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Irradiation-induced Deinococcus radiodurans genome fragmentation triggers transposition of a single resident insertion sequence.

Pasternak C, Ton-Hoang B, Coste G, Bailone A, Chandler M, Sommer S - PLoS Genet. (2010)

Bottom Line: This capacity of D. radiodurans to withstand irradiation raises important questions concerning its response to radiation-induced mutagenic lesions.A recent study analyzed the mutational profile in the thyA gene following irradiation.ISDra2 is a member of a newly recognised class of ISs, the IS200/IS605 family of insertion sequences.

View Article: PubMed Central - PubMed

Affiliation: Université Paris-Sud, Centre National de Recherche Scientifique, Unité Mixte de Recherche 8621, LRC CEA 42V, Institut de Génétique et Microbiologie, Bât. 409, Orsay, France.

ABSTRACT
Stress-induced transposition is an attractive notion since it is potentially important in creating diversity to facilitate adaptation of the host to severe environmental conditions. One common major stress is radiation-induced DNA damage. Deinococcus radiodurans has an exceptional ability to withstand the lethal effects of DNA-damaging agents (ionizing radiation, UV light, and desiccation). High radiation levels result in genome fragmentation and reassembly in a process which generates significant amounts of single-stranded DNA. This capacity of D. radiodurans to withstand irradiation raises important questions concerning its response to radiation-induced mutagenic lesions. A recent study analyzed the mutational profile in the thyA gene following irradiation. The majority of thyA mutants resulted from transposition of one particular Insertion Sequence (IS), ISDra2, of the many different ISs in the D. radiodurans genome. ISDra2 is a member of a newly recognised class of ISs, the IS200/IS605 family of insertion sequences.

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A genetic assay for ISDra2 transposition.(A) Derivatives of ISDra2 (1736 bp). Orfs are indicated as boxes with arrowheads showing the direction of translation; LE and RE, red and blue boxes, respectively. In ISDra2-113 (1509 bp) both tnpA and tnpB were replaced by a CamR cassette, while ISDra2-103 (1778 bp) was deleted only of tnpB and expresses tnpA from its natural promoter. ISDra2-104 results from replacement of the tnpA coding region of ISDra2-103 by the lacZ coding region. ISDra2-103Term116 (1889 bp) carries the Deinococcal transcription terminator Term116 [45] downstream of the cat gene. (B) In vivo genetic assay to measure excision and insertion events of a derivative of ISDra2. The ISDra2F copy was first replaced by a TetR cassette and ISDra2-113 (or ISDra2-103) was inserted at the unique TTGAT target site present in the tetA gene. The second inactive copy, ISDra2*, was replaced by the sacB gene and an accompanying hygromycin (hyg) resistance cassette as a selective marker. TnpA-dependent ISDra2 excision restores a functional tetA gene, giving rise to TetR colonies while insertion into the reporter sacB gene confers resistance to sucrose. See Figure S1 and its legend.
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pgen-1000799-g001: A genetic assay for ISDra2 transposition.(A) Derivatives of ISDra2 (1736 bp). Orfs are indicated as boxes with arrowheads showing the direction of translation; LE and RE, red and blue boxes, respectively. In ISDra2-113 (1509 bp) both tnpA and tnpB were replaced by a CamR cassette, while ISDra2-103 (1778 bp) was deleted only of tnpB and expresses tnpA from its natural promoter. ISDra2-104 results from replacement of the tnpA coding region of ISDra2-103 by the lacZ coding region. ISDra2-103Term116 (1889 bp) carries the Deinococcal transcription terminator Term116 [45] downstream of the cat gene. (B) In vivo genetic assay to measure excision and insertion events of a derivative of ISDra2. The ISDra2F copy was first replaced by a TetR cassette and ISDra2-113 (or ISDra2-103) was inserted at the unique TTGAT target site present in the tetA gene. The second inactive copy, ISDra2*, was replaced by the sacB gene and an accompanying hygromycin (hyg) resistance cassette as a selective marker. TnpA-dependent ISDra2 excision restores a functional tetA gene, giving rise to TetR colonies while insertion into the reporter sacB gene confers resistance to sucrose. See Figure S1 and its legend.

Mentions: We have explored the properties and behaviour of ISDra2 (ISDra2F; Figure 1A) in D. radiodurans and have identified the mechanism by which ISDra2 transposition is triggered by radiation. Using a genetic system to detect two principal transposition steps, transposon excision and insertion, we show that ISDra2, like other IS200/605 family members, requires TnpA but not TnpB for both and that insertion occurs 3′ to a specific pentanucleotide (as deduced from genome analyses [20]). We demonstrate genetically that both steps are significantly increased following host cell irradiation. We also show that the entire TnpA-catalysed transposition cycle including excision and insertion depends strictly on single strand DNA substrates in vitro. Finally, using a PCR-based approach, we demonstrate that, in vivo, exposure to γ-irradiation stimulates excision of the single genomic copy of ISDra2 from the genome in the form of a DNA circle. These events are closely correlated with the initiation of the process leading to genome reassembly from chromosomal fragments, which occurs mainly through a mechanism generating long stretches of single stranded DNA [18].


