Compaction and transport properties of newly replicated Caulobacter crescentus DNA

Abstract

SummaryUpon initiating replication of the Caulobacter chromosome, one copy of the parS centromere remains at the stalked pole; the other moves to the distal pole. We identified the segregation dynamics and compaction characteristics of newly replicated Caulobacter DNA during transport (highly variable from cell to cell) using time-lapse fluorescence microscopy. The parS centromere and a length (also highly variable) of parS proximal DNA on each arm of the chromosome are segregated with the same relatively slow transport pattern as the parS locus. Newly replicated DNA further than about 100 kb from parS segregates with a different and faster pattern, while loci at 48 kb from parS segregate with the slow pattern in some cells and the fast pattern in others. The observed parS-proximal DNA compaction characteristics have scaling properties that suggest the DNA is branched. HU2-deletion strains exhibited a reduced compaction phenotype except near the parS site where only the DHU1DHU2 double mutant had a compaction phenotype. The chromosome shows speed-dependent extension during translocation suggesting the DNA polymer is under tension. While DNA segregation is highly reliable and succeeds in virtually all wild-type cells, the high degree of cell to cell variation in the segregation process is noteworthy.

Caulobacter chromosome in vivo configuration matches model predictions for a supercoiled polymer in a cell-like confinement

“…We reported elsewhere (19) that (i) variable amounts of newly synthesized DNA are transported along with the parS site from the replisome to the distal pole by a mechanism involving a ParA structure extending from that pole, and (ii) newly synthesized DNA remote from the parS site is transported by a different mechanism. These differences in the history of the labeled DNA segments in the strain sets with labeled loci near parS vs. the strain sets with labeled loci near 605 kb might explain differences in the compaction characteristics of the strain sets.…”

“…In our Brownian dynamics simulations, a supercoiled polymer is injected into a cylindrical space with hemispherical caps in a scenario comparable to newly replicated DNA being transported through the template DNA and stored at a cell pole (19) (Fig. 2A).…”

“…2B shows stages in progression of the polymer stowage process (Movie S1). The in vivo DNA configuration corresponding to the start of the simulation results from action of a ParA structure that extends from the distal pole toward the replisome until it connects with ParB proteins bound to DNA in the vicinity of the parS centromere (19)(20)(21)(22) (Fig. 2A).…”

“…2A). The ParA structure is thought to be depolymerized by interaction with ParB (20)(21)(22)(23), resulting in transport of the parS site and a highly variable amount of surrounding DNA to the distal pole where it is anchored to polar PopZ (19,24,25) (Fig. 2A).…”

“…The two newly synthesized arms are transported through a crowded region as they go through the template DNA. This movement takes several minutes and affords ample opportunity for entanglement of the two strands (19). Because the two new strands come from different replisomes that operate asynchronously, there is no reason to expect that loci at similar distances (in kilobases) from the origin will be entangled in phase.…”