scholarly journals High Mobility of Flap Endonuclease 1 and DNA Polymerase η Associated with Replication Foci in Mammalian S-Phase Nucleus

2005 ◽  
Vol 16 (5) ◽  
pp. 2518-2528 ◽  
Author(s):  
Lioudmila Solovjeva ◽  
Maria Svetlova ◽  
Lioudmila Sasina ◽  
Kyoji Tanaka ◽  
Masafumi Saijo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Fluorescent Protein ◽  
High Mobility ◽  
Nuclear Antigen ◽  
Frap Assay ◽  
Flap Endonuclease 1 ◽  
Replication Foci ◽  
Mobile Fraction ◽  
Green Fluorescent

Originally detected in fixed cells, DNA replication foci (RFi) were later visualized in living cells by using green fluorescent protein (GFP)-tagged proliferating cell nuclear antigen (PCNA) and DNA ligase I. It was shown using fluorescence redistribution after photobleaching (FRAP) assay that focal GFP-PCNA slowly exchanged, suggesting the existence of a stable replication holocomplex. Here, we used the FRAP assay to study the dynamics of the GFP-tagged PCNA-binding proteins: Flap endonuclease 1 (Fen1) and DNA polymerase η (Polη). We also used the GFP-Cockayne syndrome group A (CSA) protein, which does associate with transcription foci after DNA damage. In normal cells, GFP-Polη and GFP-Fen1 are mobile with residence times at RFi (tm) ∼2 and ∼0.8 s, respectively. GFP-CSA is also mobile but does not concentrate at discrete foci. After methyl methanesulfonate (MMS) damage, the mobile fraction of focal GFP-Fen1 decreased and tm increased, but it then recovered. The mobilities of focal GFP-Polη and GFP-PCNA did not change after MMS. The mobility of GFP-CSA did not change after UV-irradiation. These data indicate that the normal replication complex contains at least two mobile subunits. The decrease of the mobile fraction of focal GFP-Fen1 after DNA damage suggests that Fen1 exchange depends on the rate of movement of replication forks.

scholarly journals Dynamics of DNA Replication Factories in Living Cells

2000 ◽  
Vol 149 (2) ◽  
pp. 271-280 ◽  
Author(s):  
Heinrich Leonhardt ◽  
Hans-Peter Rahn ◽  
Peter Weinzierl ◽  
Anje Sporbert ◽  
Thomas Cremer ◽  
...  
Keyword(s):  
Dna Replication ◽  
Fluorescent Protein ◽  
Protein Complexes ◽  
Time Lapse ◽  
Living Cells ◽  
Nuclear Antigen ◽  
Central Component ◽  
Nuclear Speckles ◽  
Replication Foci ◽  
Green Fluorescent

DNA replication occurs in microscopically visible complexes at discrete sites (replication foci) in the nucleus. These foci consist of DNA associated with replication machineries, i.e., large protein complexes involved in DNA replication. To study the dynamics of these nuclear replication foci in living cells, we fused proliferating cell nuclear antigen (PCNA), a central component of the replication machinery, with the green fluorescent protein (GFP). Imaging of stable cell lines expressing low levels of GFP-PCNA showed that replication foci are heterogeneous in size and lifetime. Time-lapse studies revealed that replication foci clearly differ from nuclear speckles and coiled bodies as they neither show directional movements, nor do they seem to merge or divide. These four dimensional analyses suggested that replication factories are stably anchored in the nucleus and that changes in the pattern occur through gradual, coordinated, but asynchronous, assembly and disassembly throughout S phase.

scholarly journals Nuclear localization of the Epstein–Barr virus EBNA3B protein

2006 ◽  
Vol 87 (4) ◽  
pp. 789-793 ◽  
Author(s):  
Anita Burgess ◽  
Marion Buck ◽  
Kenia Krauer ◽  
Tom Sculley
Keyword(s):  
Nuclear Localization ◽  
Epstein Barr Virus ◽  
Fluorescent Protein ◽  
Site Directed Mutagenesis ◽  
Nuclear Antigen ◽  
Nuclear Localization Signals ◽  
Barr Virus ◽  
Computer Analyses ◽  
Epstein Barr ◽  
Green Fluorescent

