Subcellular localization of Dna‐initiation proteins of Bacillus subtilis: evidence that chromosome replication begins at either edge of the nucleoids

Abstract

SummaryWe examined the intracellular distribution of Bacillus subtilis Dna-initiation proteins by immunofluorescence microscopy to visualize the initiation complex of replication in vivo. DnaA was distributed throughout the cytoplasm, but both DnaB and DnaI were always detected as foci during the cell-division cycle. Interaction of DnaI with the DnaC helicase by the yeast two-hybrid assay suggests that DnaI acts as a helicase loader. The number of DnaB and DnaI foci within the cell exceeded that of oriC. Although the foci were not always co-localized with oriC, they seemed to be localized near the outer or inner edges of the nucleoids at initiation of replication. When the replication cycle was synchronized in cells using a temperature-sensitive dnaA mutant, duplication of the oriC region was observed predominantly near an edge of the nucleoid. Before initiation occurred, each one of the DnaB and DnaI foci was frequently observed near there. Furthermore, DnaX±GFP (DnaX is a component of DNA polymerase III) foci were detected near either of the edges of the nucleoids at the onset of replication. These results suggest that the replisome is recruited into oriC near either edge of the nucleoids to initiate chromosome replication in B. subtilis.

An expanded view of bacterial DNA replication

“…Thus, our data suggest that the proximity of the polar MCPs with initiation proteins might play a functional role, possibly at a stage of the cell cycle when the origins are also oriented preferentially toward the poles (47). The interactions of replication proteins with massive membrane-associated protein complexes (Pdh and MCPs) might be important in determining some of the striking localization patterns that have been described (3,30,47).…”

“…Furthermore, the DnaB initiator protein (different from the E. coli DnaB helicase) interacts with McpA and YvaQ. DnaB frequently localizes near the cell poles (30). Thus, our data suggest that the proximity of the polar MCPs with initiation proteins might play a functional role, possibly at a stage of the cell cycle when the origins are also oriented preferentially toward the poles (47).…”

“…Recently it was proposed, based on the DnaI-DnaC interaction, that DnaI acts as a helicase loader (30). Interestingly, the PolC-DnaX interaction was shown to be relatively weak in Gram-positive bacteria (25).…”

The extrusion-capture model for chromosome partitioning in bacteria

“…The B. subtilis sister origins, however, are located at or near the cell quarter positions (Sharpe and Errington 1998;Lin 1999), sites that will become mid cell following cell division. Presently, there is conflicting evidence as to whether DNA replication initiates at mid cell (or positions that will become mid cell) or near a cell pole (Imai et al 2000;Lemon and Grossman 2000). However, considering the position of the DNA polymerase (see below), the simplest model is that the origin is duplicated at mid cell.…”

“…During sporulation of a spo0J soj double mutant, multiple foci of both the origin and terminus region are observed, indicating an increase in chromosome number ). More recently, flow cytometry data indicate that overreplication occurs in spo0J mutants during exponential growth (Imai et al 2000). Spo0J might influence the frequency of initiation of DNA replication by bringing the origin proximal 20% of the chromosome together in a nucleoprotein structure that resists reinitiation.…”