Nuclear distribution of PCNA during embryonic development in Xenopus laevis: a reinvestigation of early cell cycles

1992 ◽  
Vol 102 (1) ◽  
pp. 63-69 ◽  
Author(s):  
M. Leibovici ◽  
G. Monod ◽  
J. Geraudie ◽  
R. Bravo ◽  
M. Mechali
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Xenopus Laevis ◽  
Early Development ◽  
Nuclear Periphery ◽  
S Phase ◽  
Nuclear Antigen ◽  
Midblastula Transition ◽  
Cell Cycles ◽  
Cyclical Pattern

The immunocytological distribution of the proliferating cell nuclear antigen (PCNA), a protein involved in DNA replication, has been examined during the early development of Xenopus laevis. The protein is uniformly detected in nuclei during early stages up to the neurula stage. PCNA is detected by its distinctive cyclical pattern during early development, remaining detectable only during the period of S phase of each cell cycle. Immunological detection of PCNA is therefore a useful and specific non-isotopic marker of S-phase cells in the embryo. PCNA associates with typical karyomeric structures, suggesting that DNA replication starts before the nuclear compartment is entirely formed. At the midblastula transition, a new pattern of PCNA staining becomes apparent. First, a new type of PCNA staining is detected at the nuclear periphery. Second, mitotic clusters with different PCNA distributions suggest that the onset of desynchronization of the cell cycle at this stage is not random.

scholarly journals Regulation of DNA Replication Licensing and Re-Replication by Cdt1

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.

scholarly journals The distribution pattern of proliferating cell nuclear antigen in the nuclei of Leishmania donovani

Microbiology ◽  
2009 ◽  
Vol 155 (11) ◽  
pp. 3748-3757 ◽  
Author(s):  
Devanand Kumar ◽  
Neha Minocha ◽  
Kalpana Rajanala ◽  
Swati Saha
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Proliferating Cell Nuclear Antigen ◽  
Cell Cycle Progression ◽  
Leishmania Donovani ◽  
Systemic Disease ◽  
S Phase ◽  
Nuclear Antigen ◽  
M Phase ◽  
Proliferating Cell

DNA replication in eukaryotes is a highly conserved process marked by the licensing of multiple origins, with pre-replication complex assembly in G1 phase, followed by the onset of replication at these origins in S phase. The two strands replicate by different mechanisms, and DNA synthesis is brought about by the activity of the replicative DNA polymerases Pol δ and Pol ϵ. Proliferating cell nuclear antigen (PCNA) augments the processivity of these polymerases by serving as a DNA sliding clamp protein. This study reports the cloning of PCNA from the protozoan Leishmania donovani, which is the causative agent of the systemic disease visceral leishmaniasis. PCNA was demonstrated to be robustly expressed in actively proliferating L. donovani promastigotes. We found that the protein was present primarily in the nucleus throughout the cell cycle, and it was found in both proliferating procyclic and metacyclic promastigotes. However, levels of expression of PCNA varied through cell cycle progression, with maximum expression evident in G1 and S phases. The subnuclear pattern of expression of PCNA differed in different stages of the cell cycle; it formed distinct subnuclear foci in S phase, while it was distributed in a more diffuse pattern in G2/M phase and post-mitotic phase cells. These subnuclear foci are the sites of active DNA replication, suggesting that replication factories exist in Leishmania, as they do in higher eukaryotes, thus opening avenues for investigating other Leishmania proteins that are involved in DNA replication as part of these replication factories.

scholarly journals Titration of Four Replication Factors Is Essential for the Xenopus laevis Midblastula Transition

Science ◽  
2013 ◽  
Vol 341 (6148) ◽  
pp. 893-896 ◽  
Author(s):  
Clara Collart ◽  
George E. Allen ◽  
Charles R. Bradshaw ◽  
James C. Smith ◽  
Philip Zegerman
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Xenopus Laevis ◽  
Replication Initiation ◽  
Midblastula Transition ◽  
Multiple Events ◽  
Zygotic Transcription ◽  
Replication Factors

