Substrate conformational dynamics facilitate 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

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

DNA-binding proteins utilise different recognition mechanisms to locate their DNA targets; some proteins recognise specific DNA sequences, while others interact with specific DNA structures. While sequence-specific DNA binding has been studied extensively, structure-specific recognition mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I Klenow Fragment (Pol) substrates both alone and in DNA-Pol complexes. Using a docking approach based on a network of 73 distances collected using single-molecule FRET, we determined a novel solution structure of the single-nucleotide-gapped DNA-Pol binary complex. The structure resembled existing crystal structures with regards to the downstream primer-template DNA substrate, and revealed a previously unobserved sharp bend (∼120°) in the DNA substrate; this pronounced bend was present in living cells. MD simulations and single-molecule assays also revealed that 4-5 nt of downstream gap-proximal DNA are unwound in the binary complex. Further, experiments and coarse-grained modelling showed the substrate alone frequently adopts bent conformations with 1-2 nt fraying around the gap, suggesting a mechanism wherein Pol recognises a pre-bent, partially-melted conformation of gapped DNA. We propose a general mechanism for substrate recognition by structure-specific enzymes driven by protein sensing of the conformational dynamics of their DNA substrates.

Original languageEnglish
Article numbergkz797
Pages (from-to)10788-10800
Number of pages13
JournalNucleic acids research
Volume47
Issue number20
DOIs
Publication statusPublished - 18 Nov 2019

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DNA-Directed DNA Polymerase
DNA
DNA Polymerase I
Nucleic Acid Conformation
DNA Primers
DNA-Binding Proteins
Proteins
Nucleotides
Enzymes

Cite this

Craggs, T. D., Sustarsic, M., Plochowietz, A., Mosayebi, M., Kaju, H., Cuthbert, A., ... Kapanidis, A. N. (2019). Substrate conformational dynamics facilitate structure-specific recognition of gapped DNA by DNA polymerase. Nucleic acids research, 47(20), 10788-10800. [gkz797]. https://doi.org/10.1093/nar/gkz797
Craggs, Timothy D. ; Sustarsic, Marko ; Plochowietz, Anne ; Mosayebi, Majid ; Kaju, Hendrik ; Cuthbert, Andrew ; Hohlbein, Johannes ; Domicevica, Laura ; Biggin, Philip C. ; Doye, Jonathan P.K. ; Kapanidis, Achillefs N. / Substrate conformational dynamics facilitate structure-specific recognition of gapped DNA by DNA polymerase. In: Nucleic acids research. 2019 ; Vol. 47, No. 20. pp. 10788-10800.
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abstract = "DNA-binding proteins utilise different recognition mechanisms to locate their DNA targets; some proteins recognise specific DNA sequences, while others interact with specific DNA structures. While sequence-specific DNA binding has been studied extensively, structure-specific recognition mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I Klenow Fragment (Pol) substrates both alone and in DNA-Pol complexes. Using a docking approach based on a network of 73 distances collected using single-molecule FRET, we determined a novel solution structure of the single-nucleotide-gapped DNA-Pol binary complex. The structure resembled existing crystal structures with regards to the downstream primer-template DNA substrate, and revealed a previously unobserved sharp bend (∼120°) in the DNA substrate; this pronounced bend was present in living cells. MD simulations and single-molecule assays also revealed that 4-5 nt of downstream gap-proximal DNA are unwound in the binary complex. Further, experiments and coarse-grained modelling showed the substrate alone frequently adopts bent conformations with 1-2 nt fraying around the gap, suggesting a mechanism wherein Pol recognises a pre-bent, partially-melted conformation of gapped DNA. We propose a general mechanism for substrate recognition by structure-specific enzymes driven by protein sensing of the conformational dynamics of their DNA substrates.",
author = "Craggs, {Timothy D.} and Marko Sustarsic and Anne Plochowietz and Majid Mosayebi and Hendrik Kaju and Andrew Cuthbert and Johannes Hohlbein and Laura Domicevica and Biggin, {Philip C.} and Doye, {Jonathan P.K.} and Kapanidis, {Achillefs N.}",
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Craggs, TD, Sustarsic, M, Plochowietz, A, Mosayebi, M, Kaju, H, Cuthbert, A, Hohlbein, J, Domicevica, L, Biggin, PC, Doye, JPK & Kapanidis, AN 2019, 'Substrate conformational dynamics facilitate structure-specific recognition of gapped DNA by DNA polymerase', Nucleic acids research, vol. 47, no. 20, gkz797, pp. 10788-10800. https://doi.org/10.1093/nar/gkz797

