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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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Related in: MedlinePlus

Affect of mutations in the 17 bp direct repeats on precise excision of the vlsE variable region.A) DNA sequences of the wild-type 17 bp direct repeat (DR) and a mutant 17 bp direct repeat (DR*) used in this study. Mutated bases are highlighted in red. B) Schematic showing the plasmid templates carrying wild-type DRs and a mutant DR at the left, right or both sides of the variable region. C) An ethidium bromide-stained agarose gel showing amplification of a portion of vlsE with Phusion DNA polymerase using the templates shown in Panel B with the indicated primers. Gel electrophoresis conditions were as noted in Fig. 2.
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pone-0057792-g003: Affect of mutations in the 17 bp direct repeats on precise excision of the vlsE variable region.A) DNA sequences of the wild-type 17 bp direct repeat (DR) and a mutant 17 bp direct repeat (DR*) used in this study. Mutated bases are highlighted in red. B) Schematic showing the plasmid templates carrying wild-type DRs and a mutant DR at the left, right or both sides of the variable region. C) An ethidium bromide-stained agarose gel showing amplification of a portion of vlsE with Phusion DNA polymerase using the templates shown in Panel B with the indicated primers. Gel electrophoresis conditions were as noted in Fig. 2.

Mentions: A closer look at the sequence of the17 bp DR’s showed a pentanucleotide G stretch (Fig. 3A). Such sequences are known to be involved in formation of G-quadruplex structures, also referred to as G4 DNA [30], [31], [32], [33], [34]. To directly probe the importance of the G run, site directed mutagenesis was used to generate constructs having a mutant direct repeat (DR*) at either the left, right or both sides (Fig. 3B) of the variable region by changing four nucleotides of the G stretch to TATA (Fig. 3A). The ability of the DR mutants to undergo precise excision was assayed by amplifying vlsE using primers B248 and B249. As expected, none of the DR mutants displayed precise excision of the vlsE variable region (Fig. 3C). The inability of the double mutant (DR*L,R) to restore precise excision indicates that the G stretch of the DR’s is important because the double mutant has two identical DR’s, but their sequence differs from the wild-type in the absence of the G run.


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)

Affect of mutations in the 17 bp direct repeats on precise excision of the vlsE variable region.A) DNA sequences of the wild-type 17 bp direct repeat (DR) and a mutant 17 bp direct repeat (DR*) used in this study. Mutated bases are highlighted in red. B) Schematic showing the plasmid templates carrying wild-type DRs and a mutant DR at the left, right or both sides of the variable region. C) An ethidium bromide-stained agarose gel showing amplification of a portion of vlsE with Phusion DNA polymerase using the templates shown in Panel B with the indicated primers. Gel electrophoresis conditions were as noted in Fig. 2.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057792-g003: Affect of mutations in the 17 bp direct repeats on precise excision of the vlsE variable region.A) DNA sequences of the wild-type 17 bp direct repeat (DR) and a mutant 17 bp direct repeat (DR*) used in this study. Mutated bases are highlighted in red. B) Schematic showing the plasmid templates carrying wild-type DRs and a mutant DR at the left, right or both sides of the variable region. C) An ethidium bromide-stained agarose gel showing amplification of a portion of vlsE with Phusion DNA polymerase using the templates shown in Panel B with the indicated primers. Gel electrophoresis conditions were as noted in Fig. 2.
Mentions: A closer look at the sequence of the17 bp DR’s showed a pentanucleotide G stretch (Fig. 3A). Such sequences are known to be involved in formation of G-quadruplex structures, also referred to as G4 DNA [30], [31], [32], [33], [34]. To directly probe the importance of the G run, site directed mutagenesis was used to generate constructs having a mutant direct repeat (DR*) at either the left, right or both sides (Fig. 3B) of the variable region by changing four nucleotides of the G stretch to TATA (Fig. 3A). The ability of the DR mutants to undergo precise excision was assayed by amplifying vlsE using primers B248 and B249. As expected, none of the DR mutants displayed precise excision of the vlsE variable region (Fig. 3C). The inability of the double mutant (DR*L,R) to restore precise excision indicates that the G stretch of the DR’s is important because the double mutant has two identical DR’s, but their sequence differs from the wild-type in the absence of the G run.

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