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.

Multi-modal Single-molecule Imaging with Continuously Controlled Spectral-resolution (CoCoS) Microscopy

“…29,30 ). While these applications require the measurement of relative FRET efficiencies, methods for absolute distance measurement have now been established 15,31 and used to determine structural models of dynamic biomolecular complexes [32][33][34][35] .…”

Single-molecule view of coordination in a multi-functional DNA polymerase

“…The smFRET method has been previously applied to Klenow fragment (KF), which is derived from Pol I by removal of the 5' nuc domain. These studies have monitored DNA synthesis by KF (Christian et al, 2009), detected nucleotide-induced conformational transitions within KF (Santoso et al, 2010;Berezhna et al, 2012;Hohlbein et al, 2013), monitored a DNA primer switching between the pol and exo sites of KF (Lamichhane et al, 2013) and established a structural model of a DNA substrate bound to KF (Craggs et al, 2019). However, smFRET has not yet been applied to full length Pol I, or any other DNA polymerase containing a 5' nuc activity.…”

The smfBox: an open-source platform for single-molecule FRET

“…opening and closing) in unimolecular processes. At the single-molecule level FRET is sensitive to heterogeneous subpopulations, can measure kinetics of processes at equilibrium 4 , and as demonstrated by a recent interlaboratory benchmarking study 5 , absolute FRET efficiencies can be used to infer precise distances for biomolecular structure determination [6][7][8][9][10][11] . Despite these advantages, smFRET is currently rarely used outside specialist labs, largely due to the high costs of commercial instruments and lack of self-build, easy to use alternatives.…”