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1.
J Bacteriol ; 199(10)2017 05 15.
Article in English | MEDLINE | ID: mdl-28242723

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.


Subject(s)
Bacteroides/enzymology , Bacteroides/metabolism , Base Pair Mismatch , DNA, Cruciform/metabolism , Recombinases/metabolism , Bacteroides/genetics , DNA/genetics , DNA/metabolism , Integration Host Factors/metabolism
2.
Plasmid ; 70(2): 190-200, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23603449

ABSTRACT

Excision of the conjugative transposon CTnDOT from the chromosome of Bacteroides spp. involves four CTnDOT-encoded proteins: IntDOT, Xis2c, Xis2d, and Exc along with a host factor. These proteins form excisive intasomes on the attR and attL sites which undergo synapsis and recombination to form the attDOT and attB sites. We recently developed an in vitro intramolecular excision reaction where the attL and attR sites are on the same plasmid. This reaction requires IntDOT, Xis2c, Xis2d, and is stimulated by Exc. We used this reaction to identify the binding sites of the IntDOT, Xis2c, and Xis2d. In this paper, we show that three of the six arm-type sites are absolutely required for excision. Furthermore, we also identified two binding sites for Xis2d and two possible binding sites for Xis2c on the attR site. We also showed that IntDOT interacts cooperatively with the Xis2c and Xis2d proteins on the attR site.


Subject(s)
Bacterial Proteins/metabolism , Bacteroides/genetics , Conjugation, Genetic/genetics , DNA Transposable Elements/genetics , Membrane Proteins/metabolism , Plasmids/genetics , Recombination, Genetic/genetics , Binding Sites/genetics , Chromosome Pairing/genetics , Conjugation, Genetic/physiology , DNA Primers/genetics , Electrophoretic Mobility Shift Assay , Mutagenesis , Recombination, Genetic/physiology
3.
J Bacteriol ; 195(9): 1883-91, 2013 May.
Article in English | MEDLINE | ID: mdl-23335422

ABSTRACT

Integrative and conjugative elements (ICEs), formerly called conjugative transposons, have been implicated in the proliferation of antibiotic resistance genes. CTnDOT is an extensively studied ICE found in Bacteroides spp. In addition to carrying resistance genes to both erythromycin and tetracycline, CTnDOT carries a gene that encodes a tyrosine recombinase called IntDOT that catalyzes integration into and excision out of the bacterial host chromosome. CTnDOT integrates into one of several known attB sites in the bacterial chromosome that consists of a pair of inverted repeat core sites called B and B' in attB. The attDOT site contains the core sites and D and D'. These sites flank the overlap regions where strand exchanges occur. A notable feature of all known attB sites is the conservation of the B core site sequence, which is also found in the D core site of attDOT. In this study, we used a mutational analysis to establish the importance of this conserved sequence for integration and characterize the interaction of IntDOT with individual base pairs. We identified important T-A base pairs at position -5 in the B and D core sites and position +5 in the poorly conserved B' core site that are important for integrative recombination. Base analog studies suggest that IntDOT may make specific contacts with the A residues in the major groove at positions -5 and +5. IntDOT interaction with the A at position -5 in the B core site is required for the first strand exchange.


Subject(s)
Bacterial Proteins/metabolism , Bacteroides/genetics , Integrases/metabolism , Recombination, Genetic , Bacterial Proteins/genetics , Bacteroides/enzymology , Bacteroides/physiology , Base Sequence , Conjugation, Genetic , Integrases/genetics , Molecular Sequence Data
4.
J Bacteriol ; 192(15): 3934-43, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20511494

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.


Subject(s)
Attachment Sites, Microbiological , Bacteroides/enzymology , DNA, Bacterial/genetics , Integrases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial/physiology , Integrases/genetics , Mutation , Protein Binding , Recombination, Genetic
5.
Nucleic Acids Res ; 38(3): 958-69, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19952068

ABSTRACT

The Bacteroides conjugative transposon CTnDOT encodes an integrase, IntDOT, which is a member of the tyrosine recombinase family. Other members of this group share a strict requirement for sequence identity within the region of strand exchange, called the overlap region. Tyrosine recombinases catalyze recombination by making an initial cleavage, strand exchange and ligation, followed by strand swapping isomerization requiring sequence identity in the overlap region, followed by the second cleavage, strand exchange and ligation. IntDOT is of particular interest because it has been shown to utilize a three-step mechanism: a sequence identity-dependent initial strand exchange that requires two base pairs of complementary DNA at the site of cleavage; a sequence identity-independent strand swapping isomerization, followed by a sequence identity-independent cleavage, strand exchange and ligation. In addition to the sequence identity requirement in the overlap region, Lambda Int interactions with arm-type sites dictate the order of strand exchange regardless of the orientation of the overlap region. Although IntDOT has an arm-binding domain, we show here that the location of sequence identity within the overlap region dictates where the initial cleavage takes place and that IntDOT can recombine substrates containing mismatches in the overlap region so long as a single base of sequence identity exists at the site of initial cleavage.


Subject(s)
Attachment Sites, Microbiological , Integrases/metabolism , Bacteroides/enzymology , DNA/chemistry , DNA/metabolism , DNA Cleavage , Sequence Homology, Nucleic Acid
6.
Nucleic Acids Res ; 35(17): 5861-73, 2007.
Article in English | MEDLINE | ID: mdl-17720706

ABSTRACT

Although the integrase (IntDOT) of the Bacteroides conjugative transposon CTnDOT has been classified as a member of the tyrosine recombinase family, the reaction it catalyzes appears to differ in some features from reactions catalyzed by other tyrosine recombinases. We tested the ability of IntDOT to cleave and ligate activated attDOT substrates in the presence of mismatches. Unlike other tyrosine recombinases, the results revealed that IntDOT is able to perform ligation reactions even when all the bases within the crossover region are mispaired. We also show that there is a strong bias in the order of strand exchanges during integrative recombination. The top strands are exchanged first in reactions that appear to require 2 bp of homology between the partner sites adjacent to the sites of cleavage. The bottom strands are exchanged next in reactions that do not require homology between the partner sites. This mode of coordination of strand exchanges is unique among tyrosine recombinases.


Subject(s)
Bacteroides/genetics , Integrases/metabolism , Recombination, Genetic , Attachment Sites, Microbiological , Base Pair Mismatch , DNA Transposable Elements , DNA, Cruciform/chemistry , DNA, Cruciform/metabolism
7.
Science ; 301(5641): 1890-3, 2003 Sep 26.
Article in English | MEDLINE | ID: mdl-14512624

ABSTRACT

High-resolution analyses of lake sediment from southwestern Alaska reveal cyclic variations in climate and ecosystems during the Holocene. These variations occurred with periodicities similar to those of solar activity and appear to be coherent with time series of the cosmogenic nuclides 14C and 10Be as well as North Atlantic drift ice. Our results imply that small variations in solar irradiance induced pronounced cyclic changes in northern high-latitude environments. They also provide evidence that centennial-scale shifts in the Holocene climate were similar between the subpolar regions of the North Atlantic and North Pacific, possibly because of Sun-ocean-climate linkages.

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