Biology subjects

Sherratt, D.

Publications and source records attributed to Sherratt, D..

4 recordsLinked to original sources

bioRxiv2021

Competitive binding of MatP and topoisomerase IV to the MukB dimerization hinge

SMC complexes have ubiquitous roles in chromosome organisation. In Escherichia coli, the interplay between the SMC complex, MukBEF, and matS-bound MatP in the replication termination region, ter, results in depletion of MukBEF from ter, thus promoting chromosome individualisation by directing replichores to separate cell halves. MukBEF also interacts with topoisomerase IV ParC2E2 heterotetramers, to direct its chromosomal distribution to mirror that of MukBEF, thereby facilitating coordination between chromosome organisation and decatenation by topoisomerase IV. Here we demonstrate that the MukB dimerization hinge binds ParC and MatP with the same dimer to dimer stoichiometry. MatP and ParC have an overlapping binding interface on the MukB hinge, leading to their mutually exclusive binding. Furthermore, the MukB hinge fails to stably associate with matS-bound MatP, while MatP mutants deficient in matS binding are impaired in MukB hinge binding, demonstrating that mats competes with the hinge for MatP binding. Cells expressing MukBEF complexes containing a mutation in the MukB hinge interface for ParC/MatP binding are deficient in ParC binding in vivo, despite having a Muk+ topoisomerase IV+ phenotype. This mutant protein is also impaired in MatP binding in vitro, and cells expressing this variant exhibit a MukBEF cellular localisation consistent with impaired MatP binding.

biochemistry↗

bioRxiv2020

Transient non-specific DNA binding dominates the target search of bacterial DNA-binding proteins

Despite their diverse biochemical characteristics and functions, all DNA-binding proteins share the ability to accurately locate their target sites among the vast excess of non-target DNA. Towards identifying universal mechanisms of the target search, we used single-molecule tracking of 11 diverse DNA-binding proteins in living Escherichia coli. The mobility of these proteins during the target search was dictated by DNA interactions, rather than by their molecular weights. By generating cells devoid of all chromosomal DNA, we discovered that the nucleoid does not pose a physical barrier for protein diffusion, but significantly slows the motion of DNA-binding proteins through frequent short-lived DNA interactions. The representative DNA-binding proteins (irrespective of their size, concentration, or function) spend the majority (58-99%) of their search time bound to DNA and occupy as much as [~]30% of the chromosomal DNA at any time. Chromosome-crowding likely has important implications for the function of all DNA-binding proteins.

cell biology↗

bioRxiv2020

Non-random segregation of sister chromosomes by Escherichia coli MukBEF axial cores

The Escherichia coli structural maintenance of chromosomes complex, MukBEF, forms axial cores to chromosomes that determine their spatio-temporal organization. Here, we show that axial cores direct chromosome arms to opposite poles and generate the translational symmetry between newly replicated sister chromosomes. MatP, a replication terminus (ter) binding protein prevents chromosome rotation around the longitudinal cell axis by displacing MukBEF from ter, thereby maintaining the linear shape of axial cores. During DNA replication, MukBEF action directs lagging strands towards the cell center, marked by accumulation of DNA-bound {beta}2-clamps in the wake of replisomes, in a process necessary for the translational symmetry of sister chromosomes. Finally, the ancestral ( immortal) template DNA strand, propagated from previous generations, is preferentially inherited by the cell forming at the old pole, dependent on MukBEF-MatP. The work demonstrates how chromosome organization-segregation can foster non-random inheritance of genetic material and provides a framework for understanding how chromosome conformation and dynamics shape subcellular organization.

microbiology↗

bioRxiv2018

Single-molecule imaging of DNA gyrase activity in living Escherichia coli

Bacterial DNA gyrase introduces negative supercoils into chromosomal DNA and relaxes positive supercoils introduced by replication and transiently by transcription. Removal of these positive supercoils is essential for replication fork progression and for the overall unlinking of the two duplex DNA strands, as well as for ongoing transcription. To address how gyrase copes with these topological challenges, we used high-speed single-molecule fluorescence imaging in live Escherichia coli cells. We demonstrate that at least 300 gyrase molecules are stably bound to the chromosome at any time, with [~]12 enzymes enriched near each replication fork. Trapping of reaction intermediates with ciprofloxacin revealed complexes undergoing catalysis. Dwell times of [~]2 s were observed for the dispersed gyrase molecules, which we propose maintain steady-state levels of negative supercoiling of the chromosome. In contrast, the dwell time of replisome-proximal molecules was [~]8 s, consistent with these catalyzing processive positive supercoil relaxation in front of the progressing replisome.

biophysics↗