scholarly journals ATAD5 regulates the lifespan of DNA replication factories by modulating PCNA level on the chromatin

2012 ◽  
Vol 200 (1) ◽  
pp. 31-44 ◽  
Author(s):  
Kyoo-young Lee ◽  
Haiqing Fu ◽  
Mirit I. Aladjem ◽  
Kyungjae Myung
Keyword(s):  
Dna Replication ◽  
Molecular Mechanisms ◽  
S Phase ◽  
Nuclear Antigen ◽  
Spatial Regulation ◽  
Replication Factory ◽  
G2 Phase ◽  
Phase Progression ◽  
Temporal And Spatial ◽  
Proliferating Cell

Temporal and spatial regulation of the replication factory is important for efficient DNA replication. However, the underlying molecular mechanisms are not well understood. Here, we report that ATAD5 regulates the lifespan of replication factories. Reduced expression of ATAD5 extended the lifespan of replication factories by retaining proliferating cell nuclear antigen (PCNA) and other replisome proteins on the chromatin during and even after DNA synthesis. This led to an increase of inactive replication factories with an accumulation of replisome proteins. Consequently, the overall replication rate was decreased, which resulted in the delay of S-phase progression. Prevalent detection of PCNA foci in G2 phase cells after ATAD5 depletion suggests that defects in the disassembly of replication factories persist after S phase is complete. ATAD5-mediated regulation of the replication factory and PCNA required an intact ATAD5 ATPase domain. Taken together, our data imply that ATAD5 regulates the cycle of DNA replication factories, probably through its PCNA-unloading activity.

scholarly journals Dynamics of plant DNA replication based on PCNA visualization

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Ryohei Yokoyama ◽  
Takeshi Hirakawa ◽  
Seri Hayashi ◽  
Takuya Sakamoto ◽  
Sachihiro Matsunaga
Keyword(s):  
Dna Replication ◽  
Fluorescent Protein ◽  
S Phase ◽  
Nuclear Antigen ◽  
Marker Line ◽  
Sliding Clamp ◽  
G2 Phase ◽  
Living Organisms ◽  
Green Fluorescent ◽  
Proliferating Cell

Abstract DNA replication is an essential process for the copying of genomic information in living organisms. Imaging of DNA replication in tissues and organs is mainly performed using fixed cells after incorporation of thymidine analogs. To establish a useful marker line to measure the duration of DNA replication and analyze the dynamics of DNA replication, we focused on the proliferating cell nuclear antigen (PCNA), which functions as a DNA sliding clamp for replicative DNA polymerases and is an essential component of replisomes. In this study we produced an Arabidopsis thaliana line expressing PCNA1 fused with the green fluorescent protein under the control of its own promoter (pAtPCNA1::AtPCNA1-sGFP). The duration of the S phase measured using the expression line was consistent with that measured after incorporation of a thymidine analog. Live cell imaging revealed that three distinct nuclear localization patterns (whole, dotted, and speckled) were sequentially observable. These whole, dotted, and speckled patterns of subnuclear AtPCNA1 signals were indicative of the G1 or G2 phase, early S phase and late S phase, respectively. The results indicate that the pAtPCNA1::AtPCNA1-sGFP line is a useful marker line for visualization of S-phase progression in live plant organs.

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 Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites.

1987 ◽  
Vol 105 (4) ◽  
pp. 1549-1554 ◽  
Author(s):  
R Bravo ◽  
H Macdonald-Bravo
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
S Phase ◽  
Nuclear Antigen ◽  
Dna Polymerase Delta ◽  
Quiescent Cells ◽  
Replication Sites ◽  
Nuclear Structures ◽  
Proliferating Cell ◽  
Two Populations

Pulse-chase experiments have revealed that cyclin, the auxiliary protein of DNA polymerase-delta, is stable during the transition from growth to quiescence in 3T3 cells. Immunoblotting together with immunofluorescence analysis has shown that the amount of cyclin after 24 h of quiescence is 30-40% of that of growing cells and that it presents a nucleoplasmic staining. Immunofluorescence studies show the existence of two populations of cyclin during the S phase, one that is nucleoplasmic as in quiescent cells and is easily extracted by detergent, and another that is associated to specific nuclear structures. By using antibromodeoxyuridine immunofluorescence to detect the sites of DNA synthesis, it was shown that the staining patterns of the replicon clusters and their order of appearance throughout the S phase are identical to those observed for cyclin. Two-dimensional gel analysis of Triton-extracted cells show that 20-30% of cyclin remains associated with the replicon clusters. This population of cyclin could not be released from the nucleus using high-salt extractions. This demonstrates that cyclin is tightly associated to the sites of DNA replication and that it must have a fundamental role in DNA synthesis in eukaryotic cells.

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

2018 ◽  
Vol 115 (9) ◽  
pp. E2030-E2039 ◽  
Author(s):  
Ryan Janke ◽  
Grant A. King ◽  
Martin Kupiec ◽  
Jasper Rine
Keyword(s):  
Dna Replication ◽  
Proliferating Cell Nuclear Antigen ◽  
Transcriptional Silencing ◽  
S Phase ◽  
Nuclear Antigen ◽  
Histone Chaperones ◽  
Replication Forks ◽  
Nucleosome Assembly ◽  
Loading And Unloading ◽  
Proliferating Cell

In 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 proliferating cell nuclear antigen (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.

scholarly journals Etoposide Induces the Dispersal of DNA Ligase I from Replication Factories

