scholarly journals Telomerase RNA localized in the replication band and spherical subnuclear organelles in hypotrichous ciliates.

1995 ◽  
Vol 130 (2) ◽  
pp. 243-253 ◽  
Author(s):  
G Fang ◽  
T R Cech
Keyword(s):  
Dna Replication ◽  
Fluorescent Antibody ◽  
Dna Binding Protein ◽  
S Phase ◽  
Telomerase Rna ◽  
Replication Machinery ◽  
Antibody Staining ◽  
Telomere Protein ◽  
Replication Band

The intranuclear distribution of telomere DNA-binding protein and telomerase RNA in hypotrichous ciliates was revealed by indirect fluorescent antibody staining and in situ hybridization. The Oxytricha telomere protein colocalized with DNA, both being dispersed throughout the macronucleus except for numerous spherical foci that contained neither DNA nor the protein. Surprisingly, the telomerase RNA was concentrated in these foci; therefore, much of telomerase does not colocalize with telomeres. These foci persist through the cell cycle. They may represent sites of assembly, transport or stockpiling of telomerase and other ribonucleoproteins. During S phase, the macronuclear DNA replication machinery is organized into a disc-shaped structure called the replication band. Telomerase RNA is enriched in the replication band as judged by fluorescence intensity. We conclude that the localization of a subfraction of telomerase is coordinated with semiconservative DNA replication.

scholarly journals Mitotic replisome disassembly in vertebrates

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.

scholarly journals Human telomerase RNA and box H/ACA scaRNAs share a common Cajal body–specific localization signal

2004 ◽  
Vol 164 (5) ◽  
pp. 647-652 ◽  
Author(s):  
Beáta E. Jády ◽  
Edouard Bertrand ◽  
Tamás Kiss
Keyword(s):  
S Phase ◽  
Cajal Body ◽  
Sequence Motif ◽  
Telomerase Rna ◽  
Dna Repeats ◽  
Telomeric Dna ◽  
Specific Localization ◽  
Localization Signal ◽  
Human Telomerase

Telomerase is a ribonucleoprotein reverse transcriptase that uses its RNA component as a template for synthesis of telomeric DNA repeats at the ends of linear eukaryotic chromosomes. Here, fluorescence in situ hybridization demonstrates that in HeLa cancer cells, human telomerase RNA (hTR) accumulates in the nucleoplasmic Cajal bodies (CBs). Localization of transiently expressed hTR to CBs is supported by a short sequence motif (411-UGAG-414) that is located in the 3′-terminal box H/ACA RNA-like domain of hTR and that is structurally and functionally indistinguishable from the CB-specific localization signal of box H/ACA small CB-specific RNAs. In synchronized HeLa cells, hTR shows the most efficient accumulation in CBs during S phase, when telomeres are most likely synthesized. CBs may function in post-transcriptional maturation (e.g., cap hypermethylation of hTR), but they may also play a role in the assembly and/or function of telomerase holoenzyme.

scholarly journals Coordinating DNA Replication and Mitosis through Ubiquitin/SUMO and CDK1

2021 ◽  
Vol 22 (16) ◽  
pp. 8796
Author(s):  
Antonio Galarreta ◽  
Pablo Valledor ◽  
Oscar Fernandez-Capetillo ◽  
Emilio Lecona
Keyword(s):  
Dna Replication ◽  
Genomic Stability ◽  
S Phase ◽  
Replication Initiation ◽  
Cancer Therapies ◽  
Replication Machinery ◽  
Post Translational Modification ◽  
Aaa Atpase ◽  
Dna Replication Initiation ◽  
Set Up

Post-translational modification of the DNA replication machinery by ubiquitin and SUMO plays key roles in the faithful duplication of the genetic information. Among other functions, ubiquitination and SUMOylation serve as signals for the extraction of factors from chromatin by the AAA ATPase VCP. In addition to the regulation of DNA replication initiation and elongation, we now know that ubiquitination mediates the disassembly of the replisome after DNA replication termination, a process that is essential to preserve genomic stability. Here, we review the recent evidence showing how active DNA replication restricts replisome ubiquitination to prevent the premature disassembly of the DNA replication machinery. Ubiquitination also mediates the removal of the replisome to allow DNA repair. Further, we discuss the interplay between ubiquitin-mediated replisome disassembly and the activation of CDK1 that is required to set up the transition from the S phase to mitosis. We propose the existence of a ubiquitin–CDK1 relay, where the disassembly of terminated replisomes increases CDK1 activity that, in turn, favors the ubiquitination and disassembly of more replisomes. This model has important implications for the mechanism of action of cancer therapies that induce the untimely activation of CDK1, thereby triggering premature replisome disassembly and DNA damage.

