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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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ISDra2 TnpA-catalyzed cleavage and strand transfer in vitro.(A) Oligonucleotides used as DNA substrates. Length of cleavage products is indicated. The potential secondary structure in both LE and RE is indicated. Black dotted and black lines: left and right DNA flanks cleavage sites are shown as vertical black arrows. Asterisk (*) indicates radioisotope position. (B) Excision in vitro: donor joint formation and single-versus double-strand substrates. The 5′-32P-labelled oligonucleotide used was the 59-base LE composed of 39 nt of LE and 20 nt 5′ to the 5′TTGAT3′ and the unlabelled 63-base oligonucleotide RE. Lane 1: no-protein control; lane 2: TnpA alone; lane 3: TnpA and unlabelled RE; lane 4: dsLE, no-protein; lane 5: dsLE, TnpA and ssRE.
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pgen-1000799-g002: ISDra2 TnpA-catalyzed cleavage and strand transfer in vitro.(A) Oligonucleotides used as DNA substrates. Length of cleavage products is indicated. The potential secondary structure in both LE and RE is indicated. Black dotted and black lines: left and right DNA flanks cleavage sites are shown as vertical black arrows. Asterisk (*) indicates radioisotope position. (B) Excision in vitro: donor joint formation and single-versus double-strand substrates. The 5′-32P-labelled oligonucleotide used was the 59-base LE composed of 39 nt of LE and 20 nt 5′ to the 5′TTGAT3′ and the unlabelled 63-base oligonucleotide RE. Lane 1: no-protein control; lane 2: TnpA alone; lane 3: TnpA and unlabelled RE; lane 4: dsLE, no-protein; lane 5: dsLE, TnpA and ssRE.

Mentions: The DNA substrate was a 59 nt single strand DNA fragment including the first 39 nt of ISDra2 LE carrying a subterminal secondary structure which serves as the TnpA binding site (Burgess-Hickman et al., in prep) and a 20 nt 5′ flank with the conserved pentanucleotide target sequence (5′TTGAT3′) (Figure 2A). Incubation of the 5′ end-labelled fragment (Figure 2B, lane 1) with purified TnpA in the presence of Mg2+ generated a cleaved 5′ end-labelled donor flank fragment of 20 nt (Figure 2B, lane 2). When mixed with an unlabelled 63 nt ss DNA fragment composed of the terminal 43 nt of RE (including the secondary structure) and a 3′ 20 nt flank, an additional fragment of 40 nt representing the joined donor flanks was generated (Figure 2B, lane 3). In contrast, double stranded LE (Figure 2B, lane 4) was neither cleaved nor underwent a strand transfer reaction with the unlabelled RE (Figure 2B, lane 5).


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)

ISDra2 TnpA-catalyzed cleavage and strand transfer in vitro.(A) Oligonucleotides used as DNA substrates. Length of cleavage products is indicated. The potential secondary structure in both LE and RE is indicated. Black dotted and black lines: left and right DNA flanks cleavage sites are shown as vertical black arrows. Asterisk (*) indicates radioisotope position. (B) Excision in vitro: donor joint formation and single-versus double-strand substrates. The 5′-32P-labelled oligonucleotide used was the 59-base LE composed of 39 nt of LE and 20 nt 5′ to the 5′TTGAT3′ and the unlabelled 63-base oligonucleotide RE. Lane 1: no-protein control; lane 2: TnpA alone; lane 3: TnpA and unlabelled RE; lane 4: dsLE, no-protein; lane 5: dsLE, TnpA and ssRE.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1000799-g002: ISDra2 TnpA-catalyzed cleavage and strand transfer in vitro.(A) Oligonucleotides used as DNA substrates. Length of cleavage products is indicated. The potential secondary structure in both LE and RE is indicated. Black dotted and black lines: left and right DNA flanks cleavage sites are shown as vertical black arrows. Asterisk (*) indicates radioisotope position. (B) Excision in vitro: donor joint formation and single-versus double-strand substrates. The 5′-32P-labelled oligonucleotide used was the 59-base LE composed of 39 nt of LE and 20 nt 5′ to the 5′TTGAT3′ and the unlabelled 63-base oligonucleotide RE. Lane 1: no-protein control; lane 2: TnpA alone; lane 3: TnpA and unlabelled RE; lane 4: dsLE, no-protein; lane 5: dsLE, TnpA and ssRE.
Mentions: The DNA substrate was a 59 nt single strand DNA fragment including the first 39 nt of ISDra2 LE carrying a subterminal secondary structure which serves as the TnpA binding site (Burgess-Hickman et al., in prep) and a 20 nt 5′ flank with the conserved pentanucleotide target sequence (5′TTGAT3′) (Figure 2A). Incubation of the 5′ end-labelled fragment (Figure 2B, lane 1) with purified TnpA in the presence of Mg2+ generated a cleaved 5′ end-labelled donor flank fragment of 20 nt (Figure 2B, lane 2). When mixed with an unlabelled 63 nt ss DNA fragment composed of the terminal 43 nt of RE (including the secondary structure) and a 3′ 20 nt flank, an additional fragment of 40 nt representing the joined donor flanks was generated (Figure 2B, lane 3). In contrast, double stranded LE (Figure 2B, lane 4) was neither cleaved nor underwent a strand transfer reaction with the unlabelled RE (Figure 2B, lane 5).

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