cleavage and ligation are executed), and a distal arm-type site, that is brought within range by one or more DNA bending proteins. The recent determination of the patterns of these Int bridges has made it possible to think realistically about the global architecture of the recombinogenic complexes. Here, we combined the previously determined Int bridging patterns with in-gel FRET experiments and in silico modeling to characterize and differentiate the two 400-kDa multiprotein Holiday junction recombination intermediates formed during λ integration and excision. The results lead to architectural models that explain how integration and excision are regulated in λ site-specific recombination. Our confidence in the basic features of these architectures is based on the redundancy and self-consistency of the underlying data from two very different experimental approaches to establish bridging interactions, a set of strategic intracomplex distances from FRET experiments, and the model's ability to explain key aspects of the integrative and excisive recombination pathways, such as topological changes, the mechanism of capturing attB, and the features of asymmetry and flexibility within the complexes.regulation of directionality | topology | recombinogenic architectures | molecular machines H igh-precision DNA transactions responsible for a variety of fundamental processes are typically promoted and modulated by large multiprotein machines that use cooperative interactions and involve DNA bending and/or wrapping. One well-studied example is the tightly regulated and highly directional site-specific recombination by which bacteriophage λ inserts and excises its DNA into and out of the Escherichia coli host chromosome, using the phage attP and the bacterial attB DNA sequences for integration and the resulting junction sequences, attL and attR, for excision. These reactions are catalyzed by the phage-encoded Integrase protein (Int), the founding member of the tyrosine recombinase family of site-specific recombinases (1). In addition to mediating integration and excision of viral genomes, members of this family function in a variety of other cellular processes including chromosome segregation, gene regulation, and conjugative transposition (2).Int has three well-characterized domains: an N-terminal DNA-binding domain (NTD), a central core-binding domain (CB), and a C-terminal catalytic domain (CAT). The CB and CAT domains (referred to here as the CTD) are together responsible for binding to the core-type DNA sequences where strand exchange and ligation t...

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Section: Discussionmentioning

confidence: 78%

Nucleoprotein architectures regulating the directionality of viral integration and excision

Seah

Warren

Tong

et al. 2014

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

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“…TnpI-mediated cleavage of the nicked strand of either substrates generated a covalent, protease K-sensitive, recombinase-DNA complex and a short oligonucleotide that is thought to diffuse away, thereby preventing the rejoining reaction ( Fig. 3A; Nunes-Duby et al , 1987). The complex formed by the wild-type recombinase migrated slightly faster than the complex containing the His-tagged TnpI as expected from the molecular weight difference between the two proteins (Fig.…”

Section: Tnpi Cleaves Dna At 6 Bp-staggered Positions Of the Ir1-ir2 mentioning

confidence: 84%

Self‐control in DNA site‐specific recombination mediated by the tyrosine recombinase TnpI

Vanhooff

Galloy

Agaisse

et al. 2006

Molecular Microbiology

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SummaryTn 4430 is a distinctive transposon of the Tn 3 family that encodes a tyrosine recombinase (TnpI) to resolve replicative transposition intermediates. The internal resolution site of Tn 4430 (IRS, 116 bp) contains two inverted repeats (IR1 and IR2) at the crossover core site, and two additional TnpI binding motifs (DR1 and DR2) adjacent to the core. Deletion analysis demonstrated that DR1 and DR2 are not required for recombination in vivo and in vitro . Their function is to provide resolution selectivity to the reaction by stimulating recombination between directly oriented sites on a same DNA molecule. In the absence of DR1 and/ or DR2, TnpI-mediated recombination of supercoiled DNA substrates gives a mixture of topologically variable products, while deletion between two wild-type IRSs exclusively produces two-noded catenanes. This demonstrates that TnpI binding to the accessory motifs DR1 and DR2 contributes to the formation of a specific synaptic complex in which catalytically inert recombinase subunits act as architectural elements to control recombination sites pairing and strand exchange. A model for the organization of TnpI/IRS recombination complex is presented.

