The replisomes remain spatially proximal throughout the cell cycle in bacteria

The positioning of the DNA replication machinery (replisome) has been the subject of several studies. Two conflicting models for replisome localization have been proposed: In the Factory Model, sister replisomes remain spatially colocalized as the replicating DNA is translocated through a stationary replication factory. In the Track Model, sister replisomes translocate independently along a stationary DNA track and the replisomes are spatially separated for the majority of the cell cycle. Here, we used time-lapse imaging to observe and quantify the position of fluorescently labeled processivity-clamp (DnaN) complexes throughout the cell cycle in two highly-divergent bacterial model organisms: Bacillus subtilis and Escherichia coli. Because DnaN is a core component of the replication machinery, its localization patterns should be an appropriate proxy for replisome positioning in general. We present automated statistical analysis of DnaN positioning in large populations, which is essential due to the high degree of cell-to-cell variation. We find that both bacteria show remarkably similar DnaN positioning, where any potential separation of the two replication forks remains below the diffraction limit throughout the majority of the replication cycle. Additionally, the localization pattern of several other core replisome components is consistent with that of DnaN. These data altogether indicate that the two replication forks remain spatially colocalized and mostly function in close proximity throughout the replication cycle.The conservation of the observed localization patterns in these highly divergent species suggests that the subcellular positioning of the replisome is a functionally critical feature of DNA replication.Author SummaryCell proliferation depends on efficient replication of the genome. Bacteria typically have a single origin of replication on a circular chromosome. After replication initiation, two replisomes assemble at the origin and each copy one of the two arms of the chromosome until they reach the terminus. There have been conflicting reports about the subcellular positioning and putative co-localization of the two replication forks during this process. It has remained controversial whether the two replisomes remain relatively close to each other with the DNA being pulled through, or separate as they translocate along the DNA like a track. Existing studies have relied heavily on snapshot images and these experiments cannot unambiguously distinguish between these two models: i.e. two resolvable forks versus two pairs of co-localized forks. The ability of replication to re-initiate before cell division in bacterial cells further complicates the interpretation of these types of imaging studies. In this paper, we use a combination of snapshot imaging, time-lapse imaging, and quantitative analysis to measure the fraction of time forks are co-localized during each cell cycle. We find that the forks are co-localized for the majority ( 80%) of the replication cycle in two highly-divergent model organisms: B. subtilis and E. coli. Our observations are consistent with proximal localization of the two forks, but also some transient separations of sister forks during replication. The conserved behavior of sub-cellular positioning of the replisomes in these two highly divergent species implies a potential functional relevance of this feature.

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Instructive simulation of the bacterial cell division cycle

Microbiology ◽

10.1099/mic.0.049403-0 ◽

2011 ◽

Vol 157 (7) ◽

pp. 1876-1885 ◽

Cited By ~ 29

Author(s):

Arieh Zaritsky ◽

Ping Wang ◽

Norbert O. E. Vischer

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Dna Replication ◽

Cell Mass ◽

Time Lapse ◽

Replication Initiation ◽

Bacterial Cells ◽

Bacterial Cell Division ◽

Cycle Simulation ◽

New Ideas

The coupling between chromosome replication and cell division includes temporal and spatial elements. In bacteria, these have globally been resolved during the last 40 years, but their full details and action mechanisms are still under intensive study. The physiology of growth and the cell cycle are reviewed in the light of an established dogma that has formed a framework for development of new ideas, as exemplified here, using the Cell Cycle Simulation (CCSim) program. CCSim, described here in detail for the first time, employs four parameters related to time (replication, division and inter-division) and size (cell mass at replication initiation) that together are sufficient to describe bacterial cells under various conditions and states, which can be manipulated environmentally and genetically. Testing the predictions of CCSim by analysis of time-lapse micrographs of Escherichia coli during designed manipulations of the rate of DNA replication identified aspects of both coupling elements. Enhanced frequencies of cell division were observed following an interval of reduced DNA replication rate, consistent with the prediction of a minimum possible distance between successive replisomes (an eclipse). As a corollary, the notion that cell poles are not always inert was confirmed by observed placement of division planes at perpendicular planes in monstrous and cuboidal cells containing multiple, segregating nucleoids.

