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Suggested role for G4 DNA in recombinational switching at the antigenic variation locus of the Lyme disease spirochete.

Walia R, Chaconas G - PLoS ONE (2013)

Bottom Line: In the work presented here we show that G4 DNA can be formed by sequences within the B31 vlsE locus, prompting us to investigate the presence of potential G4-forming DNA throughout the vls locus of several Lyme spirochete strains and species.We found that runs of G, three nucleotides and longer occur at a very high density, with a greater than 100-fold strand-specific distribution in the vls locus of three B. burgdorferi strains as well as in B. afzelii and B. garinii, in spite of the bias for the use of A-T rich codons in Borrelia species.Our findings suggest the possibility that G4 DNA may be a mediator of recombinational switching at the vlsE locus in the Lyme spirochetes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology, Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada.

ABSTRACT
Antigenic variation through targeted DNA rearrangements provides a powerful diversity generating mechanism that allows a variety of pathogens to stay one step ahead of acquired immunity in their hosts. The Lyme disease spirochete encodes such a system that is required for persistent infection. The vls locus, carried on a 29 kb linear plasmid (lp28-1) in the type strain B31, carries 15 silent cassettes from which information is unidirectionally transferred into the expression locus, vlsE. Recent studies have surprisingly shown that, with the exception of the RuvAB branch migrase, no other known recombination/repair proteins appear to play a role in the recombinational switching process. In the work presented here we show that G4 DNA can be formed by sequences within the B31 vlsE locus, prompting us to investigate the presence of potential G4-forming DNA throughout the vls locus of several Lyme spirochete strains and species. We found that runs of G, three nucleotides and longer occur at a very high density, with a greater than 100-fold strand-specific distribution in the vls locus of three B. burgdorferi strains as well as in B. afzelii and B. garinii, in spite of the bias for the use of A-T rich codons in Borrelia species. Our findings suggest the possibility that G4 DNA may be a mediator of recombinational switching at the vlsE locus in the Lyme spirochetes.

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Methylation protection of the vlsE G4-forming sequence.A) Autoradiogram of a 25% denaturing polyacrylamide gel of dimethylsulfate treated 14-mer and 17-mer oligonucleotides. 32P-labeled oligonucleotides were annealed in 200 mM KCl and treated with 0.5% dimethylsulfate. The methylated oligos were then subjected to electrophoresis in a 20% native polyacrylamide gel to separate the single stranded (SS) oligos from the G4-DNA. The free and the G4 bands were treated with 1 M piperidine to induce strand breaks at the methylated guanine residues and the cleavage products were resolved on a 25% denaturing polyacrylamide gel. C represents the control oligo which was not treated with DMS. The arrows correspond to the DMS-protected guanine residues. B) Sequence of the oligonucleotides used in this experiment. The dots show the position of G residues. The smallest cleavage fragment was run off the bottom of the gel.
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pone-0057792-g006: Methylation protection of the vlsE G4-forming sequence.A) Autoradiogram of a 25% denaturing polyacrylamide gel of dimethylsulfate treated 14-mer and 17-mer oligonucleotides. 32P-labeled oligonucleotides were annealed in 200 mM KCl and treated with 0.5% dimethylsulfate. The methylated oligos were then subjected to electrophoresis in a 20% native polyacrylamide gel to separate the single stranded (SS) oligos from the G4-DNA. The free and the G4 bands were treated with 1 M piperidine to induce strand breaks at the methylated guanine residues and the cleavage products were resolved on a 25% denaturing polyacrylamide gel. C represents the control oligo which was not treated with DMS. The arrows correspond to the DMS-protected guanine residues. B) Sequence of the oligonucleotides used in this experiment. The dots show the position of G residues. The smallest cleavage fragment was run off the bottom of the gel.

Mentions: To confirm that the slower migrating gel bands were indeed G4 DNA, dimethyl sulfate (DMS) protection assays [35] were performed with the 14-mer and 17-mer DR. In a G-quadruplex the N7 position of the guanines are involved in Hoogsteen bonding and are inaccessible to methylation by DMS. The oligos were annealed in 200 mM KCl and methylated using 0.5% DMS. The methylated oligos were run on a 20% native polyacrylamide gel (as shown in Fig. 6) and the upper (G4) and lower (single stranded or SS) bands were excised and treated with 1 M piperidine to induce strand breaks at the methylated guanine residues. End-labeled cleavage fragments were resolved on a 25% denaturing polyacrylamide gel (Fig. 6A). Lanes 1 and 4 show the untreated 14-mer and 17-mer oligonuceotides, respectively. Lanes 2 and 5 show the DMS cleavage pattern of the faster migrating (lower gel band) single stranded oligo monomers while lanes 3 and 6 show the footprint of the slower moving G4 bands. In lanes 3 and 6 the N7 positions within the pentanucleotide G runs were protected from DMS modification, as expected for G-quadruplex. Interestingly, the two G residues at the 3′ end of the 17 bp DR were also protected, suggesting that they were also involved in the formation of G-tetrads (Fig. 6A, B). A G4 synapse of 17 bp DR’s could promote precise deletion in vitro, as shown in Fig. 2 and 3, by promoting premature termination of DNA synthesis followed by use of the incomplete product to prime DNA synthesis in a subsequent cycle.


