Publications

Measuring DNA mechanics on the genome scale

Mechanical deformations of DNA such as bending are ubiquitous and have been implicated in diverse cellular functions1. However, the lack of high-throughput tools to measure the mechanical properties of DNA has limited our understanding of how DNA mechanics influence chromatin transactions across the genome. Here we develop ‘loop-seq’—a high-throughput assay to measure the propensity for DNA looping—and determine the intrinsic cyclizabilities of 270,806 50-base-pair DNA fragments that span Saccharomyces cerevisiae chromosome V, other genomic regions, and random sequences. We found sequence-encoded regions of unusually low bendability within nucleosome-depleted regions upstream of transcription start sites (TSSs). Low bendability of linker DNA inhibits nucleosome sliding into the linker by the chromatin remodeller INO80, which explains how INO80 can define nucleosome-depleted regions in the absence of other factors2. Chromosome-wide, nucleosomes were characterized by high DNA bendability near dyads and low bendability near linkers. This contrast increases for deeper gene-body nucleosomes but disappears after random substitution of synonymous codons, which suggests that the evolution of codon choice has been influenced by DNA mechanics around gene-body nucleosomes. Furthermore, we show that local DNA mechanics affect transcription through TSS-proximal nucleosomes. Overall, this genome-scale map of DNA mechanics indicates a ‘mechanical code’ with broad functional implications.

Deciphering the mechanical code of the genome and epigenome

Diverse DNA-deforming processes are impacted by the local mechanical and structural properties of DNA, which in turn depend on local sequence and epigenetic modifications. Deciphering this mechanical code (that is, this dependence) has been challenging due to the lack of high-throughput experimental methods. Here we present a comprehensive characterization of the mechanical code. Utilizing high-throughput measurements of DNA bendability via loop-seq, we quantitatively established how the occurrence and spatial distribution of dinucleotides, tetranucleotides and methylated CpG impact DNA bendability. We used our measurements to develop a physical model for the sequence and methylation dependence of DNA bendability. We validated the model by performing loop-seq on mouse genomic sequences around transcription start sites and CTCF-binding sites. We applied our model to test the predictions of all-atom molecular dynamics simulations and to demonstrate that sequence and epigenetic modifications can mechanically encode regulatory information in diverse contexts.

Dynamic coupling between conformations and nucleotide states in DNA gyrase

Gyrase is an essential bacterial molecular motor that supercoils DNA using a conformational cycle in which chiral wrapping of > 100 base pairs confers directionality on topoisomerization. To understand the mechanism of this nucleoprotein machine, global structural transitions must be mapped onto the nucleotide cycle of ATP binding, hydrolysis and product release. Here we investigate coupling mechanisms using single-molecule tracking of DNA rotation and contraction during Escherichia coli gyrase activity under varying nucleotide conditions. We find that ADP must be exchanged for ATP to drive the rate-limiting remodeling transition that generates the chiral wrap. ATP hydrolysis accelerates subsequent duplex strand passage and is required for resetting the enzyme and recapturing transiently released DNA. Our measurements suggest how gyrase coordinates DNA rearrangements with the dynamics of its ATP-driven protein gate, how the motor minimizes futile cycles of ATP hydrolysis and how gyrase may respond to changing cellular energy levels to link gene expression with metabolism.

ATP binding controls distinct structural transitions in Escherichia Coli DNA gyrase in complex with DNA

DNA gyrase is a molecular motor that harnesses the free energy of ATP hydrolysis to introduce negative supercoils into DNA. A critical step in this reaction is the formation of a chiral DNA wrap. Here we observe gyrase structural dynamics using a single-molecule assay in which gyrase drives the processive, stepwise rotation of a nanosphere attached to the side of a stretched DNA molecule. Analysis of rotational pauses and measurements of DNA contraction reveal multiple ATP-modulated structural transitions. DNA wrapping is coordinated with the ATPase cycle and proceeds by way of an unanticipated structural intermediate that dominates the kinetics of supercoiling. Our findings reveal a conformational landscape of loosely coupled transitions funneling the motor toward productive energy transduction, a feature that may be common to the reaction cycles of other DNA and protein remodeling machines.