Tyrosine Recombinase Retrotransposons and Transposons

ABSTRACT The naphthalene-catabolic (nah) genes on the incompatibility group P-9 (IncP-9) self-transmissible plasmid NAH7 from Pseudomonas putida G7 are some of the most extensively characterized genetic determinants for bacterial aerobic catabolism of aromatic hydrocarbons. In contrast to the detailed studies of its catabolic cascade and enzymatic functions, the biological characteristics of plasmid NAH7 have remained unclear. Our sequence determination in this study together with the previously deposited sequences revealed the entire structure of NAH7 (82,232 bp). Comparison of NAH7 with two other completely sequenced IncP-9 catabolic plasmids, pDTG1 and pWW0, revealed that the three plasmids share very high nucleotide similarities in a 39-kb region encoding the basic plasmid functions (the IncP-9 backbone). The backbone of NAH7 is phylogenetically more related to that of pDTG1 than that of pWW0. These three plasmids carry their catabolic gene clusters at different positions on the IncP-9 backbone. All of the NAH7-specified nah genes are located on a class II transposon, Tn4655. Our analysis of the Tn4655-encoded site-specific recombination system revealed that (i) a novel tyrosine recombinase, TnpI, catalyzed both the intra- and intermolecular recombination between two copies of the attI site, (ii) the functional attI site was located within a 119-bp segment, and (iii) the site-specific strand exchange occurred within a 30-bp segment in the 41-bp CORE site. Our results and the sequence data of other naphthalene-catabolic plasmids, pDTG1 and pND6-1, suggest a potential role of the TnpI-attI recombination system in the establishment of these catabolic plasmids.

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DIRS retrotransposons amplify via linear, single-stranded cDNA intermediates

Nucleic Acids Research ◽

10.1093/nar/gkaa160 ◽

2020 ◽

Vol 48 (8) ◽

pp. 4230-4243 ◽

Cited By ~ 1

Author(s):

Marek Malicki ◽

Thomas Spaller ◽

Thomas Winckler ◽

Christian Hammann

Keyword(s):

Rna Interference ◽

Rna Polymerase ◽

Dictyostelium Discoideum ◽

Repeat Sequence ◽

Marker Genes ◽

Argonaute Protein ◽

Tyrosine Recombinase ◽

Rna Dependent Rna Polymerase ◽

Rna Transcripts ◽

Complementary Region

Abstract The Dictyostelium Intermediate Repeat Sequence 1 (DIRS-1) is the name-giving member of the DIRS order of tyrosine recombinase retrotransposons. In Dictyostelium discoideum, DIRS-1 is highly amplified and enriched in heterochromatic centromers of the D. discoideum genome. We show here that DIRS-1 it tightly controlled by the D. discoideum RNA interference machinery and is only mobilized in mutants lacking either the RNA dependent RNA polymerase RrpC or the Argonaute protein AgnA. DIRS retrotransposons contain an internal complementary region (ICR) that is thought to be required to reconstitute a full-length element from incomplete RNA transcripts. Using different versions of D. discoideum DIRS-1 equipped with retrotransposition marker genes, we show experimentally that the ICR is in fact essential to complete retrotransposition. We further show that DIRS-1 produces a mixture of single-stranded, mostly linear extrachromosomal cDNA intermediates. If this cDNA is isolated and transformed into D. discoideum cells, it can be used by DIRS-1 proteins to complete productive retrotransposition. This work provides the first experimental evidence to propose a general retrotransposition mechanism of the class of DIRS like tyrosine recombinase retrotransposons.

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In vitro DNA Inversions Mediated by the PsrA Site-Specific Tyrosine Recombinase of Streptococcus pneumoniae

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2020.00043 ◽

2020 ◽

Vol 7 ◽

Cited By ~ 1

Author(s):

Jingwen Li ◽

Juanjuan Wang ◽

Sofía Ruiz-Cruz ◽

Manuel Espinosa ◽

Jing-Ren Zhang ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Tyrosine Recombinase ◽

Site Specific

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CTnDOT Integrase Interactions with Attachment Site DNA and Control of Directionality of the Recombination Reaction

Journal of Bacteriology ◽

10.1128/jb.00351-10 ◽

2010 ◽

Vol 192 (15) ◽

pp. 3934-3943 ◽

Cited By ~ 12

Author(s):

Margaret M. Wood ◽

Jeanne M. DiChiara ◽

Sumiko Yoneji ◽

Jeffrey F. Gardner

Keyword(s):