The Epstein–Barr virus nuclear antigen (EBNA) 3B is a hydrophilic, proline-rich, charged protein that is thought to be involved in transcriptional regulation and is targeted exclusively to the cell nucleus, where it localizes to discrete subnuclear granules. Co-localization studies utilizing a fusion protein between enhanced green fluorescent protein (EGFP) and EBNA3B with FLAG-tagged EBNA3A and EBNA3C proteins demonstrated that EBNA3B co-localized with both EBNA3A and EBNA3C in the nuclei of cells when overexpressed. Computer analyses identified four potential nuclear-localization signals (NLSs) in the EBNA3B amino acid sequence. By utilizing fusion proteins with EGFP, deletion constructs of EBNA3B and site-directed mutagenesis, three of the four NLSs (aa 160–166, 430–434 and 867–873) were shown to be functional in truncated forms of EBNA3B, whilst an additional NLS (aa 243–246) was identified within the N-terminal region of EBNA3B. Only two of the NLSs were found to be functional in the context of the full-length EBNA3B protein.

scholarly journals Oxidative DNA Damage Bypass in Arabidopsis thaliana Requires DNA Polymerase λ and Proliferating Cell Nuclear Antigen 2

2011 ◽  
Vol 23 (2) ◽  
pp. 806-822 ◽  
Author(s):  
Alessandra Amoroso ◽  
Lorenzo Concia ◽  
Caterina Maggio ◽  
Cécile Raynaud ◽  
Catherine Bergounioux ◽  
...  
Keyword(s):  
Dna Damage ◽  
Arabidopsis Thaliana ◽  
Dna Polymerase ◽  
Proliferating Cell Nuclear Antigen ◽  
Oxidative Dna Damage ◽  
Nuclear Antigen ◽  
Dna Polymerase Λ ◽  
Proliferating Cell

The Cdk and PCNA domains on p21Cip1 both function to inhibit G1/S progression during hyperoxia

2004 ◽  
Vol 286 (3) ◽  
pp. L506-L513 ◽  
Author(s):  
Christopher E. Helt ◽  
Rhonda J. Staversky ◽  
Yi-Jang Lee ◽  
Robert A. Bambara ◽  
Peter C. Keng ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Molecular Mechanisms ◽  
Kinase Inhibitors ◽  
Fluorescent Protein ◽  
Nuclear Antigen ◽  
Cell Cycle Regulators ◽  
Amino Terminal ◽  
Binding Domains ◽  
Green Fluorescent

This study investigates molecular mechanisms underlying cell cycle arrest when cells are exposed to high levels of oxygen (hyperoxia). Hyperoxia has previously been shown to increase expression of the cell cycle regulators p53 and p21. In the current study, we found that p53-deficient human lung adenocarcinoma H1299 cells failed to induce p21 or growth arrest in G1 when exposed to 95% oxygen. Instead, cells arrested in S and G2. Stable expression of p53 restored induction of p21 and G1 arrest without affecting mRNA expression of the other Cip or INK4 G1 kinase inhibitors. To confirm the role of p21 in G1 arrest, we created H1299 cells with tetracycline-inducible expression of enhanced green fluorescent protein (EGFP), EGFP fused to p21 (EGFp21), or EGFP fused to p27 (EGFp27), a related cell cycle inhibitor. The amino terminus of p21 and p27 bind cyclin-dependent kinases (Cdk), whereas the carboxy terminus of p21 binds the sliding clamp proliferating cell nuclear antigen (PCNA). EGFp21 or EGFp27, but not EGFP by itself, restored G1 arrest during hyperoxia. When separately overexpressed, the amino-terminal Cdk and carboxy-terminal PCNA binding domains of p21 each prevented cells from exiting G1 during exposure. These findings demonstrate that exposure in vitro to hyperoxia exerts G1 arrest through p53-dependent induction of p21 that suppresses Cdk and PCNA activity. Because PCNA also participates in DNA repair, these results raise the possibility that p21 also affects repair of oxidized DNA.

scholarly journals Multisite SUMOylation restrains DNA polymerase η interactions with DNA damage sites

2020 ◽  
Vol 295 (25) ◽  
pp. 8350-8362 ◽  
Author(s):  
Claire Guérillon ◽  
Stine Smedegaard ◽  
Ivo A. Hendriks ◽  
Michael L. Nielsen ◽  
Niels Mailand
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Feedback Inhibition ◽  
Dna Lesions ◽  
Nuclear Antigen ◽  
Inhibition Mechanism ◽  
Proteomic Profiling ◽  
Lesion Bypass ◽  
Y Family ◽  
Damaged Dna

