DNA Replication Initiation Is Blocked by a Distant Chromosome–Membrane Attachment

Abstract

Summary Although it has been recognized for several decades that chromosome structure regulates the capacity of replication origins to initiate, very little is known about how or if cells actively regulate structure to direct initiation [1–3]. We report that a localized inducible protein tether between the chromosome and cell membrane in E. coli cells imparts a rapid and complete block to replication initiation. Tethers, composed of a trans-membrane and transcription repressor fusion protein bound to an array of operator sequences, can be placed up to one megabase from the origin with no loss of penetrance. Tether-induced initiation blocking has no effect on elongation at pre-existing replication forks and does not cause cell or DNA damage. Whole-genome and site-specific fluorescent DNA labeling in tethered cells indicates that global nucleoid structure and chromosome organization are disrupted. Gene expression patterns, assayed by RNA sequencing shows that tethering induces global supercoiling changes, which are likely incompatible with replication initiation. Parallels between tether-induced initiation blocking and rifampicin treatment, and the role of programmed changes in chromosome structure in replication control are discussed.

DNA gyrase activity regulates DnaA‐dependent replication initiation in Bacillus subtilis

“…For example, recent work by Magnan et al . showed that chromosome tethering at sites as far as 1 Megabases away from the origin of replication can alter global DNA topology and inhibit replication initiation (Magnan et al ., ). Furthermore, inhibition of gyrase may affect replication initiation indirectly.…”

Regulation of DNA Replication Initiation by Chromosome Structure

“…Since only a single round of replication is triggered by an increase in temperature, topological changes are likely quickly compensated for by adjustments to expression of gyrase and Topo I ( 78 ), implying that net origin energy status is under homeostatic control (below). Similarly, rapidly increasing osmotic levels (to ∼0.5 M NaCl), which results in an immediate but temporary increase in negative supercoiling ( 79 ), induces replication initiation in dnaA46 mutant cells at a restrictive temperature ( 80 ) and also in cells blocked for replication initiation by a chromosome-membrane tether ( 13 ; see also below).…”

“…Also, replication initiation in Caulobacter crescentus is regulated through cell cycle changes in chromosome structure and position ( 11 ). It is well established that chromosome condensation in early stationary phase of bacterial growth is highly refractive to initiation of replication and transcription ( 12 ), both of which require duplex melting, and there is emerging evidence that initiation in Escherichia coli is sensitive to chromosome structure changes in the cell cycle ( 13 ). In this review, we outline the key determinants of chromosome structure in bacteria and discuss the role of DNA structure in regulating replication initiation.…”

Expression of different ParE toxins results in conserved phenotypes with distinguishable classes of toxicity

“…The finding that nucleic acid is present in multiple discrete foci is consistent with defects during DNA replication caused by DNA gyrase inhibition (Nakanishi et al, ). The blebs of lipidic material noted along the inside of cells could be related to the tethering of chromosomes to the inner membrane (Magnan & Bates, ; Magnan, Joshi, Barker, Visser, & Bates, ) or could arise from effects on the regulation between peptidoglycan biosynthesis and the timing and completion of DNA replication (Harris & Theriot, ). Previous studies with the other ParE toxin from M. tuberculosis noted bleb formations visible on the cell surface by scanning electron microscopy, consistent with the current observations (Gupta et al, ).…”