Biology subjects

Sherratt, D. J.

Publications and source records attributed to Sherratt, D. J..

7 recordsLinked to original sources

bioRxiv2026

RecA structure kinetics determine SOS dynamics and predict spontaneous cell death

Even under optimal growth conditions, bacterial cells experience endogenous stresses that threaten cellular function. Individual cells sporadically activate stress responses to restore homeostasis, but some cells ultimately die. The SOS response to DNA damage is particularly heterogeneous, but the origin of its spontaneous activation and its relationship to cell fate remain unclear. Here, we imaged fluorescently labelled RecA in single Escherichia coli cells under steady-state unperturbed growth using microfluidics. We find that RecA* structures assemble frequently near replication forks and precede SOS response pulses. RecA* structure lifetime quantitatively encodes the magnitude of SOS induction: longer-lived structures promote greater LexA degradation and larger SOS pulses. Thus, cell-to-cell variability in spontaneous SOS activation reflects differences in the repair kinetics of endogenous DNA damage events. By tracking cell lineages over time, we further show that RecA* structures are strong and early predictors of cell fate. Elevated RecA* structure presence emerges up to six generations before spontaneous cell death in over 75% of cases, before other physiological disturbances become detectable. Together, our results establish spontaneous SOS activation as a quantitative readout of endogenous DNA damage processing and identify failure to resolve such damage as a major route to spontaneous bacterial cell death.

biophysics↗

bioRxiv2026

Change in topological linking number during Xer recombination at the plasmid pSC101 psi site

The XerCD recombinase functions with accessory proteins PepA, ArcA and ArgR at plasmid recombination sites such as psi and cer to ensure stable monomeric plasmid inheritance. Xer recombination acts only on directly repeated psi sites, and recombination produces a specific catenane of two product circles interlinked exactly four times. Here we measure the precise change in topological linkage ({Delta}Lk) that occurs during Xer recombination at psi. We use a DNA substrate with close-spaced psi sites that recombines to produce one circle of 398 bp and another of 3039 bp and demonstrate that the small circle is exclusively the -1 topoisomer. Using a purified topoisomer of the substrate, we show that Xer recombination proceeds with a linkage change ({Delta}Lk) of +4. Similar experiments using a substrate with equally spaced psi sites agreed with this result. The measured linkage change is consistent with a reaction mechanism for tyrosine recombinases in which the sites align antiparallel prior to recombination and recombine via a Holliday junction intermediate. Four negative supercoils are converted to catenation nodes by strand exchange, providing an energetic driving force for the reaction. We compare this result to the mechanisms of serine recombinases and the recently discovered bridge RNA-guided recombinases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/706985v2_ufig1.gif" ALT="Figure 1000"> View larger version (29K): org.highwire.dtl.DTLVardef@3ac1c5org.highwire.dtl.DTLVardef@1876da7org.highwire.dtl.DTLVardef@301ff0org.highwire.dtl.DTLVardef@180e054_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Acyl Carrier Protein is essential for MukBEF action in Escherichia coli chromosome organization-segregation

Structural Maintenance of Chromosomes (SMC) complexes contribute ubiquitously to chromosome organization-segregation. SMC proteins have a conserved architecture, with a dimerization hinge and an ATPase head domain separated by a long antiparallel intramolecular coiled-coil. Dimeric SMC proteins interact with essential accessory proteins, kleisins that bridge the two subunits of an SMC dimer, and HAWK/KITE accessory proteins that interact with kleisins. The ATPase activity of the Escherichia coli SMC protein, MukB, is essential for in vivo function and is regulated by interactions with its dimeric kleisin, MukF, and KITE, MukE. Here we demonstrate that, in addition, MukB interacts with Acyl Carrier Protein (AcpP) that has essential functions in fatty acid synthesis. We characterize the AcpP interaction site at the joint of the MukB coiled-coil and show that the interaction is essential for MukB ATPase and for MukBEF function in vivo. Therefore, AcpP is an essential co-factor for MukBEF action in chromosome organization-segregation.

biochemistry↗

bioRxiv2019

The SMC complex, MukBEF, organizes the Escherichia coli chromosome by forming an axial core