scholarly journals The p97 cofactor Ubxn7 facilitates replisome disassembly during S-phase

2021 ◽  
Author(s):  
Zeynep Tarcan ◽  
Divyasree Poovathumkadavil ◽  
Aggeliki Skagia ◽  
Agnieszka Gambus
Keyword(s):  
Dna Replication ◽  
Molecular Mechanism ◽  
Protein Complexes ◽  
Ubiquitin Ligases ◽  
S Phase ◽  
E3 Ubiquitin Ligases ◽  
Replication Machinery ◽  
Replicative Helicase ◽  
Cellular Processes ◽  
Eukaryotic Dna

Complex cellular processes are driven by the regulated assembly and disassembly of large multi-protein complexes. In eukaryotic DNA replication, whilst we are beginning to understand the molecular mechanism for assembly of the replication machinery (replisome), we still know relatively little about the regulation of its disassembly at replication termination. Over recent years, the first elements of this process have emerged, revealing that the replicative helicase, at the heart of the replisome, is polyubiquitylated prior to unloading and that this unloading requires p97 segregase activity. Two different E3 ubiquitin ligases are now known to ubiquitylate the helicase under different conditions: Cul2Lrr1 and TRAIP. Here we have found two p97 cofactors, Ubxn7 and Faf1, which can interact with p97 during replisome disassembly in S-phase. Only Ubxn7 however facilitates efficient replisome disassembly through its interaction with both Cul2Lrr1 and p97. Our data therefore characterise Ubxn7 as the first substrate-specific p97 cofactor regulating replisome disassembly in vertebrates.

Distinct roles of cdk2 and cdc2 in RP-A phosphorylation during the cell cycle

1993 ◽  
Vol 106 (3) ◽  
pp. 983-994 ◽  
Author(s):  
F. Fang ◽  
J.W. Newport
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Binding ◽  
Dna Binding Protein ◽  
Model System ◽  
S Phase ◽  
Chromosomal Dna ◽  
Cdc2 Kinase ◽  
Single Stranded Dna

RP-A is a single-stranded DNA-binding protein, which has been shown to be required for DNA replication using an SV40 model system. The protein has also been shown to be phosphorylated at the G1-S phase transition. Using Xenopus cell-free extracts we have investigated the role of RP-A in nuclear replication and characterized the kinases and conditions that lead to phosphorylation of RP-A during the cell cycle. By immunodepleting RP-A from Xenopus extracts we have shown that RP-A is essential for replication of chromosomal DNA. Our results show that, during S phase, only that RP-A which is associated with nuclei is phosphorylated. Furthermore our results indicate that during S phase RP-A is only phosphorylated when associated with single-stranded DNA. By immunodepleting cdk2 kinase we show that cdk2 kinase is required for the observed phosphorylation of RP-A in nuclei during S phase. However, using purified cdk2 kinase and RP-A we are unable to detect a direct phosphorylation of RP-A by cdk2 kinase. This observation suggests that phosphorylation of DNA-bound RP-A at S phase is carried out by a kinase distinct from cdk2. Consistent with this we find that when single-stranded DNA is added to S phase extracts depleted of cdk2 kinase, RP-A is phosphorylated. Together these results suggest that cdk2 kinase participates in the activation of DNA replication at a stage prior to the binding of RP-A to the initiation complex. In addition to RP-A phosphorylation in S phase, we have also found that at the onset of mitosis RP-A is quantitatively phosphorylated and that phosphorylation is directly mediated by cdc2 kinase. However, at this time during the cell cycle, cdc2-dependent phosphorylation of RP-A is independent of DNA binding. These observations further demonstrate the distinctions between cdk2 and cdc2 kinases.

