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

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.

Figure 1. 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 Cam^R 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 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 Tet^R 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 Tet^R colonies while insertion into the reporter sacB gene confers resistance to sucrose. See Figure S1 and its legend.

Figure 2. 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.

Figure 3. Physical evidence for irradiation-induced ISDra2 excision.

Strain GY13120 (tetAΩISDra2-113 expressing tnpA in trans) received 5 kGy of γ–irradiation and aliquots were taken to isolate genomic DNA used to prepare DNA agarose plugs and as template for PCR analysis. The time following irradiation is shown in hours above each lane. (A) Schematic representation of IS excision products. The excision products are shown together with the position of primers used in PCR analysis. Note that the transposon circle including the transposon junction is single stranded. (B) Kinetics of double-strand-break repair. DNA agarose plugs were digested with NotI prior to PFGE analyses and loaded onto a 1.06% agarose gel. L: λ Ladder. (C) Kinetics of donor joint appearance. PCR reactions were performed with primer pair P1 and P2 and loaded onto a 1% agarose gel; L: MassRuler DNA Ladder. (D) Kinetics of IS circle junction appearance. PCR reactions were performed with primer pair P3 and P4 and loaded onto a 2% agarose gel; L: O'GeneRuler 100 bp DNA Ladder.