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Paper   IPM / Physic / 16251
School of Physics
  Title:   Substrate conformational dynamics facilitate structure-specific recognition of gapped DNA by DNA polymerase
1.  T. D Craggs
2.  M. Sustarsic
3.  A. Plochowietz
4.  M. Mosayebi
5.  H. Kaju
6.  A. Cuthbert
7.  J. Hohlbein
8.  L. Domicevica
9.  Ph. C Biggin
10.  J. P K Doye
11.  A. N Kapanidis
  Status:   Published
  Journal: Nucleic Acids Research
  Vol.:  47
  Year:  2019
  Pages:   10788-10800
  Supported by:  IPM
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.

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