The rapid, reductive early divisions of many metazoan embryos are followed by the midblastula transition (MBT), during which the cell cycle elongates and zygotic transcription begins. It has been proposed that the increasing nuclear to cytoplasmic (N/C) ratio is critical for controlling the events of the MBT. We show that four DNA replication factors—Cut5, RecQ4, Treslin, and Drf1—are limiting for replication initiation at increasing N/C ratios in vitro and in vivo in Xenopus laevis. The levels of these factors regulate multiple events of the MBT, including the slowing of the cell cycle, the onset of zygotic transcription, and the developmental activation of the kinase Chk1. This work provides a mechanism for how the N/C ratio controls the MBT and shows that the regulation of replication initiation is fundamental for normal embryogenesis.

scholarly journals Dynamic properties of nuclear lamins: lamin B is associated with sites of DNA replication.

1994 ◽  
Vol 125 (6) ◽  
pp. 1201-1212 ◽  
Author(s):  
R D Moir ◽  
M Montag-Lowy ◽  
R D Goldman
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dynamic Properties ◽  
Nuclear Lamina ◽  
S Phase ◽  
Nuclear Antigen ◽  
Nuclear Staining ◽  
Lamin B ◽  
Nuclear Lamins ◽  
Proliferating Cell

The nuclear lamins form a fibrous structure, the nuclear lamina, at the periphery of the nucleus. Recent results suggest that lamins are also present as foci or spots in the nucleoplasm at various times during interphase of the cell cycle (Goldman, A. E., R. D. Moir, M. Montag-Lowy, M. Stewart, and R. D. Goldman. 1992. J. Cell Biol. 104:725-732; Bridger, J. M., I. R. Kill, M. O'Farrell, and C. J. Hutchison. 1993. J. Cell Sci. 104:297-306). In this report we demonstrate that during mid-late S-phase, nuclear foci detected with lamin B antibodies are coincident with sites of DNA replication as detected by the colocalization of sites of incorporation of bromodeoxyuridine (BrDU) or proliferating cell nuclear antigen (PCNA). The relationship between lamin B and BrDU is not maintained in the following G1 stage of the cell cycle. Furthermore, the nuclear staining patterns seen with antibodies directed against lamins A and C in mid-late S-phase do not coalign with the lamin B/BrDU-containing structures. These results imply that there is a role for lamin B in the organization of replicating chromatin during S phase.

scholarly journals Dissection of the XChk1 Signaling Pathway in Xenopus laevis Embryos

2000 ◽  
Vol 11 (9) ◽  
pp. 3101-3108 ◽  
Author(s):  
Nicholas C. Kappas ◽  
Pamela Savage ◽  
Katherine C. Chen ◽  
Allan T. Walls ◽  
Jill C. Sible
Keyword(s):  
Cell Cycle ◽  
Xenopus Laevis ◽  
Signaling Pathway ◽  
Embryonic Cell ◽  
Midblastula Transition ◽  
Cell Cycles ◽  
Cyclin Dependent Kinases ◽  
Xenopus Embryos ◽  
Egg Extracts ◽  
Sperm Nuclei

Checkpoint pathways inhibit cyclin-dependent kinases (Cdks) to arrest cell cycles when DNA is damaged or unreplicated. Early embryonic cell cycles of Xenopus laevis lack these checkpoints. Completion of 12 divisions marks the midblastula transition (MBT), when the cell cycle lengthens, acquiring gap phases and checkpoints of a somatic cell cycle. Although Xenopus embryos lack checkpoints prior to the MBT, checkpoints are observed in cell-free egg extracts supplemented with sperm nuclei. These checkpoints depend upon the Xenopus Chk1 (XChk1)-signaling pathway. To understand why Xenopus embryos lack checkpoints,xchk1 was cloned, and its expression was examined and manipulated in Xenopus embryos. Although XChk1 mRNA is degraded at the MBT, XChk1 protein persists throughout development, including pre-MBT cell cycles that lack checkpoints. However, when DNA replication is blocked, XChk1 is activated only after stage 7, two cell cycles prior to the MBT. Likewise, DNA damage activates XChk1 only after the MBT. Furthermore, overexpression of XChk1 inXenopus embryos creates a checkpoint in which cell division arrests, and both Cdc2 and Cdk2 are phosphorylated on tyrosine 15 and inhibited in catalytic activity. These data indicate that XChk1 signaling is intact but blocked upstream of XChk1 until the MBT.

