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

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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.

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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    Craggs, T. D., Sustarsic, M., Plochowietz, A., Mosayebi, M., Kaju, H., Cuthbert, A., Hohlbein, J., Domicevica, L., Biggin, P. C., Doye, J. P. K., & 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