scholarly journals DNA ligase I mediates essential functions in mammalian cells.

1995 ◽  
Vol 15 (8) ◽  
pp. 4303-4308 ◽  
Author(s):  
J H Petrini ◽  
Y Xiao ◽  
D T Weaver
Keyword(s):  
Mammalian Cells ◽  
Catalytic Domain ◽  
Embryonic Stem ◽  
Dna Ligase ◽  
Null Mutation ◽  
Dna Ligases ◽  
Dna Ligase I ◽  
The Creation ◽  
Stem Cell Lines

DNA replication, repair, and recombination are essential processes in mammalian cells. Hence, the application of gene targeting to the study of these DNA metabolic pathways requires the creation of nonnull mutations. We have developed a method for introducing partially defective mutants in murine embryonic stem cells that circumvents the problem of cellular lethality of targeted mutations at essential loci. Using this approach, we have determined that mammalian DNA ligase I is essential for cell viability. Thus, DNA ligases II and III are not redundant with DNA ligase I for the function(s) associated with cell proliferation. Partial complementation of the lethal DNA ligase I null mutation allowed the creation of deficient embryonic stem cell lines. We found that a wild-type DNA ligase I cDNA, as well as a variant DNA ligase I cDNA, was able to rescue the lethality of the homozygous null mutation, whereas an N-terminal deletion mutant consisting of the minimal DNA ligase I catalytic domain was not. This observation demonstrates that sequences outside the DNA ligase I catalytic domain are essential for DNA ligase I function in vivo.

scholarly journals Mammalian DNA ligases. Catalytic domain and size of DNA ligase I.

1990 ◽  
Vol 265 (21) ◽  
pp. 12611-12617 ◽  
Author(s):  
A E Tomkinson ◽  
D D Lasko ◽  
G Daly ◽  
T Lindahl
Keyword(s):  
Catalytic Domain ◽  
Dna Ligase ◽  
Dna Ligases ◽  
Dna Ligase I

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

1997 ◽  
Vol 139 (3) ◽  
pp. 579-587 ◽  
Author(s):  
M. Cristina Cardoso ◽  
Cuthbert Joseph ◽  
Hans-Peter Rahn ◽  
Regina Reusch ◽  
Bernardo Nadal-Ginard ◽  
...  
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Functional Organization ◽  
S Phase ◽  
Dna Ligase ◽  
Chimeric Proteins ◽  
Dna Ligase I ◽  
Replication Foci

The mammalian nucleus is highly organized, and nuclear processes such as DNA replication occur in discrete nuclear foci, a phenomenon often termed “functional organization” of the nucleus. We describe the identification and characterization of a bipartite targeting sequence (amino acids 1–28 and 111–179) that is necessary and sufficient to direct DNA ligase I to nuclear replication foci during S phase. This targeting sequence is located within the regulatory, NH2-terminal domain of the protein and is dispensable for enzyme activity in vitro but is required in vivo. The targeting domain functions position independently at either the NH2 or the COOH termini of heterologous proteins. We used the targeting sequence of DNA ligase I to visualize replication foci in vivo. Chimeric proteins with DNA ligase I and the green fluorescent protein localized at replication foci in living mammalian cells and thus show that these subnuclear functional domains, previously observed in fixed cells, exist in vivo. The characteristic redistribution of these chimeric proteins makes them unique markers for cell cycle studies to directly monitor entry into S phase in living cells.

scholarly journals Characterization of constitutive HSF2 DNA-binding activity in mouse embryonal carcinoma cells

1994 ◽  
Vol 14 (8) ◽  
pp. 5309-5317
Author(s):  
S P Murphy ◽  
J J Gorzowski ◽  
K D Sarge ◽  
B Phillips
Keyword(s):  
Transcription Factors ◽  
Heat Shock ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Embryonal Carcinoma ◽  
Embryonic Stem ◽  
Binding Activity ◽  
Hsp70 Promoter ◽  
Ec Cells

