Evidence for a Replication Function of Ffa-1, the Xenopus Orthologue of Werner Syndrome Protein

DNA replication in higher eukaryotic cells occurs at a large number of discrete sites called replication foci. We have previously purified a protein, focus-forming activity 1 (FFA-1), which is involved in the assembly of putative prereplication foci in Xenopus egg extracts. FFA-1 is the orthologue of the Werner syndrome gene product (WRN), a member of the RecQ helicase family. In this paper we show that FFA-1 colocalizes with sites of DNA synthesis and the single-stranded DNA binding protein, replication protein A (RPA), in nuclei reconstituted in the egg extract. In addition, we show that two glutathione S-transferase FFA-1 fusion proteins can inhibit DNA replication in a dominant negative manner. The dominant negative effect correlates with the incorporation of the fusion proteins into replication foci to form “hybrid foci,” which are unable to engage in DNA replication. At the biochemical level, RPA can interact with FFA-1 and specifically stimulates its DNA helicase activity. However, in the presence of the dominant negative mutant proteins, the stimulation is prevented. These results provide the first direct biochemical evidence of an important role for FFA-1 in DNA replication.

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A Dominant-negative Mutant of Human DNA Helicase B Blocks the Onset of Chromosomal DNA Replication

Journal of Biological Chemistry ◽

10.1074/jbc.m208067200 ◽

2002 ◽

Vol 277 (43) ◽

pp. 40853-40861 ◽

Cited By ~ 29

Author(s):

Poonam Taneja ◽

Jinming Gu ◽

Rui Peng ◽

Ryan Carrick ◽

Fumiaki Uchiumi ◽

...

Keyword(s):

Dna Replication ◽

Dna Helicase ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Chromosomal Dna ◽

Human Dna ◽

Negative Mutant

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Restoration of Silencing in Saccharomyces cerevisiae by Tethering of a Novel Sir2-Interacting Protein, Esc8

Genetics ◽

10.1093/genetics/162.2.633 ◽

2002 ◽

Vol 162 (2) ◽

pp. 633-645 ◽

Cited By ~ 2

Author(s):

Guido Cuperus ◽

David Shore

Keyword(s):

Protein A ◽

Dominant Negative ◽

Class I ◽

Dominant Negative Effect ◽

Rna Pol Ii ◽

Interacting Protein ◽

Nucleosome Remodeling ◽

Negative Effect ◽

Close Homolog

Abstract We previously described two classes of SIR2 mutations specifically defective in either telomeric/HM silencing (class I) or rDNA silencing (class II) in S. cerevisiae. Here we report the identification of genes whose protein products, when either overexpressed or directly tethered to the locus in question, can establish silencing in SIR2 class I mutants. Elevated dosage of SCS2, previously implicated as a regulator of both inositol biosynthesis and telomeric silencing, suppressed the dominant-negative effect of a SIR2-143 mutation. In a genetic screen for proteins that restore silencing when tethered to a telomere, we isolated ESC2 and an uncharacterized gene, (YOL017w), which we call ESC8. Both Esc2p and Esc8p interact with Sir2p in two-hybrid assays, and the Esc8p-Sir2 interaction is detected in vitro. Interestingly, Esc8p has a single close homolog in yeast, the ISW1-complex factor Ioc3p, and has also been copurified with Isw1p, raising the possibility that Esc8p is a component of an Isw1p-containing nucleosome remodeling complex. Whereas esc2 and esc8 deletion mutants alone have only marginal silencing defects, cells lacking Isw1p show a strong silencing defect at HMR but not at telomeres. Finally, we show that Esc8p interacts with the Gal11 protein, a component of the RNA pol II mediator complex.

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Mutation-specific non-canonical pathway of PTEN as a distinct therapeutic target for glioblastoma

Cell Death and Disease ◽

10.1038/s41419-021-03657-0 ◽

2021 ◽

Vol 12 (4) ◽

Author(s):

Seung Won Choi ◽

Yeri Lee ◽

Kayoung Shin ◽

Harim Koo ◽

Donggeon Kim ◽

...

