title: Single strand transposition at the host replication fork.
abstract: Members of the IS200/IS605 insertion sequence family differ fundamentally from classical IS essentially by their specific single-strand (ss) transposition mechanism, orchestrated by the Y1 transposase, TnpA, a small HuH enzyme which recognizes and processes ss DNA substrates. Transposition occurs by the 'peel and paste' pathway composed of two steps: precise excision of the top strand as a circular ss DNA intermediate; and subsequent integration into a specific ssDNA target. Transposition of family members was experimentally shown or suggested by in silico high-throughput analysis to be intimately coupled to the lagging strand template of the replication fork. In this study, we investigated factors involved in replication fork targeting and analysed DNA-binding properties of the transposase which can assist localization of ss DNA substrates on the replication fork. We showed that TnpA interacts with the β sliding clamp, DnaN and recognizes DNA which mimics replication fork structures. We also showed that dsDNA can facilitate TnpA targeting ssDNA substrates. We analysed the effect of Ssb and RecA proteins on TnpA activity in vitro and showed that while RecA does not show a notable effect, Ssb inhibits integration. Finally we discuss the way(s) in which integration may be directed into ssDNA at the replication fork.
authors.affiliation: Laboratoire de Microbiologie et Génétique Moléculaires, CBI, CNRS, 118 Route de Narbonne, F-31062 Toulouse Cedex, France.
authors.forename: Laure
authors.initials: L
authors.lastname: Lavatine
authors.affiliation: Laboratoire de Microbiologie et Génétique Moléculaires, CBI, CNRS, 118 Route de Narbonne, F-31062 Toulouse Cedex, France.
authors.forename: Susu
authors.initials: S
authors.lastname: He
authors.affiliation: Laboratoire de Microbiologie et Génétique Moléculaires, CBI, CNRS, 118 Route de Narbonne, F-31062 Toulouse Cedex, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Caumont-Sarcos
authors.affiliation: Laboratoire de Microbiologie et Génétique Moléculaires, CBI, CNRS, 118 Route de Narbonne, F-31062 Toulouse Cedex, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Guynet
authors.affiliation: Laboratoire de Microbiologie et Génétique Moléculaires, CBI, CNRS, 118 Route de Narbonne, F-31062 Toulouse Cedex, France.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Marty
authors.affiliation: Laboratoire de Microbiologie et Génétique Moléculaires, CBI, CNRS, 118 Route de Narbonne, F-31062 Toulouse Cedex, France mike@ibcg.biotoul.fr.
authors.forename: Mick
authors.initials: M
authors.lastname: Chandler
authors.affiliation: Laboratoire de Microbiologie et Génétique Moléculaires, CBI, CNRS, 118 Route de Narbonne, F-31062 Toulouse Cedex, France tonhoang@ibcg.biotoul.fr.
authors.forename: Bao
authors.initials: B
authors.lastname: Ton-Hoang
mesh_terms: D002876:Chromosomes, Bacterial / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004261:DNA Replication; D004251:DNA Transposable Elements / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli; D007700:Kinetics; D016254:Mutagenesis, Insertional / Q000235:genetics; D011938:Rec A Recombinases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D004251:DNA Transposable Elements; D004277:DNA, Single-Stranded; C022262:Okazaki fragments; D004247:DNA; D011938:Rec A Recombinases