Two distinct murine heat shock transcription factors, HSF1 and HSF2, have been identified. HSF1 mediates the transcriptional activation of heat shock genes in response to environmental stress, while the function of HSF2 is not understood. Both factors can bind to heat shock elements (HSEs) but are maintained in a non-DNA-binding state under normal growth conditions. Mouse embryonal carcinoma (EC) cells are the only mammalian cells known to exhibit HSE-binding activity, as determined by gel shift assays, even when maintained at normal physiological temperatures. We demonstrate here that the constitutive HSE-binding activity present in F9 and PCC4.aza.R1 EC cells, as well as a similar activity found to be present in mouse embryonic stem cells, is composed predominantly of HSF2. HSF2 in F9 EC cells is trimerized and is present at higher levels than in a variety of nonembryonal cell lines, suggesting a correlation of these properties with constitutive HSE-binding activity. Surprisingly, transcription run-on assays suggest that HSF2 in unstressed EC cells does not stimulate transcription of two putative target genes, hsp70 and hsp86. Genomic footprinting analysis indicates that HSF2 is not bound in vivo to the HSE of the hsp70 promoter in unstressed F9 EC cells, although HSF2 is present in the nucleus and the promoter is accessible to other transcription factors and to HSF1 following heat shock. Thus trimerization and nuclear localization of HSF2 do not appear to be sufficient for in vivo binding of HSF2 to the HSE of the hsp70 promoter in unstressed F9 EC cells.

MAMMALIAN DNA LIGASES; ALTERED DNA LIGASE I IN BLOOM'S SYNDROME

1991 ◽  
pp. 71
Author(s):  
ALAN E. TOMKINSON ◽  
DEBORAH E. BARNES ◽  
KEN-ICHI KODAMA ◽  
EEMA ROBERTS ◽  
GRAHAM DALY ◽  
...  
Keyword(s):  
Dna Ligase ◽  
Bloom's Syndrome ◽  
Dna Ligases ◽  
Bloom’S Syndrome ◽  
Dna Ligase I

scholarly journals The ligation of pol β mismatch insertion products governs the formation of promutagenic base excision DNA repair intermediates

2020 ◽  
Vol 48 (7) ◽  
pp. 3708-3721 ◽  
Author(s):  
Melike Çağlayan
Keyword(s):  
Excision Repair ◽  
Dna Ligase ◽  
Active Site Residues ◽  
Dna Ligases ◽  
Terminal Domain ◽  
Dna Ligase I ◽  
Nucleotide Insertion ◽  
Base Excision ◽  
Ligation Step

Abstract DNA ligase I and DNA ligase III/XRCC1 complex catalyze the ultimate ligation step following DNA polymerase (pol) β nucleotide insertion during base excision repair (BER). Pol β Asn279 and Arg283 are the critical active site residues for the differentiation of an incoming nucleotide and a template base and the N-terminal domain of DNA ligase I mediates its interaction with pol β. Here, we show inefficient ligation of pol β insertion products with mismatched or damaged nucleotides, with the exception of a Watson–Crick-like dGTP insertion opposite T, using BER DNA ligases in vitro. Moreover, pol β N279A and R283A mutants deter the ligation of the promutagenic repair intermediates and the presence of N-terminal domain of DNA ligase I in a coupled reaction governs the channeling of the pol β insertion products. Our results demonstrate that the BER DNA ligases are compromised by subtle changes in all 12 possible noncanonical base pairs at the 3′-end of the nicked repair intermediate. These findings contribute to understanding of how the identity of the mismatch affects the substrate channeling of the repair pathway and the mechanism underlying the coordination between pol β and DNA ligase at the final ligation step to maintain the BER efficiency.

scholarly journals Molecular cloning and expression of human cDNAs encoding a novel DNA ligase IV and DNA ligase III, an enzyme active in DNA repair and recombination.