Keyword(s):

Functional Properties ◽

Therapeutic Target ◽

Medical Center ◽

Dominant Negative ◽

The Cancer Genome Atlas ◽

Dominant Negative Effect ◽

Cell Periphery ◽

Missense Mutations ◽

Phosphatase Domain ◽

Mutant Proteins

AbstractPTEN is one of the most frequently altered tumor suppressor genes in malignant tumors. The dominant-negative effect of PTEN alteration suggests that the aberrant function of PTEN mutation might be more disastrous than deletion, the most frequent genomic event in glioblastoma (GBM). This study aimed to understand the functional properties of various PTEN missense mutations and to investigate their clinical relevance. The genomic landscape of PTEN alteration was analyzed using the Samsung Medical Center GBM cohort and validated via The Cancer Genome Atlas dataset. Several hotspot mutations were identified, and their subcellular distributions and phenotypes were evaluated. We established a library of cancer cell lines that overexpress these mutant proteins using the U87MG and patient-derived cell models lacking functional PTEN. PTEN mutations were categorized into two major subsets: missense mutations in the phosphatase domain and truncal mutations in the C2 domain. We determined the subcellular compartmentalization of four mutant proteins (H93Y, C124S, R130Q, and R173C) from the former group and found that they had distinct localizations; those associated with invasive phenotypes (‘edge mutations’) localized to the cell periphery, while the R173C mutant localized to the nucleus. Invasive phenotypes derived from edge substitutions were unaffected by an anti-PI3K/Akt agent but were disrupted by microtubule inhibitors. PTEN mutations exhibit distinct functional properties regarding their subcellular localization. Further, some missense mutations (‘edge mutations’) in the phosphatase domain caused enhanced invasiveness associated with dysfunctional cytoskeletal assembly, thus suggesting it to be a potent therapeutic target.

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Telomere repeat DNA forms a large non-covalent complex with unique cohesive properties which is dissociated by Werner syndrome DNA helicase in the presence of replication protein A

Nucleic Acids Research ◽

10.1093/nar/28.18.3642 ◽

2000 ◽

Vol 28 (18) ◽

pp. 3642-3648 ◽

Cited By ~ 16

Author(s):

I. Ohsugi

Keyword(s):

Werner Syndrome ◽

Protein A ◽

Replication Protein A ◽

Dna Helicase ◽

Replication Protein ◽

Telomere Repeat ◽

Cohesive Properties ◽

Repeat Dna ◽

Covalent Complex

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Fanconi anemia group J mutation abolishes its DNA repair function by uncoupling DNA translocation from helicase activity or disruption of protein-DNA complexes

Blood ◽

10.1182/blood-2009-11-256016 ◽

2010 ◽

Vol 116 (19) ◽

pp. 3780-3791 ◽

Cited By ~ 52

Author(s):

Yuliang Wu ◽

Joshua A. Sommers ◽

Avvaru N. Suhasini ◽

Thomas Leonard ◽

Julianna S. Deakyne ◽

...

Keyword(s):

Dna Repair ◽

Fanconi Anemia ◽

Atp Hydrolysis ◽

Bone Marrow Failure ◽

Dna Helicase ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Dna Complexes ◽

Null Cell ◽

Negative Effect

Abstract Fanconi anemia (FA) is a genetic disease characterized by congenital abnormalities, bone marrow failure, and susceptibility to leukemia and other cancers. FANCJ, one of 13 genes linked to FA, encodes a DNA helicase proposed to operate in homologous recombination repair and replicational stress response. The pathogenic FANCJ-A349P amino acid substitution resides immediately adjacent to a highly conserved cysteine of the iron-sulfur domain. Given the genetic linkage of the FANCJ-A349P allele to FA, we investigated the effect of this particular mutation on the biochemical and cellular functions of the FANCJ protein. Purified recombinant FANCJ-A349P protein had reduced iron and was defective in coupling adenosine triphosphate (ATP) hydrolysis and translocase activity to unwinding forked duplex or G-quadruplex DNA substrates or disrupting protein-DNA complexes. The FANCJ-A349P allele failed to rescue cisplatin or telomestatin sensitivity of a FA-J null cell line as detected by cell survival or γ-H2AX foci formation. Furthermore, expression of FANCJ-A349P in a wild-type background exerted a dominant-negative effect, indicating that the mutant protein interferes with normal DNA metabolism. The ability of FANCJ to use the energy from ATP hydrolysis to produce the force required to unwind DNA or destabilize protein bound to DNA is required for its role in DNA repair.

