A Two-Protein Strategy for the Functional Loading of a Cellular Replicative DNA Helicase

Velten, Marion; McGovern, Stephen; Marsin, Stéphanie; Ehrlich, S. Dusko; Naveilhan, Philippe; Polard, Patrice

doi:10.1016/s1097-2765(03)00130-8

Cited by 121 publications

(182 citation statements)

References 33 publications

(4 reference statements)

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“…To analyze vegetatively growing cells with 1-N DNA content, we inhibited replication initiation using a temperature-sensitive mutant of the helicase loader (DnaB) (29,30). Cells were grown in rich medium at the permissive temperature (30°C, τ = 57 min) to early exponential phase and then shifted to the restrictive temperature (42°C, τ = 33 min) ( Fig.…”

Section: Left-ori-right Chromosome Organization In Cells With a Singlementioning

confidence: 99%

Bacillus subtilischromosome organization oscillates between two distinct patterns

Wang

Llopis

Rudner

2014

Proc. Natl. Acad. Sci. U.S.A.

116

146

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Bacterial chromosomes have been found to possess one of two distinct patterns of spatial organization. In the first, called "ori-ter" and exemplified by Caulobacter crescentus, the chromosome arms lie side-by-side, with the replication origin and terminus at opposite cell poles. In the second, observed in slow-growing Escherichia coli ("left-ori-right"), the two chromosome arms reside in separate cell halves, on either side of a centrally located origin. These two patterns, rotated 90°relative to each other, appear to result from different segregation mechanisms. Here, we show that the Bacillus subtilis chromosome alternates between them. For most of the cell cycle, newly replicated origins are maintained at opposite poles with chromosome arms adjacent to each other, in an ori-ter configuration. Shortly after replication initiation, the duplicated origins move as a unit to midcell and the two unreplicated arms resolve into opposite cell halves, generating a left-ori-right pattern. The origins are then actively segregated toward opposite poles, resetting the cycle. Our data suggest that the condensin complex and the parABS partitioning system are the principal driving forces underlying this oscillatory cycle. We propose that the distinct organization patterns observed for bacterial chromosomes reflect a common organization-segregation mechanism, and that simple modifications to it underlie the unique patterns observed in different species.DNA replication | ParA | chromosome segregation | SMC condensin C entral to reproduction is the faithful segregation of replicated chromosomes to daughter cells. In eukaryotes, DNA replication, chromosome condensation, and sister chromatid segregation are separated into distinct steps in the cell cycle that are safeguarded by checkpoint pathways. In bacteria, these processes occur concurrently, posing unique challenges to genome integrity and inheritance (1, 2). In the absence of temporal control, bacteria take advantage of spatial organization to promote faithful and efficient chromosome segregation. The organization of the chromosome dictates where the chromosome is replicated, and the factors that organize and compact the newly replicated DNA play a central role in its segregation (1, 2).Studies in different bacteria have revealed strikingly distinct patterns of chromosome organization that appear to arise from different segregation mechanisms. In Caulobacter crescentus and Vibrio cholerae chromosome I, the origin and terminus are located at opposite cell poles, with the two replication arms between them, in a pattern referred to as "ori-ter" (3-5). After replication initiation, one of the sister origins is held in place and the other is actively translocated to the opposite cell pole, regenerating the oriter organization in both daughter cells (5-11). By contrast, in slowgrowing Escherichia coli, the origin is located in the middle of the nucleoid and the two replication arms reside in opposite cell halves, in a "left-ori-right" pattern (12, 13). Replication initiates at mid...

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Section: Left-ori-right Chromosome Organization In Cells With a Singlementioning

confidence: 99%

Bacillus subtilischromosome organization oscillates between two distinct patterns

Wang

Llopis

Rudner

2014

Proc. Natl. Acad. Sci. U.S.A.

116

146

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“…The helicase loading process was best characterized at the molecular level in two bacterial systems: Escherichia coli (3) and Bacillus subtilis (4). The E. coli replicative homohexameric DNA helicase DnaB associates with its loader, DnaC, forming a 6:6 complex that is recruited at the chromosomal replication origin (oriC) by the initiator protein DnaA (5).…”

Section: From the Istituto DI Biochimica Delle Proteine Consiglio Namentioning

confidence: 99%

A CDC6-like Factor from the Archaea Sulfolobus solfataricus Promotes Binding of the Mini-chromosome Maintenance Complex to DNA

Felice

Esposito²,

Pucci³

et al. 2004

Journal of Biological Chemistry

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The archaeal replication apparatus appears to be a simplified version of the eukaryotic one with fewer polypeptides and simpler protein complexes. Herein, we report evidence that a Cdc6-like factor from the hyperthermophilic crenarchaea Sulfolobus solfataricus stimulates binding of the homohexameric MCM-like complex to bubble-and fork-containing DNA oligonucleotides that mimic early replication intermediates. This function does not require the Cdc6 ATP and DNA binding activities. These findings may provide important clues to understanding how the DNA replication initiation process has evolved in the more complex eukaryotic organisms.Living organisms have evolved different strategies to recruit and load DNA helicases at the replication origins, and accessory factors, referred to as helicase loaders, are required to accomplish this task (1, 2). The helicase loading process was best characterized at the molecular level in two bacterial systems: Escherichia coli (3) and Bacillus subtilis (4). The E. coli replicative homohexameric DNA helicase DnaB associates with its loader, DnaC, forming a 6:6 complex that is recruited at the chromosomal replication origin (oriC) by the initiator protein DnaA (5). DnaB is then released in an active form, and this initiates formation of two replication forks that extend in opposite directions from oriC (6). Because E. coli DnaB forms stable hexamers in solution that are unable to efficiently selfload onto single-stranded DNA, it was proposed that its loading by DnaC is carried out by a ring-breaking mechanism (1). A similar ring-opening/closing mechanism was demonstrated to take place during loading of the E. coli phage T7 gp4 DNA helicase/primase, but in this case no accessory loading factor is required (7). In B. subtilis, the replicative DNA helicase DnaC forms homohexamers in solution in the presence of ATP, but without ATP the hexameric form readily dissociates into monomers. The preformed hexameric DnaC helicase is unable to unwind DNA and instead must be assembled from monomers around DNA by a loading system that consists of two proteins, DnaI and DnaB (4). A ring-forming mechanism was also proposed for the E. coli bacteriophage T4 helicase gp41 (8) and for the simian virus 40 large tumor antigen, but in this latter case a separate helicase loader is not required (1, 9).

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“…This method was relatively inefficient and timeconsuming, especially for the application to high-throughput screening [6,7]. Moreover, rigorous safety measures should be applied to prevent experimenters from radioactive contamination.…”

Section: Introductionmentioning

confidence: 99%

ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method

Yang

Wang

2016

Analytical Biochemistry

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A Two-Protein Strategy for the Functional Loading of a Cellular Replicative DNA Helicase

Cited by 121 publications

References 33 publications

Bacillus subtilischromosome organization oscillates between two distinct patterns

Bacillus subtilischromosome organization oscillates between two distinct patterns

A CDC6-like Factor from the Archaea Sulfolobus solfataricus Promotes Binding of the Mini-chromosome Maintenance Complex to DNA

ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method

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