Structural Maintenance of Chromosomes (SMC) complexes organize and individualize chromosomes ubiquitously, thereby contributing to their faithful segregation. Here we explore how Escherichia coli chromosome organization emerges from the action of the SMC complex MukBEF, using quantitative imaging in cells with increased MukBEF occupancy on the chromosome. We demonstrate that the E. coli chromosome is organized as series of loops around a thin axial MukBEF core whose length is ~1100 times shorter than the chromosomal DNA. The core is linear (1 m), or circular (1.5 m) in the absence of MatP, which displaces MukBEF from the 800 kbp replication termination region (ter). Our findings illustrate how MukBEF compacts the chromosome lengthwise and demonstrate how displacement of MukBEF from ter promotes MukBEF enrichment with the replication origin.

cell biology↗

bioRxiv2019

Dynamic architecture of the Escherichia coli structural maintenance of chromosomes (SMC) complex, MukBEF

Structural Maintenance of Chromosomes (SMC) complexes use a proteinaceous ring-shaped architecture to organise chromosomes, thereby facilitating chromosome segregation. They utilise cycles of ATP binding and hydrolysis to transport themselves rapidly with respect to DNA, a process requiring protein conformational changes and multiple DNA contacts. We have analysed changes in the architecture of the Escherichia coli SMC complex, MukBEF, as a function of nucleotide binding to MukB and subsequent ATP hydrolysis. This builds upon previous work showing that MukF kleisin directs formation of a MukBEF tripartite ring as a consequence of functional interactions between the C- and N-terminal domains of MukF with the MukB head and neck, respectively (Zawadzka et al., 2018). Using both model truncated substrates and complexes containing full length MukB, we now demonstrate formation of MukBEF dimers of dimers, dependent on MukF dimerization, MukB head-engagement and MukE, which plays an essential role in organizing MukBEF complexes.

biochemistry↗

bioRxiv2017

Pathways of DNA unlinking: A story of stepwise simplification

In Escherichia coli DNA replication yields interlinked chromosomes. Controlling topological changes associated with replication and returning the newly replicated chromosomes to an unlinked monomeric state is essential to cell survival. In the absence of the topoisomerase topoIV, the site-specific recombination complex XerCD-dif-FtsK can remove replication links by local reconnection. We previously showed mathematically that there is a unique minimal pathway of unlinking replication links by reconnection while stepwise reducing the topological complexity. However, the possibility that reconnection preserves or increases topological complexity is biologically plausible. In this case, are there other unlinking pathways? Which is the most probable? We consider these questions in an analytical and numerical study of minimal unlinking pathways. We use a Markov Chain Monte Carlo algorithm with Multiple Markov Chain sampling to model local reconnection on 491 different substrate topologies, 166 knots and 325 links, and distinguish between pathways connecting a total of 881 different topologies. We conclude that the minimal pathway of unlinking replication links that was found under more stringent assumptions is the most probable. We also present exact results on unlinking a 6-crossing replication link. These results point to a general process of topology simplification by local reconnection, with applications going beyond DNA.

bioinformatics↗

bioRxiv2017

Asymmetric MukB ATPases are regulated independently by the N- and C-terminal domains of MukF kleisin

The Escherichia coli SMC complex, MukBEF, acts in chromosome segregation. MukBEF shares the distinctive architecture of other SMC complexes, with one prominent difference; unlike other kleisins, MukF forms dimers through its N-terminal domain. We show that a 4-helix bundle adjacent to the MukF dimerization domain interacts functionally with the MukB coiled-coiled neck adjacent to the ATPase head, forming an asymmetric tripartite complex, as in other SMC complexes. Since MukF dimerization is preserved during this interaction, MukF directs the formation of dimer of dimers MukBEF complexes, observed previously in vivo. The MukF N- and C-terminal domains stimulate ATPase independently and additively, consistent with them each targeting only one of the two MukB ATPase active sites in the asymmetric complex. We demonstrate that MukF interaction with the MukB neck turns over during cycles of ATP binding and hydrolysis in vivo and that impairment of this interaction leads to MukBEF release from chromosomes.

biochemistry↗