1995 ◽  
Vol 15 (6) ◽  
pp. 3206-3216 ◽  
Author(s):  
Y F Wei ◽  
P Robins ◽  
K Carter ◽  
K Caldecott ◽  
D J Pappin ◽  
...  
Keyword(s):  
Dna Repair ◽  
Cdna Clone ◽  
Dna Ligase ◽  
Dna Ligases ◽  
Dna Ligase Iv ◽  
Ligase Iv ◽  
Dna Ligase I ◽  
Cloned Cdna ◽  
Dna Ligase Iii

Three distinct DNA ligases, I to III, have been found previously in mammalian cells, but a cloned cDNA has been identified only for DNA ligase I, an essential enzyme active in DNA replication. A short peptide sequence conserved close to the C terminus of all known eukaryotic DNA ligases was used to search for additional homologous sequences in human cDNA libraries. Two different incomplete cDNA clones that showed partial homology to the conserved peptide were identified. Full-length cDNAs were obtained and expressed by in vitro transcription and translation. The 103-kDa product of one cDNA clone formed a characteristic complex with the XRCC1 DNA repair protein and was identical with the previously described DNA ligase III. DNA ligase III appears closely related to the smaller DNA ligase II. The 96-kDa in vitro translation product of the second cDNA clone was also shown to be an ATP-dependent DNA ligase. A fourth DNA ligase (DNA ligase IV) has been purified from human cells and shown to be identical to the 96-kDa DNA ligase by unique agreement between mass spectrometry data on tryptic peptides from the purified enzyme and the predicted open reading frame of the cloned cDNA. The amino acid sequences of DNA ligases III and IV share a related active-site motif and several short regions of homology with DNA ligase I, other DNA ligases, and RNA capping enzymes. DNA ligases III and IV are encoded by distinct genes located on human chromosomes 17q11.2-12 and 13q33-34, respectively.

scholarly journals Citrinin induces apoptosis via a mitochondria-dependent pathway and inhibition of survival signals in embryonic stem cells, and causes developmental injury in blastocysts

2007 ◽  
Vol 404 (2) ◽  
pp. 317-326 ◽  
Author(s):  
Wen-Hsiung Chan
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Mammalian Cells ◽  
Cell Injury ◽  
Reactive Oxygen Species Generation ◽  
Embryonic Stem ◽  
Subsequent Development ◽  
Cytochrome C Release

The mycotoxin CTN (citrinin), a natural contaminant in foodstuffs and animal feeds, has cytotoxic and genotoxic effects on various mammalian cells. CTN is known to cause cell injury, including apoptosis, but the precise regulatory mechanisms of CTN action, particularly in stem cells and embryos, are currently unclear. In the present paper, I report that CTN has cytotoxic effects on mouse embryonic stem cells and blastocysts, and is associated with defects in their subsequent development, both in vitro and in vivo. Experiments in embryonic stem cells (ESC-B5) showed that CTN induces apoptosis via ROS (reactive oxygen species) generation, increased Bax/Bcl-2 ratio, loss of MMP (mitochondrial membrane potential), induction of cytochrome c release, and activation of caspase 3. In this model, CTN triggers cell death via inactivation of the HSP90 [a 90 kDa isoform of the HSP (heat-shock protein) family proteins]/multichaperone complex and subsequent degradation of Ras and Raf-1, further inhibiting anti-apoptotic processes, such as the Ras→ERK (extracellular-signal-regulated kinase) signal transduction pathway. In addition, CTN causes early developmental injury in mouse ESCs and blastocysts in vitro. Lastly, using an in vivo mouse model, I show that consumption of drinking water containing 10 μM CTN results in blastocyst apoptosis and early embryonic developmental injury. Collectively, these findings show for the first time that CTN induces ROS and mitochondria-dependent apoptotic processes, inhibits Ras→ERK survival signalling via inactivation of the HSP90/multichaperone complex, and causes developmental injury in vivo.

scholarly journals Mapping transcription factor occupancy using minimal numbers of cells in vitro and in vivo

