How Structure-Specific DNA-Binding Proteins Recognise their Substrates

Abstract

DNA binding proteins utilise different recognition mechanisms to locate their DNA targets. While sequence-specific enzymes recognise a particular nucleotide sequence, structure-specific enzymes have no sequence specificity, and instead interact with particular DNA structures; for example, DNA polymerase I (Pol) binds gapped DNA and Flap endonuclease-1 (FEN1) binds double-flap DNA (both substrates being generated during lagging-strand DNA replication).Bound DNA in FEN1 and DNA Polymerase Beta structures is significantly bent, suggesting a possible role for the conformational dynamics of the DNA in substrate recognition. Here, we have used single-molecule FRET in combination with all-atom and coarse-grained molecular dynamics simulations to study the structures and conformational dynamics of Pol and FEN1 substrates both alone and in DNA-protein complexes.Our FRET-restrained structure of the DNA-Pol binary complex showed a 120° bend in the gapped DNA substrate. All-atom molecular dynamics simulations on the complex revealed 4-5 nt of downstream DNA proximal to the gap to be unwound, which we verified experimentally using a single-molecule FRET quenching assay.Coarse-grained simulations on the gapped substrate alone reproduced experimental FRET values with surprising accuracy ( = −0.0025, across 34 independent distances). The gapped DNA frequently adopted bent conformations as observed in the bound state (free energy 7 kT) or duplex (>> 10 kT) DNA, consistent with weaker Pol binding to these substrates. Bent conformations were also more often frayed around the gap, consistent with a mechanism in which Pol recognises a pre-bent, partially-melted conformation of the gapped-DNA.Double-flap DNA also adopted bent conformations similar to those observed in the FEN1-bound structure. Taken together, these results suggest a general mechanism for substrate recognition by structure-specific DNA-binding proteins, in which specificity is encoded through the conformational dynamics of the DNA substrates.