scholarly journals Pivotal roles of PCNA loading and unloading on heterochromatin function

2017 ◽  
Author(s):  
Ryan Janke ◽  
Grant King ◽  
Martin Kupiec ◽  
Jasper Rine
Keyword(s):  
Dna Replication ◽  
Transcriptional Silencing ◽  
Structural Features ◽  
S Phase ◽  
Histone Chaperones ◽  
Replication Forks ◽  
Nucleosome Assembly ◽  
Replication Machinery ◽  
Loading And Unloading ◽  
Regulatory Functions

ABSTRACTIn Saccharomyces cerevisiae, heterochromatin structures required for transcriptional silencing of the HML and HMR loci are duplicated in coordination with passing DNA replication forks. Despite major reorganization of chromatin structure, the heterochromatic, transcriptionally-silent states of HML and HMR are successfully maintained throughout S-phase. Mutations of specific components of the replisome diminish the capacity to maintain silencing of HML and HMR through replication. Similarly, mutations in histone chaperones involved in replication-coupled nucleosome assembly reduce gene silencing. Bridging these observations, we determined that the PCNA unloading activity of Elg1 was important for coordinating DNA replication forks with the process of replication-coupled nucleosome assembly to maintain silencing of HML and HMR through S-phase. Collectively these data identified a mechanism by which chromatin reassembly is coordinated with DNA replication to maintain silencing through S-phase.SIGNIFICANCE STATEMENTDNA replication poses a unique logistical challenge for the cell in that structural features of chromatin and their regulatory functions must be carefully coordinated with passage of replication machinery so faithful duplication of both the genome and its chromatin structures may be achieved. Nucleosome assembly is fundamental to reestablishment of chromatin in the wake of DNA replication, and here a mechanism by which nucleosome assembly is coordinated with DNA replication to maintain silenced chromatin is described.

scholarly journals DNA polymerase ϵ links the DNA replication machinery to the S phase checkpoint

Cell ◽  
1995 ◽  
Vol 80 (1) ◽  
pp. 29-39 ◽  
Author(s):  
Tony A Navas ◽  
Zheng Zhou ◽  
Stephen J Elledge
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
S Phase ◽  
Replication Machinery ◽  
S Phase Checkpoint

Detection of S-phase cells in tissue sections by in situ DNA replication

2000 ◽  
Vol 2 (4) ◽  
pp. 244-245 ◽  
Author(s):  
Anthony D. Mills ◽  
Nicholas Coleman ◽  
Lesley S. Morris ◽  
Ronald A. Laskey
Keyword(s):  
Dna Replication ◽  
S Phase ◽  
Tissue Sections

Legionella pneumophilaGrows Adherent to Surfaces in vitro and in situ

1989 ◽  
Vol 10 (9) ◽  
pp. 408-415 ◽  
Author(s):  
J.B. Wright ◽  
I. Ruseska ◽  
M.A. Athar ◽  
S. Corbett ◽  
J.W. Costerton
Keyword(s):  
Legionella Pneumophila ◽  
Distribution Systems ◽  
Water Distribution ◽  
Fluorescent Antibody ◽  
Monitoring Program ◽  
Plate Count ◽  
Air Cooling ◽  
Antibody Staining

AbstractLegionella pneumophilacontinues to play a role in both community- and nosocomially-acquired pneumonia. We investigated the ability ofL pneumophilato adhere to various types of materials such as those found in the hospital air-cooling and potable water distribution systems. Through the use of a unique sampling apparatus, we were able to regularly acquire planktonic and sessile samples and determine the numbers of bacteria present in both populations, in vitro and in situ.Portions of these apparatuses could be aseptically removed for examination by scanning electron microscopy, or for the determination of the number of viable adherentL pneumophila.The number of bacteria present in each sample was determined by direct plate count, with presumptiveL pneumophilacolonies being positively identified by direct fluorescent antibody staining techniques.The results demonstrated that not only are legionellae capable of colonizing various metallic and nonmetallic surfaces but that they are preferentially found on surfaces. Surface-adherent bacteria may play a profound role as a reservoir of these potential pathogens in aquatic environments. Furthermore, these results suggest that any comprehensive legionella monitoring program must include not only water samples but also an examination of the adherent populations.

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