scholarly journals Different cyclin types collaborate to reverse the S-phase checkpoint and permit prompt mitosis

2012 ◽  
Vol 198 (6) ◽  
pp. 973-980 ◽  
Author(s):  
Kai Yuan ◽  
Jeffrey A. Farrell ◽  
Patrick H. O’Farrell
Keyword(s):  
Cell Cycle ◽  
Embryonic Cell ◽  
S Phase ◽  
Midblastula Transition ◽  
Cell Cycles ◽  
Mitotic Entry ◽  
G2 Phase ◽  
Embryonic Morphogenesis ◽  
Do So ◽  
S Phase Checkpoint

Precise timing coordinates cell proliferation with embryonic morphogenesis. As Drosophila melanogaster embryos approach cell cycle 14 and the midblastula transition, rapid embryonic cell cycles slow because S phase lengthens, which delays mitosis via the S-phase checkpoint. We probed the contributions of each of the three mitotic cyclins to this timing of interphase duration. Each pairwise RNA interference knockdown of two cyclins lengthened interphase 13 by introducing a G2 phase of a distinct duration. In contrast, pairwise cyclin knockdowns failed to introduce a G2 in embryos that lacked an S-phase checkpoint. Thus, the single remaining cyclin is sufficient to induce early mitotic entry, but reversal of the S-phase checkpoint is compromised by pairwise cyclin knockdown. Manipulating cyclin levels revealed that the diversity of cyclin types rather than cyclin level influenced checkpoint reversal. We conclude that different cyclin types have distinct abilities to reverse the checkpoint but that they collaborate to do so rapidly.

scholarly journals Transcription-dependent induction of G1 phase during the zebra fish midblastula transition.

1997 ◽  
Vol 17 (2) ◽  
pp. 529-536 ◽  
Author(s):  
E Zamir ◽  
Z Kam ◽  
A Yarden
Keyword(s):  
Cell Cycle ◽  
Early Development ◽  
Actinomycin D ◽  
Flow Cytometric Analysis ◽  
G1 Phase ◽  
Fish Cell ◽  
Zebra Fish ◽  
Midblastula Transition ◽  
Cell Cycles ◽  
Zygotic Transcription

The early development of the zebra fish (Danio rerio) embryo is characterized by a series of rapid and synchronous cell cycles with no detectable transcription. This period is followed by the midblastula transition (MBT), during which the cell cycle gradually lengthens, cell synchrony is lost, and zygotic transcription is initially detected. In this work, we examined the changes in the pattern of the cell cycle during MBT in zebra fish and whether these changes are dependent on the initiation of zygotic transcription. To characterize the pattern of the early zebra fish cell cycles, the embryonic DNA content was determined by flow cytometric analysis. We found that G1 phase is below detection levels during the first 10 cleavages and can be initially detected at the onset of MBT. Inhibition of zygotic transcription, by microinjection of actinomycin D, abolished the appearance of G1 phase at MBT. Premature activation of zygotic transcription, by microinjection of nonspecific DNA, induced G1 phase before the onset of MBT, while coinjection of actinomycin D and nonspecific DNA abolished this early appearance of G1 phase. We therefore suggest that during the early development of the zebra fish embryo, G1 phase appears at the onset of MBT and that the activation of transcription at MBT is essential and sufficient for the G1-phase induction.

scholarly journals CDK-Independent and PCNA-Dependent Functions of p21 in DNA Replication

Genes ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. 593 ◽  
Author(s):  
Sabrina Florencia Mansilla ◽  
María Belén De La Vega ◽  
Nicolás Luis Calzetta ◽  
Sebastián Omar Siri ◽  
Vanesa Gottifredi
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
S Phase ◽  
Nuclear Antigen ◽  
Γ Irradiation ◽  
Cancer Treatments ◽  
Origin Firing ◽  
Cycle Arrest ◽  
Independent Functions ◽  
Unstructured Protein

p21Waf/CIP1 is a small unstructured protein that binds and inactivates cyclin-dependent kinases (CDKs). To this end, p21 levels increase following the activation of the p53 tumor suppressor. CDK inhibition by p21 triggers cell-cycle arrest in the G1 and G2 phases of the cell cycle. In the absence of exogenous insults causing replication stress, only residual p21 levels are prevalent that are insufficient to inhibit CDKs. However, research from different laboratories has demonstrated that these residual p21 levels in the S phase control DNA replication speed and origin firing to preserve genomic stability. Such an S-phase function of p21 depends fully on its ability to displace partners from chromatin-bound proliferating cell nuclear antigen (PCNA). Vice versa, PCNA also regulates p21 by preventing its upregulation in the S phase, even in the context of robust p21 induction by γ irradiation. Such a tight regulation of p21 in the S phase unveils the potential that CDK-independent functions of p21 may have for the improvement of cancer treatments.

