scholarly journals The Human SETMAR Protein Preserves Most of the Activities of the Ancestral Hsmar1 Transposase

2006 ◽  
Vol 27 (3) ◽  
pp. 1125-1132 ◽  
Author(s):  
Danxu Liu ◽  
Julien Bischerour ◽  
Azeem Siddique ◽  
Nicolas Buisine ◽  
Yves Bigot ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Binding ◽  
Human Genome ◽  
Specific Binding ◽  
Protein A ◽  
Protein Coding ◽  
Specific Binding Sites ◽  
Human Genes ◽  
Starting Point ◽  
The Many

ABSTRACT Transposons have contributed protein coding sequences to a unexpectedly large number of human genes. Except for the V(D)J recombinase and telomerase, all remain of unknown function. Here we investigate the activity of the human SETMAR protein, a highly expressed fusion between a histone H3 methylase and a mariner family transposase. Although SETMAR has demonstrated methylase activity and a DNA repair phenotype, its mode of action and the role of the transposase domain remain obscure. As a starting point to address this problem, we have dissected the activity of the transposase domain in the context of the full-length protein and the isolated transposase domain. Complete transposition of an engineered Hsmar1 transposon by the transposase domain was detected, although the extent of the reaction was limited by a severe defect for cleavage at the 3′ ends of the element. Despite this problem, SETMAR retains robust activity for the other stages of the Hsmar1 transposition reaction, namely, site-specific DNA binding to the transposon ends, assembly of a paired-ends complex, cleavage of the 5′ end of the element in Mn2+, and integration at a TA dinucleotide target site. SETMAR is unlikely to catalyze transposition in the human genome, although the nicking activity may have a role in the DNA repair phenotype. The key activity for the mariner domain is therefore the robust DNA-binding and looping activity which has a high potential for targeting the histone methylase domain to the many thousands of specific binding sites in the human genome provided by copies of the Hsmar1 transposon.

scholarly journals Mitotic chromosome binding predicts transcription factor properties in interphase

2018 ◽  
Author(s):  
Mahé Raccaud ◽  
Andrea B. Alber ◽  
Elias T. Friman ◽  
Harsha Agarwal ◽  
Cédric Deluz ◽  
...  
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Mitotic Chromosome ◽  
Specific Binding ◽  
Site Occupancy ◽  
Chromatin Accessibility ◽  
Live Cells ◽  
Specific Binding Sites ◽  
Genome Wide ◽  
Chromosome Binding

SummaryMammalian transcription factors (TFs) differ broadly in their nuclear mobility and sequence-specific/non-specific DNA binding affinity. How these properties affect the ability of TFs to occupy their specific binding sites in the genome and modify the epigenetic landscape is unclear. Here we combined live cell quantitative measurements of mitotic chromosome binding (MCB) of 502 TFs, measurements of TF mobility by fluorescence recovery after photobleaching, single molecule imaging of DNA binding in live cells, and genome-wide mapping of TF binding and chromatin accessibility. MCB scaled with interphase properties such as association with DNA-rich compartments, mobility, as well as large differences in genome-wide specific site occupancy that correlated with TF impact on chromatin accessibility. As MCB is largely mediated by electrostatic, non-specific TF-DNA interactions, our data suggests that non-specific DNA binding of TFs enhances their search for specific sites and thereby their impact on the accessible chromatin landscape.

scholarly journals Design and Construction of a One-Dimensional DNA Track for an Artificial Molecular Motor

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Suzana Kovacic ◽  
Laleh Samii ◽  
Derek N. Woolfson ◽  
Paul M. G. Curmi ◽  
Heiner Linke ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Binding Sites ◽  
Specific Binding ◽  
Molecular Motor ◽  
Search Space ◽  
One Dimensional ◽  
Motor Processes ◽  
Specific Binding Sites ◽  
The Many ◽  
The Ideal

DNA is a versatile heteropolymer that shows great potential as a building block for a diverse array of nanostructures. We present here a solution to the problem of designing and synthesizing a DNA-based nanostructure that will serve as the track along which an artificial molecular motor processes. This one-dimensional DNA track exhibits periodically repeating elements that provide specific binding sites for the molecular motor. Besides these binding elements, additional sequences are necessary to label specific regions within the DNA track and to facilitate track construction. Designing an ideal DNA track sequence presents a particular challenge because of the many variable elements that greatly expand the number of potential sequences from which the ideal sequence must be chosen. In order to find a suitable DNA sequence, we have adapted a genetic algorithm which is well suited for a large but sparse search space. This algorithm readily identifies long DNA sequences that include all the necessary elements to both facilitate DNA track construction and to present appropriate binding sites for the molecular motor. We have successfully experimentally incorporated the sequence identified by the algorithm into a long DNA track meeting the criteria for observation of the molecular motor's activity.

scholarly journals The inhibition of mitochondrial calcium transport by lanthanides and Ruthenium Red