2001 ◽  
Vol 12 (7) ◽  
pp. 2109-2118 ◽  
Author(s):  
Alessandra Montecucco ◽  
Rossella Rossi ◽  
Giovanni Ferrari ◽  
A. Ivana Scovassi ◽  
Ennio Prosperi ◽  
...  
Keyword(s):  
Dna Replication ◽  
Ataxia Telangiectasia ◽  
Topoisomerase Ii ◽  
Molecular Mechanisms ◽  
Kinase Inhibitor ◽  
Protein A ◽  
S Phase ◽  
Nuclear Antigen ◽  
Dna Ligase ◽  
Dna Ligase I

In eukaryotic cells DNA replication occurs in specific nuclear compartments, called replication factories, that undergo complex rearrangements during S-phase. The molecular mechanisms underlying the dynamics of replication factories are still poorly defined. Here we show that etoposide, an anticancer drug that induces double-strand breaks, triggers the redistribution of DNA ligase I and proliferating cell nuclear antigen from replicative patterns and the ensuing dephosphorylation of DNA ligase I. Moreover, etoposide triggers the formation of RPA foci, distinct from replication factories. The effect of etoposide on DNA ligase I localization is prevented by aphidicolin, an inhibitor of DNA replication, and by staurosporine, a protein kinase inhibitor and checkpoints' abrogator. We suggest that dispersal of DNA ligase I is triggered by an intra-S-phase checkpoint activated when replicative forks meet topoisomerase II-DNA–cleavable complexes. However, etoposide treatment of ataxia telangiectasia cells demonstrated that ataxia-telangiectasia-mutated activity is not required for the disassembly of replication factories and the formation of replication protein A foci.

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 Cell proliferation and gill morphology in anoxic crucian carp

2005 ◽  
Vol 289 (4) ◽  
pp. R1196-R1201 ◽  
Author(s):  
Jørund Sollid ◽  
Aina Kjernsli ◽  
Paula M. De Angelis ◽  
Åsmund K. Røhr ◽  
Göran E. Nilsson
Keyword(s):  
Cell Proliferation ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Crucian Carp ◽  
Fold Increase ◽  
S Phase ◽  
Nuclear Antigen ◽  
Tyrosyl Radical ◽  
Radical Generation ◽  
Proliferating Cell

Is DNA replication/cell proliferation in vertebrates possible during anoxia? The oxygen dependence of ribonucleotide reductase (RNR) could lead to a stop in DNA synthesis, thereby making anoxic DNA replication impossible. We have studied this question in an anoxia-tolerant vertebrate, the crucian carp ( Carassius carassius), by examining 5′-bromo-2′-deoxyuridine incorporation and proliferating cell nuclear antigen levels in the gills, intestinal crypts, and liver. We exposed crucian carp to 1 and 7 days of anoxia followed by 7 days of reoxygenation. There was a reduced incidence of S-phase cells (from 12.2 to 5.0%) in gills during anoxia, which coincided with a concomitant increase of G0 cells. Anoxia also decreased the number of S-phase cells in intestine (from 8.1 to 1.8%). No change in the fraction of S-phase cells (∼1%) in liver was found. Thus new S-phase cells after 7 days of anoxia were present in all tissues, revealing a considerable rate of DNA synthesis. Subsequently, the oxygen-dependent subunit of crucian carp RNR (RNRR2) was cloned. We found no differences in amino acids involved in radical generation and availability of the iron center compared with mouse, which could have explained reduced oxygen dependence. Furthermore, the amount of RNRR2 mRNA in gills did not decrease throughout anoxia exposure. These results indicate that crucian carp is able to sustain some cell proliferation in anoxia, possibly because RNRR2 retains its tyrosyl radical in anoxia, and that the replication machinery is still maintained. Although hypoxia triggers a 7.5-fold increase of respiratory surface area in crucian carp, this response was not triggered in anoxia.

Nuclear distribution of proliferating cell nuclear antigen (PCNA) in fertilized eggs of the starfish Asterina pectinifera

1994 ◽  
Vol 107 (12) ◽  
pp. 3291-3300 ◽  
Author(s):  
A. Nomura
Keyword(s):  
Dna Replication ◽  
Proliferating Cell Nuclear Antigen ◽  
Three Dimensional ◽  
S Phase ◽  
Nuclear Antigen ◽  
Dimensional Structure ◽  
Confocal Laser ◽  
Simultaneous Occurrence ◽  
Limited Region ◽  
Proliferating Cell

Previous studies (Nomura et al. (1991) Dev. Biol. 143, 289–296 (1993) Dev. Biol. 159, 288–297) determined the time of DNA replication period (S phase) in starfish eggs fertilized either during or after oocyte maturation. Here proliferating cell nuclear antigen (PCNA) localized within nuclei of starfish eggs was detected with an anti-PCNA human antiserum. Using a confocal laser scanning microscope, a three-dimensional structure of the PCNA region was analyzed. In eggs fertilized during maturation, PCNA started to localize within the nuclei at the same time as the initiation of the first S phase. During the S phase, the distribution of localized PCNA in a three-dimensional view coincided with the chromatin distribution. After the S phase, PCNA remained localized within the nuclei, but its distribution no longer coincided with the chromatin distribution. In eggs fertilized after maturation, however, PCNA started to localize within the female pronuclei about 10 minutes ahead of the first S phase. Localized PCNA occupied only a limited region of the nuclei without diffusing over the whole nuclear area. Chromatin distributed around the peripheral region of the nuclei mostly outside the PCNA region. When the first S phase was initiated, the chromatin distribution became coincident with the PCNA region. Later behavior of PCNA was the same as that of the eggs fertilized during maturation. The precocious localization of PCNA in those eggs fertilized after maturation simply demonstrates that the ‘postactivation process’ for preparing DNA replication is triggered by fertilization and PCNA localization and S phase are sequentially initiated with a time-lapse. On the other hand, the simultaneous occurrence of them seen in those eggs fertilized during maturation indicates that the postactivation process must be going on in parallel with the maturation process.

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.

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