scholarly journals The Effect of Dia2 Protein Deficiency on the Cell Cycle, Cell Size, and Recruitment of Ctf4 Protein in Saccharomyces cerevisiae

Molecules ◽  
2021 ◽  
Vol 27 (1) ◽  
pp. 97
Author(s):  
Aneliya Ivanova ◽  
Aleksandar Atemin ◽  
Sonya Uzunova ◽  
Georgi Danovski ◽  
Radoslav Aleksandrov ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Replication ◽  
Cell Size ◽  
Ubiquitin Ligase ◽  
Driving Forces ◽  
S Phase ◽  
Cycle Duration ◽  
Cell Cycles ◽  
Cell Cycle Duration

Cells have evolved elaborate mechanisms to regulate DNA replication machinery and cell cycles in response to DNA damage and replication stress in order to prevent genomic instability and cancer. The E3 ubiquitin ligase SCFDia2 in S. cerevisiae is involved in the DNA replication and DNA damage stress response, but its effect on cell growth is still unclear. Here, we demonstrate that the absence of Dia2 prolongs the cell cycle by extending both S- and G2/M-phases while, at the same time, activating the S-phase checkpoint. In these conditions, Ctf4—an essential DNA replication protein and substrate of Dia2—prolongs its binding to the chromatin during the extended S- and G2/M-phases. Notably, the prolonged cell cycle when Dia2 is absent is accompanied by a marked increase in cell size. We found that while both DNA replication inhibition and an absence of Dia2 exerts effects on cell cycle duration and cell size, Dia2 deficiency leads to a much more profound increase in cell size and a substantially lesser effect on cell cycle duration compared to DNA replication inhibition. Our results suggest that the increased cell size in dia2∆ involves a complex mechanism in which the prolonged cell cycle is one of the driving forces.

scholarly journals A Genetic Screen for Suppressors and Enhancers of the Drosophila PAN GU Cell Cycle Kinase Identifies Cyclin B as a Target

Genetics ◽  
2001 ◽  
Vol 158 (4) ◽  
pp. 1545-1556 ◽  
Author(s):  
Laura A Lee ◽  
Lisa K Elfring ◽  
Giovanni Bosco ◽  
Terry L Orr-Weaver
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Genetic Screen ◽  
S Phase ◽  
Cyclin B ◽  
Cell Cycles ◽  
Protein Levels ◽  
Drosophila Embryogenesis ◽  
Giant Nuclei ◽  
Cell Cycle Kinase

Abstract The early cell cycles of Drosophila embryogenesis involve rapid oscillations between S phase and mitosis. These unique S-M cycles are driven by maternal stockpiles of components necessary for DNA replication and mitosis. Three genes, pan gu (png), plutonium (plu), and giant nuclei (gnu) are required to control the cell cycle specifically at the onset of Drosophila development by inhibiting DNA replication and promoting mitosis. PNG is a protein kinase that is in a complex with PLU. We employed a sensitized png mutant phenotype to screen for genes that when reduced in dosage would dominantly suppress or enhance png. We screened deficiencies covering over 50% of the autosomes and identified both enhancers and suppressors. Mutations in eIF-5A and PP1 87B dominantly suppress png. Cyclin B was shown to be a key PNG target. Mutations in cyclin B dominantly enhance png, whereas png is suppressed by cyclin B overexpression. Suppression occurs via restoration of Cyclin B protein levels that are decreased in png mutants. The plu and gnu phenotypes are also suppressed by cyclin B overexpression. These studies demonstrate that a crucial function of PNG in controlling the cell cycle is to permit the accumulation of adequate levels of Cyclin B protein.

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