Mapping and Use of a Sequence that Targets DNA Ligase I to Sites of DNA Replication In Vivo

Cardoso, M. Cristina; Joseph, Cuthbert; Rahn, Hans-Peter; Reusch, Regina; Nadal‐Ginard, Bernardo; Leonhardt, Heinrich

doi:10.1083/jcb.139.3.579

Cited by 91 publications

(117 citation statements)

References 63 publications

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“…DNMT1 has a diffuse nuclear staining which redistributes to replication foci during S-phase where it co-localizes with several other replication proteins that have been identified in the synthesome, including PCNA and DNA ligase I. 16,18,19,45 Association of DNMT1 with the synthesome thus may be cell-cycle regulated, and the pool of DNMT1 purifying with the synthesome representative of the fraction of cells in S-phase.…”

Section: Discussionmentioning

confidence: 99%

DNMT1 is a Component of a Multiprotein DNA Replication Complex

Vertino¹,

Sekowski²,

Coll³

et al. 2002

Cell Cycle

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DNA methylation is a major determinant of epigenetic inheritance and plays an important role in genome stability. The accurate propagation of DNA methylation patterns with cell division requires that methylation be closely coupled to DNA replication, however the precise molecular determinants of this interaction have not been defined. In the present study, we show that the predominant DNA methyltransferase species in somatic cells, DNMT1, is a component of a multiprotein DNA replication complex termed the DNA synthesome that fully supports semi-conservative DNA replication in a cell-free system. DNMT1 protein and activity were found to co-purify with the human DNA synthesome through a series of subcellular fractionation and chromatography steps, resulting in an enrichment of methyltransferase specific activity from two human cell lines. DNA methyltransferase activity co-eluted with in vitro replication activity and DNA polymerase α activity on sucrose density gradients suggesting that DNMT1 is a tightly bound, core component of the replication complex. The synthesome-associated pool of DNA methyltransferase exhibited both maintenance and de novo methyltransferase activity and the ratio of the two was similar to that observed in whole cell lysates and for recombinant DNMT1. These data indicate that interactions within the synthesome complex do not influence the intrinsic preference of DNMT1 for hemimethylated DNA, but suggest that newly replicated DNA may be subject to low level de novo methylation. The data indicate that DNA methylation is tightly coupled to replication through physical interaction of DNMT1 and core components of the replication machinery. The definition of the molecular interactions between DNMT1 and other proteins in the replication complex in normal and neoplastic cells will provide further insight into the regulation of DNA methylation and the mechanisms underlying the alteration of DNA methylation patterns during carcinogenesis.

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Section: Discussionmentioning

confidence: 99%

DNMT1 is a Component of a Multiprotein DNA Replication Complex

Vertino¹,

Sekowski²,

Coll³

et al. 2002

Cell Cycle

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“…Cell numbers were small, however. Pmi28 primary mouse myoblasts were kindly provided by A. StarzinskiPowitz (Kaufmann et al 1999) and cultured as described (Cardoso et al 1997). For microscopic preparations, glass coverslips with a thickness of 170 μm were used (Karl Hecht KG, Sondheim/Rhön, Germany).…”

Section: Cellsmentioning

confidence: 99%

Three-dimensional positioning of genes in mouse cell nuclei

et al. 2008

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To understand the regulation of the genome, it is necessary to understand its three-dimensional organization in the nucleus. We investigated the positioning of eight gene loci on four different chromosomes, including the β-globin gene, in mouse embryonic stem cells and in in vitro differentiated macrophages by fluorescence in situ hybridization on structurally preserved nuclei, confocal microscopy, and 3D quantitative image analysis. We found that gene loci on the same chromosome can significantly differ from each other and from their chromosome territory in their average radial nuclear position. Radial distribution of a given gene locus can change significantly between cell types, excluding the possibility that positioning is determined solely by the DNA sequence. For the set of investigated gene loci, we found no relationship between radial distribution and local gene density, as it was described for human cell nuclei. We did find, however, correlation with other genomic properties such as GC content and certain repetitive elements such as long terminal repeats or long interspersed nuclear elements. Our results suggest that gene density itself is not a driving force in nuclear positioning. Instead, we propose that other genomic properties play a role in determining nuclear chromatin distribution.

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“…The transfection of cells with plasmid constructs was performed using the CaPO 4 -DNA co-precipitation protocol as described before, 37 except for HEK 293-EBNA cells, which were transfected using poly-ethylenimine (1 mg/mL in ddH 2 O, pH 7; Sigma-Aldrich, St. Louis, MO, USA) as described before. 38 Cell fixation and antibody staining Cells grown on glass coverslips for 24 h were incubated with 10 mM FITC-R10 for 1 h. After washing with 1xPBS, cells were fixed in 3.7% formaldehyde in PBS for 10 min and permeabilized with 0.25% Triton X100 in PBS for 10 min.…”

Section: Methodsmentioning

confidence: 99%

“…The microscope was placed in an incubation chamber heated to 37 C to maintain the cell incubation conditions (Okolab, Italy). For the measurements of plasmid transfected cells, a Leica TCS SP confocal microscope was used.…”

Section: Microscopy Image Acquisition and Analysismentioning

confidence: 99%

Principles of protein targeting to the nucleolus

Martin

Ter-Avetisyan

Herce

et al. 2015

Nucleus

114

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The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of rRNA synthesis and assembly of ribosomes. Nucleolar proteins dynamically localize and accumulate in this nuclear compartment relative to the surrounding nucleoplasm. In this study, we have assessed the molecular requirements that are necessary and sufficient for the localization and accumulation of peptides and proteins inside the nucleoli of living cells. The data showed that positively charged peptide entities composed of arginines alone and with an isoelectric point at and above 12.6 are necessary and sufficient for mediating significant nucleolar accumulation. A threshold of 6 arginines is necessary for peptides to accumulate in nucleoli, but already 4 arginines are sufficient when fused within 15 amino acid residues of a nuclear localization signal of a protein. Using a pH sensitive dye, we found that the nucleolar compartment is particularly acidic when compared to the surrounding nucleoplasm and, hence, provides the ideal electrochemical environment to bind poly-arginine containing proteins. In fact, we found that oligo-arginine peptides and GFP fusions bind RNA in vitro. Consistent with RNA being the main binding partner for arginines in the nucleolus, we found that the same principles apply to cells from insects to man, indicating that this mechanism is highly conserved throughout evolution.

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Mapping and Use of a Sequence that Targets DNA Ligase I to Sites of DNA Replication In Vivo

Cited by 91 publications

References 63 publications

DNMT1 is a Component of a Multiprotein DNA Replication Complex

DNMT1 is a Component of a Multiprotein DNA Replication Complex

Three-dimensional positioning of genes in mouse cell nuclei

Principles of protein targeting to the nucleolus

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