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Structural basis for broad DNA-specificity in integron recombination

Abstract

Lateral DNA transfer—the movement of genetic traits between bacteria—has a profound impact on genomic evolution and speciation. The efficiency with which bacteria incorporate genetic information reflects their capacity to adapt to changing environmental conditions. Integron integrases are proteins that mediate site-specific DNA recombination between a proximal primary site (attI) and a secondary target site (attC) found within mobile gene cassettes encoding resistance or virulence factors. The lack of sequence conservation among attC sites has led to the hypothesis that a sequence-independent structural recognition determinant must exist within attC. Here we report the crystal structure of an integron integrase bound to an attC substrate. The structure shows that DNA target site recognition and high-order synaptic assembly are not dependent on canonical DNA but on the position of two flipped-out bases that interact in cis and in trans with the integrase. These extrahelical bases, one of which is required for recombination in vivo, originate from folding of the bottom strand of attC owing to its imperfect internal dyad symmetry. The mechanism reported here supports a new paradigm for how sequence-degenerate single-stranded genetic material is recognized and exchanged between bacteria.

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Acknowledgements

This work was supported by laboratory start-up funds from the Pasteur Institute to D.N.G., research grants from the CNRS to D.N.G., research grants from the Institut Pasteur and from the CNRS on transposable elements to D. Mazel, and a French Ministry of Research (MENESR) grant to D. Mazel and D.N.G. D. MacDonald is a Florence Gould Scholar funded by the Pasteur Foundation of New York. G.D. and M.B. are doctoral fellows from the Fondation pour la Recherche Médicale and MENESR, respectively. We also thank T. Ellenberger and G. Liou for critical review of this manuscript. Author Contributions D. MacDonald carried out the in vitro assays, structural determination and wrote the paper with editing from D.N.G. D. Mazel provided the conceptual framework for the substrate and invaluable background. G.D. and M.B. performed the in vivo excision frequency assays. All authors discussed the results and commented on the manuscript.

Author information

Correspondence to Deshmukh N. Gopaul.

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Competing interests

Coordinates and structure factors are deposited in the Protein Data Bank under accession code 2A3V. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

This figure contains the predicted secondary structures of attC bottom strands. (PDF 94 kb)

Supplementary Figure 2

Supplementary Figure 2 nature04643-s02.pdf This figure contains stereo views of the VchIntIA–VCRbs active sites. (PDF 941 kb)

Supplementary Figure 3

This figure contains a stereo model of a VchIntIA attacking subunit and the sequence alignment of VchIntIA and Cre recombinase with their corresponding secondary structure elements. (PDF 259 kb)

Supplementary Figure 4

This figure contains a schematic representation of the protein-DNA contacts between VchIntIA and VCRbs. (PDF 225 kb)

Supplementary Figure 5

This figure contains a model of the proposed movement of the β 4,5 hairpin. (PDF 841 kb)

Supplementary Figure 6

This figure contains data from EMSA and in vivo excision assays. (PDF 124 kb)

Supplementary Figure 7

This figure contains proposed IntI double strand exchange pathway via a single-stranded DNA substrate. (PDF 141 kb)

Supplementary Figure 8

This figure contains the rotation of the C-terminal domain within the non-attacking subunits. (PDF 633 kb)

Supplementary Table 1

Data collection and refinement statistics (PDF 125 kb)

Supplementary Movie 1

This movie shows the final model of the VchIntIA–VCRbs synaptic complex. (WMV 4614 kb)

Supplementary Notes

This file contains Supplementary Methods, Supplementary Figure Legends and additional references. (DOC 56 kb)

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Further reading

Figure 1: Pathways of IntI-mediated cassette excision.
Figure 2: Architecture of the VchIntIA–VCR bs synapse.
Figure 3: Stereo-model of half the VchIntIA–VCR bs synapse.
Figure 4: Cis and trans extrahelical interactions.

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