Substrate conformational dynamics facilitate structure-specific recognition of gapped DNA by DNA polymerase. / Craggs, Timothy D.; Sustarsic, Marko; Plochowietz, Anne; Mosayebi, Majid; Kaju, Hendrik; Cuthbert, Andrew; Hohlbein, Johannes; Domicevica, Laura; Biggin, Philip C.; Doye, Jonathan P.K.; Kapanidis, Achillefs N.

In: Nucleic acids research, Vol. 47, No. 20, gkz797, 18.11.2019, p. 10788-10800.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Substrate conformational dynamics facilitate structure-specific recognition of gapped DNA by DNA polymerase

AU - Craggs, Timothy D.

AU - Sustarsic, Marko

AU - Plochowietz, Anne

AU - Mosayebi, Majid

AU - Kaju, Hendrik

AU - Cuthbert, Andrew

AU - Hohlbein, Johannes

AU - Domicevica, Laura

AU - Biggin, Philip C.

AU - Doye, Jonathan P.K.

AU - Kapanidis, Achillefs N.

PY - 2019/11/18

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N2 - DNA-binding proteins utilise different recognition mechanisms to locate their DNA targets; some proteins recognise specific DNA sequences, while others interact with specific DNA structures. While sequence-specific DNA binding has been studied extensively, structure-specific recognition mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I Klenow Fragment (Pol) substrates both alone and in DNA-Pol complexes. Using a docking approach based on a network of 73 distances collected using single-molecule FRET, we determined a novel solution structure of the single-nucleotide-gapped DNA-Pol binary complex. The structure resembled existing crystal structures with regards to the downstream primer-template DNA substrate, and revealed a previously unobserved sharp bend (∼120°) in the DNA substrate; this pronounced bend was present in living cells. MD simulations and single-molecule assays also revealed that 4-5 nt of downstream gap-proximal DNA are unwound in the binary complex. Further, experiments and coarse-grained modelling showed the substrate alone frequently adopts bent conformations with 1-2 nt fraying around the gap, suggesting a mechanism wherein Pol recognises a pre-bent, partially-melted conformation of gapped DNA. We propose a general mechanism for substrate recognition by structure-specific enzymes driven by protein sensing of the conformational dynamics of their DNA substrates.

AB - DNA-binding proteins utilise different recognition mechanisms to locate their DNA targets; some proteins recognise specific DNA sequences, while others interact with specific DNA structures. While sequence-specific DNA binding has been studied extensively, structure-specific recognition mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I Klenow Fragment (Pol) substrates both alone and in DNA-Pol complexes. Using a docking approach based on a network of 73 distances collected using single-molecule FRET, we determined a novel solution structure of the single-nucleotide-gapped DNA-Pol binary complex. The structure resembled existing crystal structures with regards to the downstream primer-template DNA substrate, and revealed a previously unobserved sharp bend (∼120°) in the DNA substrate; this pronounced bend was present in living cells. MD simulations and single-molecule assays also revealed that 4-5 nt of downstream gap-proximal DNA are unwound in the binary complex. Further, experiments and coarse-grained modelling showed the substrate alone frequently adopts bent conformations with 1-2 nt fraying around the gap, suggesting a mechanism wherein Pol recognises a pre-bent, partially-melted conformation of gapped DNA. We propose a general mechanism for substrate recognition by structure-specific enzymes driven by protein sensing of the conformational dynamics of their DNA substrates.

U2 - 10.1093/nar/gkz797

DO - 10.1093/nar/gkz797

M3 - Article

VL - 47

SP - 10788

EP - 10800

JO - Nucleic acids research

JF - Nucleic acids research

SN - 0305-1048

IS - 20

M1 - gkz797

ER -

Craggs TD, Sustarsic M, Plochowietz A, Mosayebi M, Kaju H, Cuthbert A et al. Substrate conformational dynamics facilitate structure-specific recognition of gapped DNA by DNA polymerase. Nucleic acids research. 2019 Nov 18;47(20):10788-10800. gkz797. https://doi.org/10.1093/nar/gkz797