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trans-dominant inhibition of poly(ADP-ribosyl)ation sensitizes cells against gamma-irradiation and N-methyl-N'-nitro-N-nitrosoguanidine but does not limit DNA replication of a polyomavirus replicon.

Molecular and Cellular Biology ◽

10.1128/mcb.15.6.3154 ◽

1995 ◽

Vol 15 (6) ◽

pp. 3154-3163 ◽

Cited By ~ 72

Author(s):

J H Küpper ◽

M Müller ◽

M K Jacobson ◽

J Tatsumi-Miyajima ◽

D L Coyle ◽

...

Keyword(s):

Dna Replication ◽

Dna Binding ◽

Gamma Irradiation ◽

Cellular Proliferation ◽

Molecular Genetic ◽

Genetic System ◽

Binding Domain ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Dna Binding Domain

Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins catalyzed by poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30), with NAD+ serving as the substrate. PARP is strongly activated upon recognition of DNA strand breaks by its DNA-binding domain. Experiments with low-molecular-weight inhibitors of PARP have led to the view that PARP activity plays a role in DNA repair and possibly also in DNA replication, cell proliferation, and differentiation. Accumulating evidence for nonspecific inhibitor effects prompted us to develop a molecular genetic system to inhibit PARP in living cells, i.e., to overexpress selectively the DNA-binding domain of PARP as a dominant negative mutant. Here we report on a cell culture system which allows inducible, high-level expression of the DNA-binding domain. Induction of this domain leads to about 90% reduction of poly(ADP-ribose) accumulation after gamma-irradiation and sensitizes cells to the cytotoxic effect of gamma-irradiation and of N-methyl-N'-nitro-N-nitrosoguanidine. In contrast, induction does not affect normal cellular proliferation or the replication of a transfected polyomavirus replicon. Thus, trans-dominant inhibition of the poly(ADP-ribose) accumulation occurring after gamma-irradiation or N-methyl-N'-nitro-N-nitrosoguanidine is specifically associated with a disturbance of the cellular recovery from the inflicted damage.

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A Mutant Allele of MRE11 Found in Mismatch Repair-deficient Tumor Cells Suppresses the Cellular Response to DNA Replication Fork Stress in a Dominant Negative Manner

Molecular Biology of the Cell ◽

10.1091/mbc.e07-09-0975 ◽

2008 ◽

Vol 19 (4) ◽

pp. 1693-1705 ◽

Cited By ~ 46

Author(s):

Qin Wen ◽

Jennifer Scorah ◽

Geraldine Phear ◽

Gary Rodgers ◽

Sheila Rodgers ◽

...

Keyword(s):

Dna Replication ◽

Mutant Allele ◽

Cellular Response ◽

Replication Fork ◽

Protein A ◽

Cancer Cell Line ◽

Replication Stress ◽

Dominant Negative ◽

Mutant Protein ◽

In Cells

The interaction of ataxia-telangiectasia mutated (ATM) and the Mre11/Rad50/Nbs1 (MRN) complex is critical for the response of cells to DNA double-strand breaks; however, little is known of the role of these proteins in response to DNA replication stress. Here, we report a mutant allele of MRE11 found in a colon cancer cell line that sensitizes cells to agents causing replication fork stress. The mutant Mre11 weakly interacts with Rad50 relative to wild type and shows little affinity for Nbs1. The mutant protein lacks 3′-5′ exonuclease activity as a result of loss of part of the conserved nuclease domain; however, it retains binding affinity for single-stranded DNA (ssDNA), double-stranded DNA with a 3′ single-strand overhang, and fork-like structures containing ssDNA regions. In cells, the mutant protein shows a time- and dose-dependent accumulation in chromatin after thymidine treatment that corresponds with increased recruitment and hyperphosphorylation of replication protein A. ATM autophosphorylation, Mre11 foci, and thymidine-induced homologous recombination are suppressed in cells expressing the mutant allele. Together, our results suggest that the mutant Mre11 suppresses the cellular response to replication stress by binding to ssDNA regions at disrupted forks and impeding replication restart in a dominant negative manner.

