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


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.

SUBMITTER: Craggs TD 

PROVIDER: S-EPMC6846080 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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

Craggs Timothy D TD   Sustarsic Marko M   Plochowietz Anne A   Mosayebi Majid M   Kaju Hendrik H   Cuthbert Andrew A   Hohlbein Johannes J   Domicevica Laura L   Biggin Philip C PC   Doye Jonathan P K JPK   Kapanidis Achillefs N AN  

Nucleic acids research 20191101 20


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  ...[more]

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