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Interdomain communication in the endonuclease/motor subunit of type I restriction-modification enzyme EcoR124I

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Abstract

Restriction-modification systems protect bacteria from foreign DNA. Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA-cleavage and ATP-dependent DNA translocation activities located on endonuclease/motor subunit HsdR. The recent structure of the first intact motor subunit of the type I restriction enzyme from plasmid EcoR124I suggested a mechanism by which stalled translocation triggers DNA cleavage via a lysine residue on the endonuclease domain that contacts ATP bound between the two helicase domains. In the present work, molecular dynamics simulations are used to explore this proposal. Molecular dynamics simulations suggest that the Lys–ATP contact alternates with a contact with a nearby loop housing the conserved QxxxY motif that had been implicated in DNA cleavage. This model is tested here using in vivo and in vitro experiments. The results indicate how local interactions are transduced to domain motions within the endonuclease/motor subunit.

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Acknowledgments

We gratefully acknowledge support from the Czech Science Foundation (P207/12/2323 to RE and MW), the institutional research project RVO 61388971, and joint Czech–US National Science Foundation international research cooperation (DBI10-04830).

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Authors and Affiliations

  1. Institute of Nanobiology and Structural Biology, Global Change Research Center, Academy of Sciences of the Czech Republic, Zamek 136, 373 33, Nove Hrady, Czech Republic

    Dhiraj Sinha, Katsiaryna Shamayeva, David Řeha, Vitali Bialevich, Morteza Khabiri, Eva Csefalvay, Jannette Carey & Rüdiger Ettrich

  2. Faculty of Sciences, University of South Bohemia in Ceske Budejovice, Zamek 136, 373 33, Nove Hrady, Czech Republic

    Dhiraj Sinha, Katsiaryna Shamayeva, David Řeha, Vitali Bialevich & Rüdiger Ettrich

  3. Chemistry Department, Princeton University, Princeton, NJ, 08544-1009, USA

    Vyas Ramasubramani, Niv Milbar & Jannette Carey

  4. Laboratory of Molecular Genetics of Bacteria, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20, Praha 4, Czech Republic

    Alena Guzanová & Marie Weiserová

Authors
  1. Dhiraj Sinha
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  2. Katsiaryna Shamayeva
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  12. Rüdiger Ettrich
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Corresponding author

Correspondence to Rüdiger Ettrich.

Additional information

Dhiraj Sinha and Katsiaryna Shamayeva contributed equally.

This paper belongs to Topical Collection MIB 2013 (Modeling Interactions in Biomolecules VI)

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Supplemental Fig. 1a–c

RMSD relative to the starting structure. The root-mean-square deviation (RMSD, Å) of Cα positions is derived every 1 ps during the trajectory by overlaying each simulation structure with its corresponding initial structure by Cα superposition. a WT HsdR. b Restrained WT. c Arg182Ala mutant. (GIF 30 kb)

High-resolution image (TIFF 87 kb)

Supplemental Fig. 2a–c

Root-mean-square fluctuation (RMSF, Å) calculated during the last 20 ns of each simulation for each Cα atom is plotted vs. residue number. a WT HsdR. b Restrained WT. c Arg182Ala mutant. (GIF 45 kb)

High-resolution image (TIFF 132 kb)

Supplemental Fig. 3a–c

Root-mean-square fluctuation (RMSF, Å) calculated during the last 20 ns of each simulation for each Cα atom of the endonuclease domain is plotted vs. residue number. a WT HsdR. b Restrained WT. c Arg182Ala mutant. The secondary structures assigned in the PDB file by DSSP are shown under each panel, with red bars representing helical segments, yellow bars representing strand segments, and gray bars representing irregular segments. (GIF 43 kb)

High-resolution image (TIFF 134 kb)

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Sinha, D., Shamayeva, K., Ramasubramani, V. et al. Interdomain communication in the endonuclease/motor subunit of type I restriction-modification enzyme EcoR124I. J Mol Model 20, 2334 (2014). https://doi.org/10.1007/s00894-014-2334-1

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