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Both the paired domain and homeodomain are required for in vivo function of Drosophila Paired

Development ◽

10.1242/dev.122.9.2709 ◽

1996 ◽

Vol 122 (9) ◽

pp. 2709-2718 ◽

Cited By ~ 4

Author(s):

P. Miskiewicz ◽

D. Morrissey ◽

Y. Lan ◽

L. Raj ◽

S. Kessler ◽

...

Keyword(s):

Dna Binding ◽

Target Genes ◽

Ectopic Expression ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Paired Domain ◽

Mutant Proteins ◽

Segment Polarity ◽

Negative Effect

Drosophila paired, a homolog of mammalian Pax-3, is key to the coordinated regulation of segment-polarity genes during embryogenesis. The paired gene and its homologs are unusual in encoding proteins with two DNA-binding domains, a paired domain and a homeodomain. We are using an in vivo assay to dissect the functions of the domains of this type of molecule. In particular, we are interested in determining whether one or both DNA-binding activities are required for individual in vivo functions of Paired. We constructed point mutants in each domain designed to disrupt DNA binding and tested the mutants with ectopic expression assays in Drosophila embryos. Mutations in either domain abolished the normal regulation of the target genes engrailed, hedgehog, gooseberry and even-skipped, suggesting that these in vivo functions of Paired require DNA binding through both domains rather than either domain alone. However, when the two mutant proteins were placed in the same embryo, Paired function was restored, indicating that the two DNA-binding activities need not be present in the same molecule. Quantitation of this effect shows that the paired domain mutant has a dominant-negative effect consistent with the observations that Paired protein can bind DNA as a dimer.

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Functional analysis of the BMP9 response of ALK1 mutants from HHT2 patients: a diagnostic tool for novel ACVRL1 mutations

Blood ◽

10.1182/blood-2010-03-276881 ◽

2010 ◽

Vol 116 (9) ◽

pp. 1604-1612 ◽

Cited By ~ 49

Author(s):

Nicolas Ricard ◽

Marie Bidart ◽

Christine Mallet ◽

Gaetan Lesca ◽

Sophie Giraud ◽

...

Keyword(s):

Diagnostic Tool ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Novel Mutations ◽

Mutant Proteins ◽

Genes Encoding ◽

Negative Effect ◽

Bone Morphogenetic Protein 9

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant genetically inheritable vascular dysplasia caused by mutations in genes encoding receptors of the transforming growth factor-β (TGF-β) family: ENG, encoding endoglin (HHT1), and ACVRL1, encoding activin receptor-like kinase-1 (ALK1; HHT2). Our recent discovery of bone morphogenetic protein 9 (BMP9) as the specific ligand for ALK1 allowed us to reevaluate the functional significance of ACVRL1 mutations. We generated 19 ALK1 mutants reproducing HHT2 mutations (4 were novel mutations) found throughout the protein. We show that all ALK1 mutant proteins were expressed by transfected cells; most of them were present at the cell surface and retained their ability to bind BMP9 (except for the extracellular mutants). However, most were defective in BMP9 signaling. None of the ALK1 mutants had a dominant negative effect on wild-type ALK1 activity. These data demonstrate that mutations of ACVRL1 fit with a functional haploinsufficiency model affecting BMP9 signaling. Our study also identified 4 ACVRL1 mutations (D179A, R386C, R454W, and A482V) that did not alter the BMP9 responses that are polymorphisms and 2 novel mutations that are pathogenic (L381P and I485F). This demonstrates that the analysis of BMP9 responses can be used as a diagnostic tool by geneticists confronted with novel or conflicting ACVRL1 mutations.

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