Attachment Site ◽

Strand Exchange ◽

Recombination Reaction ◽

Tyrosine Recombinase ◽

Conjugative Transposon ◽

The Core ◽

Type Site ◽

Gel Shift ◽

And Control

ABSTRACT IntDOT is a tyrosine recombinase encoded by the conjugative transposon CTnDOT. The core binding (CB) and catalytic (CAT) domains of IntDOT interact with core-type sites adjacent to the regions of strand exchange, while the N-terminal arm binding (N) domain interacts with arm-type sites distal to the core. Previous footprinting experiments identified five arm-type sites, but how the arm-type sites participate in the integration and excision of CTnDOT was not known. In vitro integration assays with substrates containing arm-type site mutants demonstrated that attDOT sequences containing mutations in the L1 arm-type site or in the R1 and R2 or R1 and R2′ arm-type sites were dramatically defective in integration. Substrates containing mutations in the L1 and R1 arm-type sites showed a 10- to 20-fold decrease in detectable in vitro excision, but introduction of multiple arm-type site mutations in attR did not have an effect on the excision frequency. A sixth arm-type site, the R1′ site, was also identified and shown to be required for integration and important for efficient excision. These results suggest that intramolecular IntDOT interactions are required for integration, while the actions of accessory factors are more important for excision. Gel shift assays performed in the presence of core- and arm-type site DNAs showed that IntDOT affinity for the attDOT core was enhanced when IntDOT was simultaneously bound to arm-type site DNA.

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The Evolution of Tyrosine-Recombinase Elements in Nematoda

PLoS ONE ◽

10.1371/journal.pone.0106630 ◽

2014 ◽

Vol 9 (9) ◽

pp. e106630 ◽

Cited By ~ 2

Author(s):

Amir Szitenberg ◽

Georgios Koutsovoulos ◽

Mark L. Blaxter ◽

David H. Lunt

Keyword(s):

Tyrosine Recombinase

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Tyrosine Recombinase Retrotransposons and Transposons

Mobile DNA III ◽

10.1128/9781555819217.ch55 ◽

2015 ◽

pp. 1271-1291 ◽

Cited By ~ 1

Author(s):

Russell T. M. Poulter ◽

Margi I Butler

Keyword(s):

Tyrosine Recombinase

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

Journal of Bacteriology ◽

10.1128/jb.00873-16 ◽

2017 ◽

Vol 199 (10) ◽

Cited By ~ 1

Author(s):

Kenneth Ringwald ◽

Sumiko Yoneji ◽

Jeffrey Gardner

Keyword(s):

Dna Bending ◽

Antibiotic Resistance Genes ◽

Product Formation ◽

Holliday Junctions ◽

Strand Exchange ◽

Holliday Junction ◽

Tyrosine Recombinase ◽

Content Type ◽

Tyrosine Recombinases ◽

Overlap Region

ABSTRACT 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 amino-terminal (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 arm-type sites and a DNA-bending protein. We constructed synthetic HJs with the arm-type 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. IMPORTANCE CTnDOT 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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Integration Site Selection by the Bacteroides Conjugative Transposon CTnBST

Journal of Bacteriology ◽

10.1128/jb.00668-07 ◽

2007 ◽

Vol 189 (18) ◽

pp. 6594-6601 ◽

Cited By ~ 9

Author(s):

Bo Song ◽

Nadja B. Shoemaker ◽

Jeffrey F. Gardner ◽

Abigail A. Salyers

Keyword(s):

Amino Acids ◽

Integration Site ◽

Consensus Sequence ◽

Base Change ◽

The Other ◽

Tyrosine Recombinase ◽

Crossover Region ◽

Conjugative Transposon ◽

Frequency Changes ◽

Conserved Amino Acids

ABSTRACT A newly discovered Bacteroides conjugative transposon (CTn), CTnBST, integrates more site specifically than two other well-studied CTns, the Bacteroides CTn CTnDOT and the enterococcal CTn Tn916. Moreover, the integrase of CTnBST, IntBST, had the C-terminal 6-amino-acid signature that is associated with the catalytic regions of members of the tyrosine recombinase family, most of which integrate site specifically. Also, in most of these integrases, all of the conserved amino acids are required for integration. In the case of IntBST, however, we found that changing three of the six conserved amino acids in the signature, one of which was the presumed catalytic tyrosine, resulted in a 1,000-fold decrease in integration frequency. Changes in the other amino acids had little or no effect. Thus, although the CTnBST integrase still seems to be a member of the tyrosine recombinase family, it clearly differs to some extent from other members of the family in its catalytic site. We also determined the sequence requirements for CTnBST integration in the 18-bp region where the crossover occurs preferentially during integration. We found that CTnBST integrates in this preferred site about one-half of the time but can also use other sites. A consensus sequence was tentatively derived by comparison of a few secondary sites: AATCTGNNAAAT. We report here that within the consensus region, no single base change affected the frequency of integration. However, 3 bp at one end of the consensus sequence (CTG) proved to be essential for integration into the preferred site. This sequence appeared to be at one end of a 7-bp crossover region, CTGNNAA. The other bases could vary without affecting either integration frequency or specificity. Thus, in contrast to well-studied site-specific recombinases which require homology throughout the crossover region, integration of CTnBST requires homology at one end of the crossover region but not at the other end.

