Substrate conformational dynamics facilitate structure-specific recognition of gapped DNA by DNA polymerase. Issue 20 (23rd September 2019)

• Record Type: Journal Article
• Authors: 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
• Is Part Of: Nucleic acids research. Volume 47:Issue 20(2019)
• Page Start: 10788
• Page End: 10800
• Publication Date: 2019-09-23
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkz797
• Languages: English
• ISSNs: 0305-1048

Abstract

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.