Irradiation-induced Deinococcus radiodurans genome fragmentation triggers transposition of a single resident insertion sequence.

Pasternak C, Ton-Hoang B, Coste G, Bailone A, Chandler M, Sommer S - PLoS Genet. (2010)

A genetic assay for ISDra2 transposition.(A) Derivatives of ISDra2 (1736 bp). Orfs are indicated as boxes with arrowheads showing the direction of translation; LE and RE, red and blue boxes, respectively. In ISDra2-113 (1509 bp) both tnpA and tnpB were replaced by a CamR cassette, while ISDra2-103 (1778 bp) was deleted only of tnpB and expresses tnpA from its natural promoter. ISDra2-104 results from replacement of the tnpA coding region of ISDra2-103 by the lacZ coding region. ISDra2-103Term116 (1889 bp) carries the Deinococcal transcription terminator Term116 [45] downstream of the cat gene. (B) In vivo genetic assay to measure excision and insertion events of a derivative of ISDra2. The ISDra2F copy was first replaced by a TetR cassette and ISDra2-113 (or ISDra2-103) was inserted at the unique TTGAT target site present in the tetA gene. The second inactive copy, ISDra2*, was replaced by the sacB gene and an accompanying hygromycin (hyg) resistance cassette as a selective marker. TnpA-dependent ISDra2 excision restores a functional tetA gene, giving rise to TetR colonies while insertion into the reporter sacB gene confers resistance to sucrose. See Figure S1 and its legend.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2806898&req=5

pgen-1000799-g001: A genetic assay for ISDra2 transposition.(A) Derivatives of ISDra2 (1736 bp). Orfs are indicated as boxes with arrowheads showing the direction of translation; LE and RE, red and blue boxes, respectively. In ISDra2-113 (1509 bp) both tnpA and tnpB were replaced by a CamR cassette, while ISDra2-103 (1778 bp) was deleted only of tnpB and expresses tnpA from its natural promoter. ISDra2-104 results from replacement of the tnpA coding region of ISDra2-103 by the lacZ coding region. ISDra2-103Term116 (1889 bp) carries the Deinococcal transcription terminator Term116 [45] downstream of the cat gene. (B) In vivo genetic assay to measure excision and insertion events of a derivative of ISDra2. The ISDra2F copy was first replaced by a TetR cassette and ISDra2-113 (or ISDra2-103) was inserted at the unique TTGAT target site present in the tetA gene. The second inactive copy, ISDra2*, was replaced by the sacB gene and an accompanying hygromycin (hyg) resistance cassette as a selective marker. TnpA-dependent ISDra2 excision restores a functional tetA gene, giving rise to TetR colonies while insertion into the reporter sacB gene confers resistance to sucrose. See Figure S1 and its legend.
Mentions: We have explored the properties and behaviour of ISDra2 (ISDra2F; Figure 1A) in D. radiodurans and have identified the mechanism by which ISDra2 transposition is triggered by radiation. Using a genetic system to detect two principal transposition steps, transposon excision and insertion, we show that ISDra2, like other IS200/605 family members, requires TnpA but not TnpB for both and that insertion occurs 3′ to a specific pentanucleotide (as deduced from genome analyses [20]). We demonstrate genetically that both steps are significantly increased following host cell irradiation. We also show that the entire TnpA-catalysed transposition cycle including excision and insertion depends strictly on single strand DNA substrates in vitro. Finally, using a PCR-based approach, we demonstrate that, in vivo, exposure to γ-irradiation stimulates excision of the single genomic copy of ISDra2 from the genome in the form of a DNA circle. These events are closely correlated with the initiation of the process leading to genome reassembly from chromosomal fragments, which occurs mainly through a mechanism generating long stretches of single stranded DNA [18].

Bottom Line: This capacity of D. radiodurans to withstand irradiation raises important questions concerning its response to radiation-induced mutagenic lesions.A recent study analyzed the mutational profile in the thyA gene following irradiation.ISDra2 is a member of a newly recognised class of ISs, the IS200/IS605 family of insertion sequences.

View Article: PubMed Central - PubMed

Affiliation: Université Paris-Sud, Centre National de Recherche Scientifique, Unité Mixte de Recherche 8621, LRC CEA 42V, Institut de Génétique et Microbiologie, Bât. 409, Orsay, France.

ABSTRACT
Stress-induced transposition is an attractive notion since it is potentially important in creating diversity to facilitate adaptation of the host to severe environmental conditions. One common major stress is radiation-induced DNA damage. Deinococcus radiodurans has an exceptional ability to withstand the lethal effects of DNA-damaging agents (ionizing radiation, UV light, and desiccation). High radiation levels result in genome fragmentation and reassembly in a process which generates significant amounts of single-stranded DNA. This capacity of D. radiodurans to withstand irradiation raises important questions concerning its response to radiation-induced mutagenic lesions. A recent study analyzed the mutational profile in the thyA gene following irradiation. The majority of thyA mutants resulted from transposition of one particular Insertion Sequence (IS), ISDra2, of the many different ISs in the D. radiodurans genome. ISDra2 is a member of a newly recognised class of ISs, the IS200/IS605 family of insertion sequences.

Show MeSH
Related in: MedlinePlus