2017 ◽  
Author(s):  
Luca Tosti ◽  
James Ashmore ◽  
Boon Siang Nicholas Tan ◽  
Benedetta Carbone ◽  
Tapan K Mistri ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Regulatory Networks ◽  
Embryonic Stem ◽  
Specific Cell ◽  
Dna Interactions

AbstractThe identification of transcription factor (TF) binding sites in the genome is critical to understanding gene regulatory networks (GRNs). While ChIP-seq is commonly used to identify TF targets, it requires specific ChIP-grade antibodies and high cell numbers, often limiting its applicability. DNA adenine methyltransferase identification (DamID), developed and widely used in Drosophila, is a distinct technology to investigate protein-DNA interactions. Unlike ChIP-seq, it does not require antibodies, precipitation steps or chemical protein-DNA crosslinking, but to date it has been seldom used in mammalian cells due to technical impediments. Here we describe an optimised DamID method coupled with next generation sequencing (DamID-seq) in mouse cells, and demonstrate the identification of the binding sites of two TFs, OCT4 and SOX2, in as few as 1,000 embryonic stem cells (ESCs) and neural stem cells (NSCs), respectively. Furthermore, we have applied this technique in vivo for the first time in mammals. Oct4 DamID-seq in the gastrulating mouse embryo at 7.5 days post coitum (dpc) successfully identified multiple Oct4 binding sites proximal to genes involved in embryo development, neural tube formation, mesoderm-cardiac tissue development, consistent with the pivotal role of this TF in post-implantation embryo. This technology paves the way to unprecedented investigations of TF-DNA interactions and GRNs in specific cell types with limited availability in mammals including in vivo samples.

Identification of a novel human DNA ligase I inhibitor that promotes cellular apoptosis in DLD-1 cells: an in silico and in vitro mechanistic study

RSC Advances ◽  
2016 ◽  
Vol 6 (97) ◽  
pp. 94574-94587 ◽  
Author(s):  
Deependra Kumar Singh ◽  
Mohd. Kamil Hussain ◽  
Shagun Krishna ◽  
Amit Laxmikant Deshmukh ◽  
Mohammad Shameem ◽  
...  
Keyword(s):  
In Silico ◽  
Specific Inhibitor ◽  
Mechanistic Study ◽  
Dna Ligase ◽  
Dna Ligases ◽  
Human Dna ◽  
Dna Ligase I ◽  
Cellular Apoptosis

The compound S-097/98 is a specific inhibitor of hLig1. As shown in the figure, the compound inhibits only hLig1 while other human and non-human DNA ligases are not inhibited.

scholarly journals DNA-ligase activity in axolotl early development: evidence for a multilevel regulation of gene expression

Development ◽  
1986 ◽  
Vol 97 (Supplement) ◽  
pp. 85-95
Author(s):  
J. Signoret ◽  
J. C. David
Keyword(s):  
Molecular Form ◽  
Regulation Of Gene Expression ◽  
Molecular Forms ◽  
Dna Ligase ◽  
Amphibian Embryo ◽  
Dna Ligases ◽  
Level Of Activity ◽  
Dna Ligase I ◽  
Basic Features ◽  
Multilevel Regulation

DNA-ligase activity in eukaryotic cells is carried out by two different molecular forms of the enzyme. This molecular duality, first described in mammals (Söderhäll & Lindhal, 1973), has been reported in chicken (David, 1977), amphibians (Carré, Signoret, Lefresne & David, 1981), fish, sea urchin and crab (Signoret & David, 1986). Depending on the developmental stage and the tissue considered, types of cell are available that express exclusively either one or the other of the two DNA-ligases with a defined level of activity. DNA-ligase I and DNA-ligase II, encoded by distinct structural genes (Thiebaud et al. 1985), can be unambiguously characterized and the advantages offered by the early amphibian embryo have made possible the identification of some basic features of their regulation. The Gene for DNA-ligase I exists in two Regulated States The heavy molecular form of the enzyme, referred to as DNA-ligase I, is generally present in proliferating tissues.

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