Suggested role for G4 DNA in recombinational switching at the antigenic variation locus of the Lyme disease spirochete.

Walia R, Chaconas G - PLoS ONE (2013)

Methylation protection of the vlsE G4-forming sequence.A) Autoradiogram of a 25% denaturing polyacrylamide gel of dimethylsulfate treated 14-mer and 17-mer oligonucleotides. 32P-labeled oligonucleotides were annealed in 200 mM KCl and treated with 0.5% dimethylsulfate. The methylated oligos were then subjected to electrophoresis in a 20% native polyacrylamide gel to separate the single stranded (SS) oligos from the G4-DNA. The free and the G4 bands were treated with 1 M piperidine to induce strand breaks at the methylated guanine residues and the cleavage products were resolved on a 25% denaturing polyacrylamide gel. C represents the control oligo which was not treated with DMS. The arrows correspond to the DMS-protected guanine residues. B) Sequence of the oligonucleotides used in this experiment. The dots show the position of G residues. The smallest cleavage fragment was run off the bottom of the gel.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585125&req=5

pone-0057792-g006: Methylation protection of the vlsE G4-forming sequence.A) Autoradiogram of a 25% denaturing polyacrylamide gel of dimethylsulfate treated 14-mer and 17-mer oligonucleotides. 32P-labeled oligonucleotides were annealed in 200 mM KCl and treated with 0.5% dimethylsulfate. The methylated oligos were then subjected to electrophoresis in a 20% native polyacrylamide gel to separate the single stranded (SS) oligos from the G4-DNA. The free and the G4 bands were treated with 1 M piperidine to induce strand breaks at the methylated guanine residues and the cleavage products were resolved on a 25% denaturing polyacrylamide gel. C represents the control oligo which was not treated with DMS. The arrows correspond to the DMS-protected guanine residues. B) Sequence of the oligonucleotides used in this experiment. The dots show the position of G residues. The smallest cleavage fragment was run off the bottom of the gel.
Mentions: To confirm that the slower migrating gel bands were indeed G4 DNA, dimethyl sulfate (DMS) protection assays [35] were performed with the 14-mer and 17-mer DR. In a G-quadruplex the N7 position of the guanines are involved in Hoogsteen bonding and are inaccessible to methylation by DMS. The oligos were annealed in 200 mM KCl and methylated using 0.5% DMS. The methylated oligos were run on a 20% native polyacrylamide gel (as shown in Fig. 6) and the upper (G4) and lower (single stranded or SS) bands were excised and treated with 1 M piperidine to induce strand breaks at the methylated guanine residues. End-labeled cleavage fragments were resolved on a 25% denaturing polyacrylamide gel (Fig. 6A). Lanes 1 and 4 show the untreated 14-mer and 17-mer oligonuceotides, respectively. Lanes 2 and 5 show the DMS cleavage pattern of the faster migrating (lower gel band) single stranded oligo monomers while lanes 3 and 6 show the footprint of the slower moving G4 bands. In lanes 3 and 6 the N7 positions within the pentanucleotide G runs were protected from DMS modification, as expected for G-quadruplex. Interestingly, the two G residues at the 3′ end of the 17 bp DR were also protected, suggesting that they were also involved in the formation of G-tetrads (Fig. 6A, B). A G4 synapse of 17 bp DR’s could promote precise deletion in vitro, as shown in Fig. 2 and 3, by promoting premature termination of DNA synthesis followed by use of the incomplete product to prime DNA synthesis in a subsequent cycle.

Bottom Line: In the work presented here we show that G4 DNA can be formed by sequences within the B31 vlsE locus, prompting us to investigate the presence of potential G4-forming DNA throughout the vls locus of several Lyme spirochete strains and species.We found that runs of G, three nucleotides and longer occur at a very high density, with a greater than 100-fold strand-specific distribution in the vls locus of three B. burgdorferi strains as well as in B. afzelii and B. garinii, in spite of the bias for the use of A-T rich codons in Borrelia species.Our findings suggest the possibility that G4 DNA may be a mediator of recombinational switching at the vlsE locus in the Lyme spirochetes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology, Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada.

ABSTRACT
Antigenic variation through targeted DNA rearrangements provides a powerful diversity generating mechanism that allows a variety of pathogens to stay one step ahead of acquired immunity in their hosts. The Lyme disease spirochete encodes such a system that is required for persistent infection. The vls locus, carried on a 29 kb linear plasmid (lp28-1) in the type strain B31, carries 15 silent cassettes from which information is unidirectionally transferred into the expression locus, vlsE. Recent studies have surprisingly shown that, with the exception of the RuvAB branch migrase, no other known recombination/repair proteins appear to play a role in the recombinational switching process. In the work presented here we show that G4 DNA can be formed by sequences within the B31 vlsE locus, prompting us to investigate the presence of potential G4-forming DNA throughout the vls locus of several Lyme spirochete strains and species. We found that runs of G, three nucleotides and longer occur at a very high density, with a greater than 100-fold strand-specific distribution in the vls locus of three B. burgdorferi strains as well as in B. afzelii and B. garinii, in spite of the bias for the use of A-T rich codons in Borrelia species. Our findings suggest the possibility that G4 DNA may be a mediator of recombinational switching at the vlsE locus in the Lyme spirochetes.

Show MeSH
Related in: MedlinePlus