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Hin-mediated DNA knotting and recombining promote replicon dysfunction and mutation.

Deibler RW, Mann JK, Sumners de WL, Zechiedrich L - BMC Mol. Biol. (2007)

Bottom Line: Here we analyze the effects of recombined and knotted plasmids in E. coli using the Hin site-specific recombination system.We show that Hin-mediated DNA knotting and recombination (i) promote replicon loss by blocking DNA replication; (ii) block gene transcription; and (iii) cause genetic rearrangements at a rate three to four orders of magnitude higher than the rate for an unknotted, unrecombined plasmid.These results show that DNA reactivity leading to recombined and knotted DNA is potentially toxic and may help drive genetic evolution.

View Article: PubMed Central - HTML - PubMed

Affiliation: Interdepartmental Program in Cell and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030-3411, USA. richard_deibler@hms.harvard.edu <richard_deibler@hms.harvard.edu>

ABSTRACT

Background: The genetic code imposes a dilemma for cells. The DNA must be long enough to encode for the complexity of an organism, yet thin and flexible enough to fit within the cell. The combination of these properties greatly favors DNA collisions, which can knot and drive recombination of the DNA. Despite the well-accepted propensity of cellular DNA to collide and react with itself, it has not been established what the physiological consequences are.

Results: Here we analyze the effects of recombined and knotted plasmids in E. coli using the Hin site-specific recombination system. We show that Hin-mediated DNA knotting and recombination (i) promote replicon loss by blocking DNA replication; (ii) block gene transcription; and (iii) cause genetic rearrangements at a rate three to four orders of magnitude higher than the rate for an unknotted, unrecombined plasmid.

Conclusion: These results show that DNA reactivity leading to recombined and knotted DNA is potentially toxic and may help drive genetic evolution.

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Related in: MedlinePlus

Physiological effects of Hin-mediated recombination/knotting. (A) Assay for the effect of knotting on the function of a gene. The ovals represent E. coli cells. The Hin expression vector, pHIN, and plasmid substrates pBR, pREC and pKNOT containing the bla gene (encoding β-lactamase) are depicted. Wild-type recombination sites are depicted as black arrows. The mutant hix site is shown as a grey arrow. (B) Effect of DNA knotting on ampicillin sensitivity of E. coli strain W3110 containing pHIN and either pBR, pREC or pKNOT. Single colonies were streaked from left to right across LB-agar that contained an ampicillin gradient and constant IPTG (1 mM) and spectinomycin (50 μg/ml) for Hin overexpression and maintenance. The experiment was repeated five times in either strain C600 or W3110, and was carried out either from high to low or from low to high ampicillin concentration with identical results. (C) Ampicillin sensitivity (MIC50) was quantified using the plate dilution method.
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Figure 1: Physiological effects of Hin-mediated recombination/knotting. (A) Assay for the effect of knotting on the function of a gene. The ovals represent E. coli cells. The Hin expression vector, pHIN, and plasmid substrates pBR, pREC and pKNOT containing the bla gene (encoding β-lactamase) are depicted. Wild-type recombination sites are depicted as black arrows. The mutant hix site is shown as a grey arrow. (B) Effect of DNA knotting on ampicillin sensitivity of E. coli strain W3110 containing pHIN and either pBR, pREC or pKNOT. Single colonies were streaked from left to right across LB-agar that contained an ampicillin gradient and constant IPTG (1 mM) and spectinomycin (50 μg/ml) for Hin overexpression and maintenance. The experiment was repeated five times in either strain C600 or W3110, and was carried out either from high to low or from low to high ampicillin concentration with identical results. (C) Ampicillin sensitivity (MIC50) was quantified using the plate dilution method.

Mentions: The experimental approach we use here to study the cellular effect of recombining and knotting DNA is outlined in Figure 1A. We have shown previously that Hin recombines and knots plasmid DNA in E. coli that topoisomerase IV unties [21]. The Hin site-specific recombination system models two in vivo processes: it tangles the DNA to create knots identical to those formed inside the cell and shuffles the DNA sequence to model DNA recombination [6-10]. The hin recombinase gene is provided by the plasmid pKH66 (hereafter referred to as pHIN) and is expressed from the tac promoter following induction by isopropyl-β-D-thiogalactopyranoside (IPTG). pHIN also encodes for spectinomycin resistance. E. coli cells harboring pHIN also contained either pBR322 (pBR), which lacks recombination sites and serves as a negative control, or one of two pBR22-derived plasmids pTGSE4 (pREC) or pRJ862 (pKNOT) that carry sites recognized by the Hin recombinase. All three plasmids contain the bla gene, which encodes β-lactamase and provides resistance to ampicillin. We used the bla gene as a reporter to assess the effects of recombining and knotting the DNA.


