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. Induction of transposition in prokaryotes under cell stress conditions is potentially important in creating diversity facilitating adaptation to severe environments. In Deinococcus radiodurans, the most radiation-resistant organism known, despite abundance of resident insertion sequences (IS), transposition of a single IS, ISDra2, was found to be strongly induced by irradiation. We show that both steps involved in transposition, IS excision, and insertion, increase significantly following host cell irradiation and, using PCR analysis of genomic DNA, that exposure to γ-irradiation stimulates massive excision of the single genomic ISDra2 copy as a DNA circle and reclosure of the empty site. 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. Consistent with this, we also demonstrate a requirement for single strand DNA substrates in TnpA-catalysed cleavage and strand transfer in vitro. Since we find no evidence for irradiation-induced expression of the ISDra2 transposase, we infer that transposition is triggered by the increase in its single-strand DNA substrate. The potential impact on genome reassembly and in creating genome host diversity by triggering transposition in this way is discussed.