Reconstitution of a functional IS608 single-strand transpososome: role of non-canonical base pairing

Single-stranded (ss) transposition, a recently identified mechanism adopted by members of the widespread IS200/IS605 family of insertion sequences (IS), is catalysed by the transposase, TnpA. The transposase of IS608, recognizes subterminal imperfect palindromes (IP) at both IS ends and cleaves at sites located at some distance. The cleavage sites, C, are not recognized directly by the protein but by short sequences 5′ to the foot of each IP, guide (G) sequences, using a network of canonical (‘Watson–Crick’) base interactions. In addition a set of non-canonical base interactions similar to those found in RNA structures are also involved. We have reconstituted a biologically relevant complex, the transpososome, including both left and right ends and TnpA, which catalyses excision of a ss DNA circle intermediate. We provide a detailed picture of the way in which the IS608 transpososome is assembled and demonstrate that both C and G sequences are essential for forming a robust transpososome detectable by EMSA. We also address several questions central to the organization and function of the ss transpososome and demonstrate the essential role of non-canonical base interactions in the IS608 ends for its stability by using point mutations which destroy individual non-canonical base interactions

Structuring the bacterial genome: Y1-transposases associated with REP-BIME sequences†

REPs are highly repeated intergenic palindromic sequences often clustered into structures called BIMEs including two individual REPs separated by short linker of variable length. They play a variety of key roles in the cell. REPs also resemble the sub-terminal hairpins of the atypical IS200/605 family of insertion sequences which encode Y1 transposases (TnpAIS200/IS605). These belong to the HUH endonuclease family, carry a single catalytic tyrosine (Y) and promote single strand transposition. Recently, a new clade of Y1 transposases (TnpAREP) was found associated with REP/BIME in structures called REPtrons. It has been suggested that TnpAREP is responsible for REP/BIME proliferation over genomes. We analysed and compared REP distribution and REPtron structure in numerous available E. coli and Shigella strains. Phylogenetic analysis clearly indicated that tnpAREP was acquired early in the species radiation and was lost later in some strains. To understand REP/BIME behaviour within the host genome, we also studied E. coli K12 TnpAREP activity in vitro and demonstrated that it catalyses cleavage and recombination of BIMEs. While TnpAREP shared the same general organization and similar catalytic characteristics with TnpAIS200/IS605 transposases, it exhibited distinct properties potentially important in the creation of BIME variability and in their amplification. TnpAREP may therefore be one of the first examples of transposase domestication in prokaryotes

Irradiation-Induced Deinococcus radiodurans Genome Fragmentation Triggers Transposition of a Single Resident Insertion Sequence

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

Spatiotemporal evolution of SARS-CoV-2 Alpha and Delta variants during large nationwide outbreak of COVID-19, Vietnam, 2021

We analyzed 1,303 SARS-CoV-2 whole-genome sequences from Vietnam, and found the Alpha and Delta variants were responsible for a large nationwide outbreak of COVID-19 in 2021. The Delta variant was confined to the AY.57 lineage and caused >1.7 million infections and >32,000 deaths. Viral transmission was strongly affected by nonpharmaceutical interventions

Safety and efficacy of fluoxetine on functional outcome after acute stroke (AFFINITY): a randomised, double-blind, placebo-controlled trial

Background Trials of fluoxetine for recovery after stroke report conflicting results. The Assessment oF FluoxetINe In sTroke recoverY (AFFINITY) trial aimed to show if daily oral fluoxetine for 6 months after stroke improves functional outcome in an ethnically diverse population. Methods AFFINITY was a randomised, parallel-group, double-blind, placebo-controlled trial done in 43 hospital stroke units in Australia (n=29), New Zealand (four), and Vietnam (ten). Eligible patients were adults (aged ≥18 years) with a clinical diagnosis of acute stroke in the previous 2–15 days, brain imaging consistent with ischaemic or haemorrhagic stroke, and a persisting neurological deficit that produced a modified Rankin Scale (mRS) score of 1 or more. Patients were randomly assigned 1:1 via a web-based system using a minimisation algorithm to once daily, oral fluoxetine 20 mg capsules or matching placebo for 6 months. Patients, carers, investigators, and outcome assessors were masked to the treatment allocation. The primary outcome was functional status, measured by the mRS, at 6 months. The primary analysis was an ordinal logistic regression of the mRS at 6 months, adjusted for minimisation variables. Primary and safety analyses were done according to the patient's treatment allocation. The trial is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12611000774921. Findings Between Jan 11, 2013, and June 30, 2019, 1280 patients were recruited in Australia (n=532), New Zealand (n=42), and Vietnam (n=706), of whom 642 were randomly assigned to fluoxetine and 638 were randomly assigned to placebo. Mean duration of trial treatment was 167 days (SD 48·1). At 6 months, mRS data were available in 624 (97%) patients in the fluoxetine group and 632 (99%) in the placebo group. The distribution of mRS categories was similar in the fluoxetine and placebo groups (adjusted common odds ratio 0·94, 95% CI 0·76–1·15; p=0·53). Compared with patients in the placebo group, patients in the fluoxetine group had more falls (20 [3%] vs seven [1%]; p=0·018), bone fractures (19 [3%] vs six [1%]; p=0·014), and epileptic seizures (ten [2%] vs two [<1%]; p=0·038) at 6 months. Interpretation Oral fluoxetine 20 mg daily for 6 months after acute stroke did not improve functional outcome and increased the risk of falls, bone fractures, and epileptic seizures. These results do not support the use of fluoxetine to improve functional outcome after stroke

Transposition simple brin de la séquence d'insertion bactérienne IS608 (étude du ciblage de la transposase TnpA sur les fourches de réplication arrêtées)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Etude du mécanisme de transposition de la séquence d'insertion bactérienne IS608

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

First Biochemical Steps on Bacterial Transposition Pathways

International audienc

Breaking and joining single-stranded DNA: the HUH endonuclease superfamily

HUH endonucleases are numerous and widespread in all three domains of life. The major function of these enzymes is processing a range of mobile genetic elements by catalysing cleavage and rejoining of single-stranded DNA using an active-site Tyr residue to make a transient 5′-phosphotyrosine bond with the DNA substrate. These enzymes have a key role in rolling-circle replication of plasmids and bacteriophages, in plasmid transfer, in the replication of several eukaryotic viruses and in various types of transposition. They have also been appropriated for cellular processes such as intron homing and the processing of bacterial repeated extragenic palindromes. Here, we provide an overview of these fascinating enzymes and their functions, using well-characterized examples of Rep proteins, relaxases and transposases, and we explore the molecular mechanisms used in their diverse activities. © 2013 Macmillan Publishers Limited. All rights reserved.This work was supported by intramural funding from the French Centre National de la Recherche Scientifique (to M.C. and B.T.H.) and the Intramural Program of the US National Institute of Diabetes and Digestive and Kidney Diseases (to A.B.H. and F.D.), and by grants from the French Agence National de Recherche (ANR-12-BSV8-0009-01; to B.T.H.), the Spanish Ministry of Science and Innovation (BIO2010-14809; to G.M.), the Spanish Ministry of Education (BFU2011-26608; to F.d.l.C.) and the European Seventh Framework Program (248919/FP7-ICT-2009-4 and 282004/FP7-HEALTH.2011.2.3.1-2; to F.d.l.C.).Peer Reviewe