Hin-mediated DNA knotting and recombining promote replicon dysfunction and mutation.

Deibler RW, Mann JK, Sumners de WL, Zechiedrich L - BMC Mol. Biol. (2007)

Physiological effects of Hin-mediated recombination/knotting. (A) Assay for the effect of knotting on the function of a gene. The ovals represent E. coli cells. The Hin expression vector, pHIN, and plasmid substrates pBR, pREC and pKNOT containing the bla gene (encoding β-lactamase) are depicted. Wild-type recombination sites are depicted as black arrows. The mutant hix site is shown as a grey arrow. (B) Effect of DNA knotting on ampicillin sensitivity of E. coli strain W3110 containing pHIN and either pBR, pREC or pKNOT. Single colonies were streaked from left to right across LB-agar that contained an ampicillin gradient and constant IPTG (1 mM) and spectinomycin (50 μg/ml) for Hin overexpression and maintenance. The experiment was repeated five times in either strain C600 or W3110, and was carried out either from high to low or from low to high ampicillin concentration with identical results. (C) Ampicillin sensitivity (MIC50) was quantified using the plate dilution method.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1904230&req=5

Figure 1: Physiological effects of Hin-mediated recombination/knotting. (A) Assay for the effect of knotting on the function of a gene. The ovals represent E. coli cells. The Hin expression vector, pHIN, and plasmid substrates pBR, pREC and pKNOT containing the bla gene (encoding β-lactamase) are depicted. Wild-type recombination sites are depicted as black arrows. The mutant hix site is shown as a grey arrow. (B) Effect of DNA knotting on ampicillin sensitivity of E. coli strain W3110 containing pHIN and either pBR, pREC or pKNOT. Single colonies were streaked from left to right across LB-agar that contained an ampicillin gradient and constant IPTG (1 mM) and spectinomycin (50 μg/ml) for Hin overexpression and maintenance. The experiment was repeated five times in either strain C600 or W3110, and was carried out either from high to low or from low to high ampicillin concentration with identical results. (C) Ampicillin sensitivity (MIC50) was quantified using the plate dilution method.
Mentions: The experimental approach we use here to study the cellular effect of recombining and knotting DNA is outlined in Figure 1A. We have shown previously that Hin recombines and knots plasmid DNA in E. coli that topoisomerase IV unties [21]. The Hin site-specific recombination system models two in vivo processes: it tangles the DNA to create knots identical to those formed inside the cell and shuffles the DNA sequence to model DNA recombination [6-10]. The hin recombinase gene is provided by the plasmid pKH66 (hereafter referred to as pHIN) and is expressed from the tac promoter following induction by isopropyl-β-D-thiogalactopyranoside (IPTG). pHIN also encodes for spectinomycin resistance. E. coli cells harboring pHIN also contained either pBR322 (pBR), which lacks recombination sites and serves as a negative control, or one of two pBR22-derived plasmids pTGSE4 (pREC) or pRJ862 (pKNOT) that carry sites recognized by the Hin recombinase. All three plasmids contain the bla gene, which encodes β-lactamase and provides resistance to ampicillin. We used the bla gene as a reporter to assess the effects of recombining and knotting the DNA.

Bottom Line: Here we analyze the effects of recombined and knotted plasmids in E. coli using the Hin site-specific recombination system.We show that Hin-mediated DNA knotting and recombination (i) promote replicon loss by blocking DNA replication; (ii) block gene transcription; and (iii) cause genetic rearrangements at a rate three to four orders of magnitude higher than the rate for an unknotted, unrecombined plasmid.These results show that DNA reactivity leading to recombined and knotted DNA is potentially toxic and may help drive genetic evolution.

View Article: PubMed Central - HTML - PubMed

Affiliation: Interdepartmental Program in Cell and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030-3411, USA. richard_deibler@hms.harvard.edu <richard_deibler@hms.harvard.edu>

ABSTRACT

Background: The genetic code imposes a dilemma for cells. The DNA must be long enough to encode for the complexity of an organism, yet thin and flexible enough to fit within the cell. The combination of these properties greatly favors DNA collisions, which can knot and drive recombination of the DNA. Despite the well-accepted propensity of cellular DNA to collide and react with itself, it has not been established what the physiological consequences are.

Results: Here we analyze the effects of recombined and knotted plasmids in E. coli using the Hin site-specific recombination system. We show that Hin-mediated DNA knotting and recombination (i) promote replicon loss by blocking DNA replication; (ii) block gene transcription; and (iii) cause genetic rearrangements at a rate three to four orders of magnitude higher than the rate for an unknotted, unrecombined plasmid.

Conclusion: These results show that DNA reactivity leading to recombined and knotted DNA is potentially toxic and may help drive genetic evolution.

Show MeSH
Related in: MedlinePlus