Translesion DNA synthesis (TLS) mediated by low-fidelity DNA polymerases is an essential cellular mechanism for bypassing DNA lesions that obstruct DNA replication progression. However, the access of TLS polymerases to the replication machinery must be kept tightly in check to avoid excessive mutagenesis. Recruitment of DNA polymerase η (Pol η) and other Y-family TLS polymerases to damaged DNA relies on proliferating cell nuclear antigen (PCNA) monoubiquitylation and is regulated at several levels. Using a microscopy-based RNAi screen, here we identified an important role of the SUMO modification pathway in limiting Pol η interactions with DNA damage sites in human cells. We found that Pol η undergoes DNA damage- and protein inhibitor of activated STAT 1 (PIAS1)-dependent polySUMOylation upon its association with monoubiquitylated PCNA, rendering it susceptible to extraction from DNA damage sites by SUMO-targeted ubiquitin ligase (STUbL) activity. Using proteomic profiling, we demonstrate that Pol η is targeted for multisite SUMOylation, and that collectively these SUMO modifications are essential for PIAS1- and STUbL-mediated displacement of Pol η from DNA damage sites. These findings suggest that a SUMO-driven feedback inhibition mechanism is an intrinsic feature of TLS-mediated lesion bypass functioning to curtail the interaction of Pol η with PCNA at damaged DNA to prevent harmful mutagenesis.

scholarly journals Recruitment of the Nucleotide Excision Repair Endonuclease XPG to Sites of UV-Induced DNA Damage Depends on Functional TFIIH

2006 ◽  
Vol 26 (23) ◽  
pp. 8868-8879 ◽  
Author(s):  
Angelika Zotter ◽  
Martijn S. Luijsterburg ◽  
Daniël O. Warmerdam ◽  
Shehu Ibrahim ◽  
Alex Nigg ◽  
...  
Keyword(s):  
Dna Damage ◽  
Nucleotide Excision Repair ◽  
Fluorescent Protein ◽  
Core Protein ◽  
Excision Repair ◽  
Mammalian Cell Lines ◽  
Nucleotide Excision ◽  
Green Fluorescent ◽  
Damaged Dna

ABSTRACT The structure-specific endonuclease XPG is an indispensable core protein of the nucleotide excision repair (NER) machinery. XPG cleaves the DNA strand at the 3′ side of the DNA damage. XPG binding stabilizes the NER preincision complex and is essential for the 5′ incision by the ERCC1/XPF endonuclease. We have studied the dynamic role of XPG in its different cellular functions in living cells. We have created mammalian cell lines that lack functional endogenous XPG and stably express enhanced green fluorescent protein (eGFP)-tagged XPG. Life cell imaging shows that in undamaged cells XPG-eGFP is uniformly distributed throughout the cell nucleus, diffuses freely, and is not stably associated with other nuclear proteins. XPG is recruited to UV-damaged DNA with a half-life of 200 s and is bound for 4 min in NER complexes. Recruitment requires functional TFIIH, although some TFIIH mutants allow slow XPG recruitment. Remarkably, binding of XPG to damaged DNA does not require the DDB2 protein, which is thought to enhance damage recognition by NER factor XPC. Together, our data present a comprehensive view of the in vivo behavior of a protein that is involved in a complex chromatin-associated process.

Genotoxicity of Chemical Compounds Identification and Assessment by Yeast Cells Transformed With GFP Reporter Constructs Regulated by the PLM2 or DIN7 Promoter

2015 ◽  
Vol 34 (1) ◽  
pp. 31-43 ◽  
Author(s):  
Van Ngoc Bui ◽  
Thi Thu Huyen Nguyen ◽  
Yvan Bettarel ◽  
Thi Hoai Thu Nguyen ◽  
Thuy Linh Pham ◽  
...  
Keyword(s):  
Dna Damage ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Chemical Compounds ◽  
Cost Effective ◽  
Yeast Cells ◽  
Fluorescence Signal ◽  
Inducible Promoters ◽  
Dna Damaging Agents ◽  
Green Fluorescent

Yeast cells transformed with high-copy number plasmids comprising a green fluorescent protein (GFP)-encoding gene optimized for yeast under the control of the new DIN7 or PLM2 and the established RNR2 and RAD54 promoters were used to assess the genotoxic potential of chemical compounds. The activity of potential DNA-damaging agents was investigated by genotoxicity assays and by OxoPlate assay in the presence of various test compounds. The fluorescence signal generated by GFP in response to DNA damage was related to the different concentrations of analytes and the analyte-dependent GFP synthesis. The use of distinct DNA damage-inducible promoters presents alternative genotoxicity testing strategies by selective induction of promoters in response to DNA damage. The new DIN7 and PLM2 systems show higher sensitivity than the RNR2 and RAD54 systems in detecting 4-nitroquinoline- N-oxide and actinomycin D. Both DIN7 and PLM2 systems are able to detect camptothecin while RNR2 and RAD54 systems are not. Automated laboratory systems with assay performance on 384-well microplates provide for cost-effective high-throughput screening of DNA-damaging agents, reducing compound consumption to about 53% as compared with existing eukaryotic genotoxicity bioassays.

scholarly journals Novel subfamily of mitochondrial HMG box-containing proteins: functional analysis of Gcf1p from Candida albicans

