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Substrate conformational dynamics drive structure-specific recognition of gapped DNA by DNA polymerase

View ORCID ProfileTimothy D. Craggs, Marko Sustarsic, Anne Plochowietz, Majid Mosayebi, Hendrik Kaju, Andrew Cuthbert, Johannes Hohlbein, Laura Domicevica, Philip C. Biggin, Jonathan P. K. Doye, Achillefs N. Kapanidis
doi: https://doi.org/10.1101/263038
Timothy D. Craggs
aBiological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, United Kingdom
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  • ORCID record for Timothy D. Craggs
  • For correspondence: t.craggs@sheffield.ac.uk a.kapanidis1@physics.ox.ac.uk
Marko Sustarsic
aBiological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, United Kingdom
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Anne Plochowietz
aBiological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, United Kingdom
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Majid Mosayebi
bPhysical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, United Kingdom
cSchool of Physics, Institute for Research in Fundamental Sciences (IPM), Tehran 19538-33511, Iran
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Hendrik Kaju
bPhysical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, United Kingdom
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Andrew Cuthbert
aBiological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, United Kingdom
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Johannes Hohlbein
dLaboratory of Biophysics, Wageningen University and Research, Wageningen 6708 WE HA, The Netherlands
eMicrospectroscopy Centre Wageningen, Wageningen University and Research, Wageningen 6708 WE, The Netherlands
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Laura Domicevica
fDepartment of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom
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Philip C. Biggin
fDepartment of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom
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Jonathan P. K. Doye
bPhysical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, United Kingdom
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Achillefs N. Kapanidis
aBiological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, United Kingdom
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  • For correspondence: t.craggs@sheffield.ac.uk a.kapanidis1@physics.ox.ac.uk
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Abstract

DNA-binding proteins utilise different recognition mechanisms to locate their DNA targets. Some proteins recognise specific nucleotide sequences, while many DNA repair proteins interact with specific (often bent) DNA structures. While sequence-specific DNA binding mechanisms have been studied extensively, structure-specific mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I (Pol) substrates both alone and in Pol-DNA complexes. Using a rigid-body docking approach based on a network of 73 distance restraints collected using single-molecule FRET, we determined a novel solution structure of the singlenucleotide-gapped DNA-Pol binary complex. The structure was highly consistent with previous crystal structures with regards to the downstream primer-template DNA substrate; further, our structure showed a previously unobserved sharp bend (~120°) in the DNA substrate; we also showed that this pronounced bending of the substrate is present in living bacteria. All-atom molecular dynamics simulations and single-molecule quenching assays revealed that 4-5 nt of downstream gap-proximal DNA are unwound in the binary complex. Coarsegrained simulations on free gapped substrates reproduced our experimental FRET values with remarkable accuracy (<ΔFRET> = -0.0025 across 34 independent distances) and revealed that the one-nucleotide-gapped DNA frequently adopted highly bent conformations similar to those in the Pol-bound state (ΔG < 4 kT); such conformations were much less accessible to nicked (> 7 kT) or duplex (>> 10 kT) DNA. Our results suggest a mechanism by which Pol and other structure-specific DNA-binding proteins locate their DNA targets through sensing of the conformational dynamics of DNA substrates.

Significance Statement Most genetic processes, including DNA replication, repair and transcription, rely on DNA-binding proteins locating specific sites on DNA; some sites contain a specific sequence, whereas others present a specific structure. While sequence-specific recognition has a clear physical basis, structure-specific recognition mechanisms remain obscure. Here, we use single-molecule FRET and computer simulations to show that the conformational dynamics of an important repair intermediate (1nt-gapped DNA) act as central recognition signals for structure-specific binding by DNA polymerase I (Pol). Our conclusion is strongly supported by a novel solution structure of the Pol-DNA complex wherein the gapped-DNA is significantly bent. Our iterative approach combining precise single-molecule measurements with molecular modelling is general and can elucidate the structure and dynamics for many large biomachines.

Footnotes

  • ↵1 Co-first authors

  • Present addresses: TDC: Department of Chemistry, University of Sheffield, S3 7HF, United Kingdom, MM: School of Mathematics, University of Bristol, University Walk, Bristol BS8 1TW, United Kingdom. AP: Palo Alto Research Center, 3333 Coyote Hill Rd, Palo Alto, CA 94304, U. S. A.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted February 10, 2018.
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Substrate conformational dynamics drive structure-specific recognition of gapped DNA by DNA polymerase
Timothy D. Craggs, Marko Sustarsic, Anne Plochowietz, Majid Mosayebi, Hendrik Kaju, Andrew Cuthbert, Johannes Hohlbein, Laura Domicevica, Philip C. Biggin, Jonathan P. K. Doye, Achillefs N. Kapanidis
bioRxiv 263038; doi: https://doi.org/10.1101/263038
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Substrate conformational dynamics drive structure-specific recognition of gapped DNA by DNA polymerase
Timothy D. Craggs, Marko Sustarsic, Anne Plochowietz, Majid Mosayebi, Hendrik Kaju, Andrew Cuthbert, Johannes Hohlbein, Laura Domicevica, Philip C. Biggin, Jonathan P. K. Doye, Achillefs N. Kapanidis
bioRxiv 263038; doi: https://doi.org/10.1101/263038

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