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A novel structure of a DNA polymerase - gapped DNA complex reveals a role for substrate conformational dynamics in structure-specific DNA recognition

Timothy D. Craggs, Marko Sustarsic, Anne Plochowietz, Majid Mosayebi, Hendrik Kaju, Andrew Cuthbert, Johannes Hohlbein, Laura Domicevica, Phillip C. Biggin, Jonathan P. K. Doye and Achillefs N. Kapanidis


DNA-binding proteins utilise different recognition mechanisms to locate their DNA targets. While some proteins recognise specific nucleotide sequences, others interact with specific DNA structures; DNA polymerase I (Pol), for example, binds gapped DNA during lagging-strand synthesis and DNA repair. Here, we present a novel structure of the 1-nt gapped DNA-Pol binary complex derived from single-molecule FRET experiments and computational simulations. The complex shows that the DNA substrate is bent substantially (by 120o); further, 4-5 basepairs immediately downstream of the gap appear melted. Using in vivo singlemolecule FRET, we confirmed the presence of bent gapped DNA in live bacteria. 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 for structure-specific enzymes driven by conformational dynamics of their DNA substrates.