Microbiology ◽  
2009 ◽  
Vol 155 (4) ◽  
pp. 1226-1240 ◽  
Author(s):  
Katarina Visacka ◽  
Joachim M. Gerhold ◽  
Jana Petrovicova ◽  
Slavomir Kinsky ◽  
Priit Jõers ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Dna Binding ◽  
Fluorescent Protein ◽  
Coiled Coil ◽  
High Mobility ◽  
Pathogenic Yeast ◽  
Hmg Box ◽  
Biochemical Analyses ◽  
Green Fluorescent ◽  
Eukaryotic Organisms

Mitochondria of eukaryotic organisms contain populations of DNA molecules that are packed into higher-order structures called mitochondrial nucleoids (mt-nucleoids). In Saccharomyces cerevisiae, the compaction of mitochondrial DNA (mtDNA) into mt-nucleoids is mediated primarily by the high-mobility group (HMG) box-containing protein Abf2, which is an important player in stabilization and metabolism of mtDNA. Although it is evident that analogous proteins must exist in other yeast species, an apparently fast divergence rate has precluded their identification, characterization and comparative analysis. Using in silico analysis of the complete genome sequence of the pathogenic yeast Candida albicans we predicted that the ORF 19.400/19.8030 assigned as GCF1 encodes a putative mitochondrial HMG box-containing protein. In contrast to Abf2p, which contains two HMG boxes, Gcf1p contains only one C-terminal HMG box. In addition, it contains one putative coiled-coil domain with a potential role in protein dimerization. Fluorescence microscopy analysis of a C-terminally tagged Gcf1p with green fluorescent protein (GFP) revealed its mitochondrial localization in both heterologous (S. cerevisiae) and native (C. albicans) hosts. Biochemical analyses of DNA-binding properties indicate that Gcf1p is, similarly to Abf2p, a non-specific DNA-binding protein. To analyse the role of Gcf1p in mtDNA metabolism, we constructed strains lacking one functional allele of the GCF1 gene and carrying one GCF1 allele under the control of the MET3 promoter. Under repressible conditions this strain exhibited a more than 3000-fold decrease in levels of GCF1 mRNA, which was correlated with a substantial decrease in the number of mtDNA copies as well as recombination intermediates. The dramatic effect of reduced levels of Gcf1p on mtDNA metabolism indicates that the protein is involved in essential molecular transactions that relate to the mitochondrial genome.

scholarly journals Nuclear Dynamics of PCNA in DNA Replication and Repair

2005 ◽  
Vol 25 (21) ◽  
pp. 9350-9359 ◽  
Author(s):  
Jeroen Essers ◽  
Arjan F. Theil ◽  
Céline Baldeyron ◽  
Wiggert A. van Cappellen ◽  
Adriaan B. Houtsmuller ◽  
...  
Keyword(s):  
Dna Replication ◽  
Residence Time ◽  
Fluorescent Protein ◽  
De Novo ◽  
Dynamic Equilibrium ◽  
Excision Repair ◽  
S Phase ◽  
Nuclear Antigen ◽  
Dna Replication And Repair ◽  
Replication Foci

ABSTRACT The DNA polymerase processivity factor proliferating cell nuclear antigen (PCNA) is central to both DNA replication and repair. The ring-shaped homotrimeric PCNA encircles and slides along double-stranded DNA, acting as a “sliding clamp” that localizes proteins to DNA. We determined the behavior of green fluorescent protein-tagged human PCNA (GFP-hPCNA) in living cells to analyze its different engagements in DNA replication and repair. Photobleaching and tracking of replication foci revealed a dynamic equilibrium between two kinetic pools of PCNA, i.e., bound to replication foci and as a free mobile fraction. To simultaneously monitor PCNA action in DNA replication and repair, we locally inflicted UV-induced DNA damage. A surprisingly longer residence time of PCNA at damaged areas than at replication foci was observed. Using DNA repair mutants, we showed that the initial recruitment of PCNA to damaged sites was dependent on nucleotide excision repair. Local accumulation of PCNA at damaged regions was observed during all cell cycle stages but temporarily disappeared during early S phase. The reappearance of PCNA accumulation in discrete foci at later stages of S phase likely reflects engagements of PCNA in distinct genome maintenance processes dealing with stalled replication forks, such as translesion synthesis (TLS). Using a ubiquitination mutant of GFP-hPCNA that is unable to participate in TLS, we noticed a significantly shorter residence time in damaged areas. Our results show that changes in the position of PCNA result from de novo assembly of freely mobile replication factors in the nucleoplasmic pool and indicate different binding affinities for PCNA in DNA replication and repair.

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