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Mitotic replisome disassembly in vertebrates

10.1101/418368 ◽

2018 ◽

Author(s):

Sara Priego Moreno ◽

Rebecca M. Jones ◽

Divyasree Poovathumkadavil ◽

Agnieszka Gambus

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Ubiquitin Ligase ◽

S Phase ◽

Replication Forks ◽

Replication Machinery ◽

Replicative Helicase ◽

Egg Extract ◽

Eukaryotic Dna ◽

Higher Eukaryotes

ABSTRACTRecent years have brought a breakthrough in our understanding of the process of eukaryotic DNA replication termination. We have shown that the process of replication machinery (replisome) disassembly at the termination of DNA replication forks in S-phase of the cell cycle is driven through polyubiquitylation of one of the replicative helicase subunits Mcm7. Our previous work in C.elegans embryos suggested also an existence of a back-up pathway of replisome disassembly in mitosis. Here we show, that in Xenopus laevis egg extract, any replisome retained on chromatin after S-phase is indeed removed from chromatin in mitosis. This mitotic disassembly pathway depends on formation of K6 and K63 ubiquitin chains on Mcm7 by TRAIP ubiquitin ligase and activity of p97/VCP protein segregase. The mitotic replisome pathway is therefore conserved through evolution in higher eukaryotes. However, unlike in lower eukaryotes it does not require SUMO modifications. This process can also remove any helicases from chromatin, including “active” stalled ones, indicating a much wider application of this pathway than just a “back-up” for terminated helicases.

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Caulobacterrequires a dedicated mechanism to initiate chromosome segregation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0807448105 ◽

2008 ◽

Vol 105 (40) ◽

pp. 15435-15440 ◽

Cited By ~ 100

Author(s):

Esteban Toro ◽

Sun-Hae Hong ◽

Harley H. McAdams ◽

Lucy Shapiro

Keyword(s):

Chromosome Segregation ◽

Caulobacter Crescentus ◽

Time Lapse ◽

Replication Initiation ◽

Circular Chromosome ◽

Chromosomal Inversions ◽

Initiation Of Replication ◽

Time Lapse Imaging ◽

Single Circular Chromosome

Chromosome segregation in bacteria is rapid and directed, but the mechanisms responsible for this movement are still unclear. We show thatCaulobacter crescentusmakes use of and requires a dedicated mechanism to initiate chromosome segregation.Caulobacterhas a single circular chromosome whose origin of replication is positioned at one cell pole. Upon initiation of replication, an 8-kb region of the chromosome containing both the origin andparSmoves rapidly to the opposite pole. This movement requires the highly conservedParABSlocus that is essential inCaulobacter.We use chromosomal inversions andin vivotime-lapse imaging to show thatparSis theCaulobactersite of force exertion, independent of its position in the chromosome. WhenparSis moved farther from the origin, the cell waits forparSto be replicated before segregation can begin. Also, a mutation in the ATPase domain of ParA halts segregation without affecting replication initiation. Chromosome segregation inCaulobactercannot occur unless a dedicatedparSguiding mechanism initiates movement.

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Regulation of DNA Replication Licensing and Re-Replication by Cdt1

International Journal of Molecular Sciences ◽

10.3390/ijms22105195 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5195

Author(s):

Hui Zhang

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Replication ◽

Genome Stability ◽

E3 Ligase ◽

S Phase ◽

Replication Initiation ◽

Nuclear Antigen ◽

Repair Synthesis ◽

Ubiquitin E3 Ligase

In eukaryotic cells, DNA replication licensing is precisely regulated to ensure that the initiation of genomic DNA replication in S phase occurs once and only once for each mitotic cell division. A key regulatory mechanism by which DNA re-replication is suppressed is the S phase-dependent proteolysis of Cdt1, an essential replication protein for licensing DNA replication origins by loading the Mcm2-7 replication helicase for DNA duplication in S phase. Cdt1 degradation is mediated by CRL4Cdt2 ubiquitin E3 ligase, which further requires Cdt1 binding to proliferating cell nuclear antigen (PCNA) through a PIP box domain in Cdt1 during DNA synthesis. Recent studies found that Cdt2, the specific subunit of CRL4Cdt2 ubiquitin E3 ligase that targets Cdt1 for degradation, also contains an evolutionarily conserved PIP box-like domain that mediates the interaction with PCNA. These findings suggest that the initiation and elongation of DNA replication or DNA damage-induced repair synthesis provide a novel mechanism by which Cdt1 and CRL4Cdt2 are both recruited onto the trimeric PCNA clamp encircling the replicating DNA strands to promote the interaction between Cdt1 and CRL4Cdt2. The proximity of PCNA-bound Cdt1 to CRL4Cdt2 facilitates the destruction of Cdt1 in response to DNA damage or after DNA replication initiation to prevent DNA re-replication in the cell cycle. CRL4Cdt2 ubiquitin E3 ligase may also regulate the degradation of other PIP box-containing proteins, such as CDK inhibitor p21 and histone methylase Set8, to regulate DNA replication licensing, cell cycle progression, DNA repair, and genome stability by directly interacting with PCNA during DNA replication and repair synthesis.