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“…A number of site-specific recombination systems have been characterized in vitro. These include the bacteriophage A integration system (2), the resolvases derived from the Tn3 class of bacterial transposons (3, 4), the bacterial invertases (5, 6), the Cre-lox system of bacteriophage P1 (7), and the FLP system of the 2-gm plasmid of Saccharomyces cerevisiae (8, 9).Studies of these systems have provided detailed information about the architecture of the recombination sites, protein-DNA interactions, juxtaposition of two recombination sites, and key reaction intermediates (1,(10)(11)(12)(13)(14). A potentially useful .approach to many questions that remain is the application of classical enzyme kinetic methods.…”

mentioning

confidence: 99%

“…Studies of these systems have provided detailed information about the architecture of the recombination sites, protein-DNA interactions, juxtaposition of two recombination sites, and key reaction intermediates (1,(10)(11)(12)(13)(14). A potentially useful .approach to many questions that remain is the application of classical enzyme kinetic methods.…”

mentioning

confidence: 99%

FLP recombinase is an enzyme.

Gates

Cox²

1988

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

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The FLP protein of the yeast 2-jum plasmid catalyzes intermolecular site-specific recombination with a turnover number of 0.12 min-(per FLP monomer) for relaxed DNA substrates. Under conditions that enhance its stability, the protein can be used in catalytic rather than stoichiometric amounts. The reaction rate exhibits a strong dependence on FLP protein concentration even when the protein is present in excess relative to available recombination sites.Site-specific recombination events play crucial roles in DNA transposition, partitioning of extrachromosomal elements, gene regulation, and generation of genetic diversity (1). A number of site-specific recombination systems have been characterized in vitro. These include the bacteriophage A integration system (2), the resolvases derived from the Tn3 class of bacterial transposons (3, 4), the bacterial invertases (5, 6), the Cre-lox system of bacteriophage P1 (7), and the FLP system of the 2-gm plasmid of Saccharomyces cerevisiae (8, 9).Studies of these systems have provided detailed information about the architecture of the recombination sites, protein-DNA interactions, juxtaposition of two recombination sites, and key reaction intermediates (1,(10)(11)(12)(13)(14). A potentially useful .approach to many questions that remain is the application of classical enzyme kinetic methods. No detailed kinetic description of a site-specific recombination reaction has appeared. A number of factors contribute to the lack of information about kinetic parameters in these systems. The assays are often tedious, instability of some recombinases may affect reproducibility, and the reactions themselves are complex. Perhaps most important is the fact that it is not clear that these proteins are enzymes. The "recombinase" in each system appears to be required in stoichiometric rather than catalytic amounts (1,3,(15)(16)(17). Enzymatic turnover has not been demonstrated in any case. The focus of this report is turnover in the yeast FLP recombination system.The yeast 2-,um plasmid encodes a site-specific recombination system (FLP) that inverts the two unique regions of the plasmid (18). This inversion event is central to the strategy by which the plasmid amplifies its copy number (19-21). The only protein required is the plasmid-encoded FLP protein (22), which has recently been purified to near homogeneity (23,24). The relatively small recombination site (FLP recombination target or FRT) has been characterized extensively. The FLP protein will promote inversions, deletions, or intermolecular recombination efficiently if appropriate substrates are provided. Supercoiled and relaxed substrates react efficiently in vitro.The minimal FRT site consists of two 13-base-pair (bp) repeats separated by an 8-bp spacer (Fig. 1) (31)(32)(33). The alignment of two sites during recombination is also mediated by asymmetry in the spacer. FRT sites with symmetrical spacers remain functional but permit either of the two possible alignments of two sites during recombination (32). Several of the prop...

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Site-specific recombination intermediates trapped with suicide substrates

Cited by 229 publications

References 29 publications

Nucleoprotein architectures regulating the directionality of viral integration and excision

Nucleoprotein architectures regulating the directionality of viral integration and excision

Self‐control in DNA site‐specific recombination mediated by the tyrosine recombinase TnpI

FLP recombinase is an enzyme.

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