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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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G-quadruplex (G4) formation by the vlsE 17 bp direct repeat.A) DNA sequences of the oligonucleotides used to assay for intermolecular G-quadruplex formation. The shortened oligos are all deleted forms of the full length 17-mer direct repeat. B. Electrophoretic mobility shift assay for G-quadruplex formation using 5′ 32P-labeled oligonucleotides from Panel A. The 5′ end-labeled oligos were annealed by heating at 95°C for 5 minutes followed by slow cooling for 15 hours to room temperature in presence of 200 mM LiCl or KCl. The products of the annealing reaction were run at 40 volts on a 10 cm, 20% native polyacrylamide gel in TBE buffer containing 25 mM K2B4O7 run at 4°C. Separated products were detected on a Perkin-Elmer Cyclone phosphorimager. SS represents single stranded oligonucleotide and G4 indicates the position of intermolecular G-quadruplex DNA.
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pone-0057792-g004: G-quadruplex (G4) formation by the vlsE 17 bp direct repeat.A) DNA sequences of the oligonucleotides used to assay for intermolecular G-quadruplex formation. The shortened oligos are all deleted forms of the full length 17-mer direct repeat. B. Electrophoretic mobility shift assay for G-quadruplex formation using 5′ 32P-labeled oligonucleotides from Panel A. The 5′ end-labeled oligos were annealed by heating at 95°C for 5 minutes followed by slow cooling for 15 hours to room temperature in presence of 200 mM LiCl or KCl. The products of the annealing reaction were run at 40 volts on a 10 cm, 20% native polyacrylamide gel in TBE buffer containing 25 mM K2B4O7 run at 4°C. Separated products were detected on a Perkin-Elmer Cyclone phosphorimager. SS represents single stranded oligonucleotide and G4 indicates the position of intermolecular G-quadruplex DNA.

Mentions: To directly probe the ability of the 17 bp DR’s to participate in G-quadruplex formation we synthesized the top (G-rich) strand of the 17 bp DR as well as several deleted forms of the oligo (Fig. 4A). The oligos were labeled at their 5′ ends and used in gel shift assays to monitor their ability to form G4 DNA (Fig. 4B). The oligonucleotides were self annealed either in 200 mM KCl to promote G4 formation or in 200 mM LiCl to inhibit it. All four oligos formed about 50% G4 DNA based upon reduced gel mobility in the presence of K+ but not Li+. The pentanucleotide G stretch appeared to be the only region required for G4 formation. To further probe the role of the G run in quadruplex formation, we incorporated the previously used DR* mutations (see Fig. 3A) in the G-motif of the top strands of the 14-mer and the 17-mer by substituting four successive G’s with TATA (Fig. 5A, C). We also synthesized the bottom strands of the 14-mer and 17-mer. Neither the mutant top strands nor the C-rich bottom strands formed G4 DNA (Fig. 5B, D).


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)

G-quadruplex (G4) formation by the vlsE 17 bp direct repeat.A) DNA sequences of the oligonucleotides used to assay for intermolecular G-quadruplex formation. The shortened oligos are all deleted forms of the full length 17-mer direct repeat. B. Electrophoretic mobility shift assay for G-quadruplex formation using 5′ 32P-labeled oligonucleotides from Panel A. The 5′ end-labeled oligos were annealed by heating at 95°C for 5 minutes followed by slow cooling for 15 hours to room temperature in presence of 200 mM LiCl or KCl. The products of the annealing reaction were run at 40 volts on a 10 cm, 20% native polyacrylamide gel in TBE buffer containing 25 mM K2B4O7 run at 4°C. Separated products were detected on a Perkin-Elmer Cyclone phosphorimager. SS represents single stranded oligonucleotide and G4 indicates the position of intermolecular G-quadruplex DNA.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057792-g004: G-quadruplex (G4) formation by the vlsE 17 bp direct repeat.A) DNA sequences of the oligonucleotides used to assay for intermolecular G-quadruplex formation. The shortened oligos are all deleted forms of the full length 17-mer direct repeat. B. Electrophoretic mobility shift assay for G-quadruplex formation using 5′ 32P-labeled oligonucleotides from Panel A. The 5′ end-labeled oligos were annealed by heating at 95°C for 5 minutes followed by slow cooling for 15 hours to room temperature in presence of 200 mM LiCl or KCl. The products of the annealing reaction were run at 40 volts on a 10 cm, 20% native polyacrylamide gel in TBE buffer containing 25 mM K2B4O7 run at 4°C. Separated products were detected on a Perkin-Elmer Cyclone phosphorimager. SS represents single stranded oligonucleotide and G4 indicates the position of intermolecular G-quadruplex DNA.
Mentions: To directly probe the ability of the 17 bp DR’s to participate in G-quadruplex formation we synthesized the top (G-rich) strand of the 17 bp DR as well as several deleted forms of the oligo (Fig. 4A). The oligos were labeled at their 5′ ends and used in gel shift assays to monitor their ability to form G4 DNA (Fig. 4B). The oligonucleotides were self annealed either in 200 mM KCl to promote G4 formation or in 200 mM LiCl to inhibit it. All four oligos formed about 50% G4 DNA based upon reduced gel mobility in the presence of K+ but not Li+. The pentanucleotide G stretch appeared to be the only region required for G4 formation. To further probe the role of the G run in quadruplex formation, we incorporated the previously used DR* mutations (see Fig. 3A) in the G-motif of the top strands of the 14-mer and the 17-mer by substituting four successive G’s with TATA (Fig. 5A, C). We also synthesized the bottom strands of the 14-mer and 17-mer. Neither the mutant top strands nor the C-rich bottom strands formed G4 DNA (Fig. 5B, D).

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