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Live-cell time-lapse imaging and single-cell tracking of in vitro cultured neural stem cells – Tools for analyzing dynamics of cell cycle, migration, and lineage selection

Methods ◽

10.1016/j.ymeth.2017.10.003 ◽

2018 ◽

Vol 133 ◽

pp. 81-90 ◽

Cited By ~ 11

Author(s):

Katja M. Piltti ◽

Brian J. Cummings ◽

Krystal Carta ◽

Ayla Manughian-Peter ◽

Colleen L. Worne ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Neural Stem Cells ◽

Single Cell ◽

Cell Tracking ◽

Time Lapse ◽

Live Cell ◽

Time Lapse Imaging

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Control of DNA Rereplication via Cdc2 Phosphorylation Sites in the Origin Recognition Complex

Molecular and Cellular Biology ◽

10.1128/mcb.21.17.5767-5777.2001 ◽

2001 ◽

Vol 21 (17) ◽

pp. 5767-5777 ◽

Cited By ~ 54

Author(s):

Amit Vas ◽

Winnie Mok ◽

Janet Leatherwood

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Origin Recognition Complex ◽

Safety Net ◽

Replication Initiation ◽

Initiation Factor ◽

Phosphorylation Sites ◽

Cdc2 Kinase ◽

Origin Recognition ◽

Dna Rereplication

ABSTRACT Cdc2 kinase is a master regulator of cell cycle progression in the fission yeast Schizosaccharomyces pombe. Our data indicate that Cdc2 phosphorylates replication factor Orp2, a subunit of the origin recognition complex (ORC). Cdc2 phosphorylation of Orp2 appears to be one of multiple mechanisms by which Cdc2 prevents DNA rereplication in a single cell cycle. Cdc2 phosphorylation of Orp2 is not required for Cdc2 to activate DNA replication initiation. Phosphorylation of Orp2 appears first in S phase and becomes maximal in G2 and M when Cdc2 kinase activity is required to prevent reinitiation of DNA replication. A mutant lacking Cdc2 phosphorylation sites in Orp2 (orp2-T4A) allowed greater rereplication of DNA than congenic orp2 wild-type strains when the limiting replication initiation factor Cdc18 was deregulated. Thus, Cdc2 phosphorylation of Orp2 may be redundant with regulation of Cdc18 for preventing reinitiation of DNA synthesis. Since Cdc2 phosphorylation sites are present in Orp2 (also known as Orc2) from yeasts to metazoans, we propose that cell cycle-regulated phosphorylation of the ORC provides a safety net to prevent DNA rereplication and resulting genetic instability.

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Initiation and termination of DNA replication in human rRNA genes

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.6600-6613.1993 ◽

1993 ◽

Vol 13 (10) ◽

pp. 6600-6613

Author(s):

R D Little ◽

T H Platt ◽

C L Schildkraut

Keyword(s):

Dna Replication ◽

Ribosomal Dna ◽

Regulatory Elements ◽

Transcription Unit ◽

Replication Initiation ◽

Rrna Genes ◽

Rrna Gene ◽

Replication Forks ◽

Nontranscribed Spacer ◽

Dna Repeat

We have used the multicopy human rRNA genes as a model system to study replication initiation and termination in mammalian chromosomes. Enrichment for replicating molecules was achieved by isolating S-phase enriched populations of cells by centrifugal elutriation, purification of DNA associated with the nuclear matrix, and a chromatographic procedure that enriches for molecules containing single-stranded regions, a characteristic of replication forks. Two-dimensional agarose gel electrophoresis techniques were used to demonstrate that replication appears to initiate at multiple sites throughout most of the 31-kb nontranscribed spacer (NTS) of human ribosomal DNA but not within the 13-kb transcription unit or adjacent regulatory elements. Although initiation events were detected throughout the majority of the NTS, some regions may initiate more frequently than others. Termination of replication, the convergence of opposing replication forks, was found throughout the ribosomal DNA repeat units, and, in some repeats, specifically at the junction of the 3' end of the transcription unit and the NTS. This site-specific termination of replication is the result of pausing of replication forks near the sites of transcription termination. The naturally occurring multicopy rRNA gene family offers a unique system to study mammalian DNA replication without the use of chemical synchronization agents.

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