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Challenging a Paradigm: the Role of DNA Homology in Tyrosine Recombinase Reactions

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.00038-08 ◽

2009 ◽

Vol 73 (2) ◽

pp. 300-309 ◽

Cited By ~ 59

Author(s):

Lara Rajeev ◽

Karolina Malanowska ◽

Jeffrey F. Gardner

Keyword(s):

Dna Homology ◽

Tyrosine Recombinase ◽

Site Specific ◽

Site Specific Recombination ◽

Conjugative Transposons ◽

The Core ◽

Sequence Identity ◽

Tyrosine Recombinases ◽

Recent Developments

SUMMARY A classical feature of the tyrosine recombinase family of proteins catalyzing site-specific recombination, as exemplified by the phage lambda integrase and the Cre and Flp recombinases, is the ability to recombine substrates sharing very limited DNA sequence identity. Decades of research have established the importance of this short stretch of identity within the core regions of the substrates. Since then, several new enzymes that challenge this paradigm have been discovered and require the role of sequence identity in site-specific recombination to be reconsidered. The integrases of the conjugative transposons such as Tn916, Tn1545, and CTnDOT recombine substrates with heterologous core sequences. The integrase of the mobilizable transposon NBU1 performs recombination more efficiently with certain core mismatches. The integration of CTX phage and capture of gene cassettes by integrons also occur by altered mechanisms. In these systems, recombination occurs between mismatched sequences by a single strand exchange. In this review, we discuss literature that led to the formulation of the current strand-swapping isomerization model for tyrosine recombinases. The review then focuses on recent developments on the recombinases that challenged the paradigm that was derived from the studies of early systems.

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Single stranded DNA annealing is a conserved activity of telomere resolvases

PLoS ONE ◽

10.1371/journal.pone.0246212 ◽

2021 ◽

Vol 16 (2) ◽

pp. e0246212

Author(s):

Siobhan L. McGrath ◽

Shu Hui Huang ◽

Kerri Kobryn

Keyword(s):

Dna Binding ◽

Dna Cleavage ◽

Bacterial Species ◽

Dna Binding Protein ◽

Tyrosine Recombinase ◽

Single Stranded Dna ◽

Terminal Domain ◽

Topoisomerase Ib ◽

Replication Intermediates

Bacterial species of the genera Agrobacterium and Borrelia possess chromosomes terminated by hairpin telomeres. Replication produces dimeric replication intermediates fused via replicated telomere junctions. A specialized class of enzymes, referred to as telomere resolvases, promotes the resolution of the replicated intermediate into linear monomers terminated by hairpin telomeres. Telomere resolution is catalyzed via DNA cleavage and rejoining events mechanistically similar to those promoted by topoisomerase-IB and tyrosine recombinase enzymes. Examination of the borrelial telomere resolvase, ResT, revealed unanticipated multifunctionality; aside from its expected telomere resolution activity ResT possessed a singled-stranded DNA (ssDNA) annealing activity that extended to both naked ssDNA and ssDNA complexed with its cognate single-stranded DNA binding protein (SSB). At present, the role this DNA annealing activity plays in vivo remains unknown. We have demonstrated here that single-stranded DNA annealing is also a conserved property of the agrobacterial telomere resolvase, TelA. This activity in TelA similarly extends to both naked ssDNA and ssDNA bound by its cognate SSB. TelA’s annealing activity was shown to stem from the N-terminal domain; removal of this domain abolished annealing without affecting telomere resolution. Further, independent expression of the N-terminal domain of TelA produced a functional annealing protein. We suggest that the apparent conservation of annealing activity in two telomere resolvases, from distantly related bacterial species, implies a role for this activity in hairpin telomere metabolism. Our demonstration of the separation of the telomere resolution and annealing activities of TelA provides a platform for future experiments aimed at identifying the role DNA annealing performs in vivo.

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