Structural heterogeneity of attC integron recombination sites revealed by optical tweezers.

Autor:	Mukhortava A; B CUBE - Center for Molecular Bioengineering, TU Dresden, Tatzberg 41, 01307 Dresden, Germany., Pöge M; B CUBE - Center for Molecular Bioengineering, TU Dresden, Tatzberg 41, 01307 Dresden, Germany., Grieb MS; B CUBE - Center for Molecular Bioengineering, TU Dresden, Tatzberg 41, 01307 Dresden, Germany., Nivina A; Institut Pasteur, Unité de Plasticité du Génome Bactérien, Département Génomes et Génétique, 28 Rue du Dr Roux, 75015 Paris, France.; CNRS, UMR3525, 28 Rue du Dr Roux, 75015 Paris, France.; Paris Descartes University, 75006 Paris, France., Loot C; Institut Pasteur, Unité de Plasticité du Génome Bactérien, Département Génomes et Génétique, 28 Rue du Dr Roux, 75015 Paris, France.; CNRS, UMR3525, 28 Rue du Dr Roux, 75015 Paris, France., Mazel D; Institut Pasteur, Unité de Plasticité du Génome Bactérien, Département Génomes et Génétique, 28 Rue du Dr Roux, 75015 Paris, France.; CNRS, UMR3525, 28 Rue du Dr Roux, 75015 Paris, France., Schlierf M; B CUBE - Center for Molecular Bioengineering, TU Dresden, Tatzberg 41, 01307 Dresden, Germany.
Jazyk:	angličtina
Zdroj:	Nucleic acids research [Nucleic Acids Res] 2019 Feb 28; Vol. 47 (4), pp. 1861-1870.
DOI:	10.1093/nar/gky1258
Abstrakt:	A predominant tool for adaptation in Gram-negative bacteria is the functional genetic platform called integron. Integrons capture and rearrange promoterless gene cassettes in a unique recombination process involving the recognition of folded single-stranded DNA hairpins-so-called attC sites-with a strong preference for the attC bottom strand. While structural elements have been identified to promote this preference, their mechanistic action remains incomplete. Here, we used high-resolution single-molecule optical tweezers (OT) to characterize secondary structures formed by the attC bottom (${{att}}{{{C}}_{{\rm{bs}}}}$) and top (${{att}}{{{C}}_{{\rm{ts}}}}$) strands of the paradigmatic attCaadA7 site. We found for both sequences two structures-a straight, canonical hairpin and a kinked hairpin. Remarkably, the recombination-preferred ${{att}}{{{C}}_{{\rm{bs}}}}$ predominantly formed the straight hairpin, while the ${{att}}{{{C}}_{{\rm{ts}}}}$ preferentially adopted the kinked structure, which exposes only one complete recombinase binding box. By a mutational analysis, we identified three bases in the unpaired central spacer, which could invert the preferred conformations and increase the recombination frequency of the ${{att}}{{{C}}_{{\rm{ts}}}}$in vivo. A bioinformatics screen revealed structural bias toward a straight, canonical hairpin conformation in the bottom strand of many antibiotic resistance cassettes attC sites. Thus, we anticipate that structural fine tuning could be a mechanism in many biologically active DNA hairpins. (© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.)
Databáze:	MEDLINE
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