Interactions of the excision proteins of CTnDOT in the attR intasome

Keeton, Carolyn M.; Hopp, Crystal M.; Yoneji, Sumiko; Gardner, Jeffrey F.

doi:10.1016/j.plasmid.2013.03.009

Cited by 9 publications

(15 citation statements)

References 26 publications

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“…Two of the CTnDOT arm-type sites (the L1 and R1= sites) are required for both integration and excision. Three additional sites act cooperatively in integration (R1, R2, and R2=) but appear to be dispensable in excision (23,24). While lambda Int has a higher affinity for the arm-type sites than the core-type sites, IntDOT has a higher affinity for the core-type sites (24,25).…”

Section: Resultsmentioning

confidence: 99%

“…In order to evaluate the importance of the individual arm-type sites in resolution of HJ intermediates, we constructed additional versions of the arm-type HJ intermediates. The L1, R1=, and R2-R2= arm-type sites were changed based on previous studies indicating their importance in integration and excision of CTnDOT (23,24). The L1 and R1= sites are each required for integration and excision.…”

Section: Resultsmentioning

confidence: 99%

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Resolution of Mismatched Overlap Holliday Junction Intermediates by the Tyrosine Recombinase IntDOT

2017

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CTnDOT is an integrated conjugative element found in Bacteroides species. CTnDOT contains and transfers antibiotic resistance genes. The element integrates into and excises from the host chromosome via a Holliday junction (HJ) intermediate as part of a site-specific recombination mechanism. The CTnDOT integrase, IntDOT, is a tyrosine recombinase with core-binding, catalytic, and aminoterminal (N) domains. Unlike well-studied tyrosine recombinases, such as lambda integrase (Int), IntDOT is able to resolve Holliday junctions containing heterology (mismatched bases) between the sites of strand exchange. All known natural isolates of CTnDOT contain mismatches in the overlap region between the sites of strand exchange. Previous work showed that IntDOT was unable to resolve synthetic Holliday junctions containing mismatched bases to products in the absence of the armtype sites and a DNA-bending protein. We constructed synthetic HJs with the armtype sites and tested them with the Bacteroides host factor (BHFa). We found that the addition of BHFa stimulated resolution of HJ intermediates with mismatched overlap regions to products. In addition, the L1 site is required for directionality of the reaction, particularly when the HJ contains mismatches. BHFa is required for product formation when the overlap region contains mismatches, and it stimulates resolution to products when the overlap region is identical. Without this DNA bending, the N domain of IntDOT is likely unable to bind the L1 arm-type site. These findings suggest that BHFa bends DNA into the necessary conformation for the higher-order complexes, including the L1 site, that are required for product formation. IMPORTANCECTnDOT is a mobile element that carries antibiotic resistance genes and moves by site-selective recombination and subsequent conjugation. The recombination reaction is catalyzed by an integrase IntDOT that is a member of the tyrosine recombinase family. The reaction proceeds through ordered strand exchanges that generate a Holliday junction (HJ) intermediate. Unlike other tyrosine recombinases, IntDOT can resolve HJs containing mismatched bases in the overlap region in vivo, as is the case under natural conditions. However, HJ intermediates including only IntDOT core-type sites cannot be resolved to products if the HJ intermediate contains mismatched bases. We added arm-type sites in cis and in trans to the HJ intermediates and the protein BHFa to study the requirements for higher-order nucleoprotein complexes.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Resolution of Mismatched Overlap Holliday Junction Intermediates by the Tyrosine Recombinase IntDOT

2017

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“…They are small, basic proteins that resemble other Recombination Directionality Factors (RDFs) such as Xis from the bacteriophage lambda system (33, 48, 49). The role of Orf3 in CTnDOT excision or conjugative transfer (if any) has not been elucidated, as a phenotype for an orf3 deletion has not been detected (6, 10, 14).…”

Section: Accessory Factors Involved In the Ctndot Excision Reactionmentioning

confidence: 99%

“…When arm-type site mutants were tested in the in vitro intramolecular excision assay, the R1′, R1, and L1 arm-type sites were shown to be essential for CTnDOT excision (Figure 9) (48). It is somewhat surprising that the same arm-type sites were shown to be important for both the integration and excision of CTnDOT.…”

Section: The Roles Of the Intdot Arm-type Sites In Ctndot Integrationmentioning

confidence: 99%

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The Integration and Excision of CTnDOT

Wood

Gardner

2015

Microbiol Spectr

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Bacteroides species are one of the most prevalent groups of bacteria present in the human colon. Many strains carry large, integrated elements including integrative and conjugative elements (ICEs). One such ICE is CTnDOT, which is 65 kb in size and encodes resistances to tetracycline and erythromycin. CTnDOT has been increasing in prevalence in Bacteroides spp., and is now found in greater than 80% of natural isolates. In recent years, CTnDOT has been implicated in the spread of antibiotic resistance among gut microbiota. Interestingly, the excision and transfer of CTnDOT is stimulated in the presence of tetracycline. The tyrosine recombinase IntDOT catalyzes the integration and excision reactions of CTnDOT. Unlike the well-characterized lambda Int, IntDOT tolerates heterology in the overlap region between the sites of cleavage and strand exchange. IntDOT also appears to have a different arrangement of active site catalytic residues. It is missing one of the arginine residues that is conserved in other tyrosine recombinases. The excision reaction of CTnDOT is complex, involving excision proteins Xis2c, Xis2d, and Exc, as well as IntDOT and a Bacteroides host factor. Xis2c and Xis2d are small, basic proteins like other recombination directionality factors (RDFs). Exc is a topoisomerase; however, the topoisomerase function is not required for the excision reaction. Exc has been shown to stimulate excision frequencies when there are mismatches in the overlap regions, suggesting that it may play a role in resolving Holliday junctions containing heterology. Work is currently under way to elucidate the complex interactions involved with the formation of the CTnDOT excisive intasomes.

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The Xis2d protein of CTnDOT binds to the intergenic region between the mob and tra operons

Hopp

Gardner

Salyers

2015

Plasmid

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CTnDOT is a 65kbp integrative and conjugative element (ICE) that carries genes encoding both tetracycline and erythromycin resistances. The Excision operon of this element encodes Xis2c, Xis2d, and Exc proteins involved in the excision of CTnDOT from host chromosomes. These proteins are also required in the complex transcriptional regulation of the divergently transcribed transfer (tra) and mobilization (mob) operons of CTnDOT. Transcription of the tra operon is positively regulated by Xis2c and Xis2d, whereas, transcription of the mob operon is positively regulated by Xis2d and Exc. Xis2d is the only protein that is involved in the excision reaction, as well as the transcriptional regulation of both the mob and tra operons. This paper helps establish how Xis2d binds the DNA in the mob and tra region. Unlike other excisionase proteins, Xis2d binds a region of dyad symmetry. The binding site is located in the intergenic region between the mob and tra promoters, and once bound Xis2d induces a bend in the DNA. Xis2d binding to this region could be the preliminary step for the activation of both operons. Then the other proteins, like Exc, can interact with Xis2d and form higher order complexes.

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Interactions of the excision proteins of CTnDOT in the attR intasome

Cited by 9 publications

References 26 publications

Resolution of Mismatched Overlap Holliday Junction Intermediates by the Tyrosine Recombinase IntDOT

Resolution of Mismatched Overlap Holliday Junction Intermediates by the Tyrosine Recombinase IntDOT

The Integration and Excision of CTnDOT

The Xis2d protein of CTnDOT binds to the intergenic region between the mob and tra operons

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