1974 ◽  
Vol 140 (2) ◽  
pp. 143-155 ◽  
Author(s):  
Ken C. Reed ◽  
Fyfe L. Bygrave
Keyword(s):  
Binding Sites ◽  
Calcium Transport ◽  
Specific Binding ◽  
Protein A ◽  
Liver Mitochondria ◽  
Ruthenium Red ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Calcium ◽  
Binding Studies ◽  
Specific Binding Sites

An EGTA (ethanedioxybis(ethylamine)tetra-acetic acid)-quench technique was developed for measuring initial rates of 45Ca2+ transport by rat liver mitochondria. This method was used in conjunction with studies of Ca2+-stimulated respiration to examine the mechanisms of inhibition of Ca2+ transport by the lanthanides and Ruthenium Red. Ruthenium Red inhibits Ca2+ transport non-competitively with Ki 3×10-8m; there are 0.08nmol of carrier-specific binding sites/mg of protein. The inhibition by La3+ is competitive (Ki=2×10-8m); the concentration of lanthanide-sensitive sites is less than 0.001nmol/mg of protein. A further difference between their modes of action is that lanthanide inhibition diminishes with time whereas that by Ruthenium Red does not. Binding studies showed that both classes of inhibitor bind to a relatively large number of external sites (probably identical with the ‘low-affinity’ Ca2+-binding sites). La3+ competes with Ruthenium Red for most of these sites, but a small fraction of the bound Ruthenium Red (less than 2nmol/mg of protein) is not displaced by La3+. The results are discussed briefly in relation to possible models for a Ca2+ carrier.

scholarly journals Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins

2016 ◽  
Vol 113 (9) ◽  
pp. E1170-E1179 ◽  
Author(s):  
Logan R. Myler ◽  
Ignacio F. Gallardo ◽  
Yi Zhou ◽  
Fade Gong ◽  
Soo-Hyun Yang ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Binding ◽  
Single Molecule ◽  
Cell Biology ◽  
Binding Proteins ◽  
Critical Role ◽  
Protein A ◽  
Dna Binding Proteins ◽  
Single Stranded Dna ◽  
Binding Domains

Exonuclease 1 (Exo1) is a 5′→3′ exonuclease and 5′-flap endonuclease that plays a critical role in multiple eukaryotic DNA repair pathways. Exo1 processing at DNA nicks and double-strand breaks creates long stretches of single-stranded DNA, which are rapidly bound by replication protein A (RPA) and other single-stranded DNA binding proteins (SSBs). Here, we use single-molecule fluorescence imaging and quantitative cell biology approaches to reveal the interplay between Exo1 and SSBs. Both human and yeast Exo1 are processive nucleases on their own. RPA rapidly strips Exo1 from DNA, and this activity is dependent on at least three RPA-encoded single-stranded DNA binding domains. Furthermore, we show that ablation of RPA in human cells increases Exo1 recruitment to damage sites. In contrast, the sensor of single-stranded DNA complex 1—a recently identified human SSB that promotes DNA resection during homologous recombination—supports processive resection by Exo1. Although RPA rapidly turns over Exo1, multiple cycles of nuclease rebinding at the same DNA site can still support limited DNA processing. These results reveal the role of single-stranded DNA binding proteins in controlling Exo1-catalyzed resection with implications for how Exo1 is regulated during DNA repair in eukaryotic cells.

The Constitutional Principle of Accountability: A Study of Contemporary South African Case Law

2018 ◽  
Vol 33 (1) ◽  
Author(s):  
Chuks Okpaluba
Keyword(s):  
Judicial Review ◽  
Police Officers ◽  
Public Procurement ◽  
Case Law ◽  
The State ◽  
Constitutional Principle ◽  
Starting Point ◽  
The Law ◽  
Constitutional Right ◽  
The Many

‘Accountability’ is one of the democratic values entrenched in the Constitution of South Africa, 1996. It is a value recognised throughout the Constitution and imposed upon the law-making organs of state, the Executive, the Judiciary and all public functionaries. This constitutional imperative is given pride of place among the other founding values: equality before the law, the rule of law and the supremacy of the Constitution. This study therefore sets out to investigate how the courts have grappled with the interpretation and application of the principle of accountability, the starting point being the relationship between accountability and judicial review. Therefore, in the exercise of its judicial review power, a court may enquire whether the failure of a public functionary to comply with a constitutional duty of accountability renders the decision made illegal, irrational or unreasonable. One of the many facets of the principle of accountability upon which this article dwells is to ascertain how the courts have deployed that expression in making the state and its agencies liable for the delictual wrongs committed against an individual in vindication of a breach of the individual’s constitutional right in the course of performing a public duty. Here, accountability and breach of public duty; the liability of the state for detaining illegal immigrants contrary to the prescripts of the law; the vicarious liability of the state for the criminal acts of the police and other law-enforcement officers (as in police rape cases and misuse of official firearms by police officers), and the liability of the state for delictual conduct in the context of public procurement are discussed. Having carefully analysed the available case law, this article concludes that no public functionary can brush aside the duty of accountability wherever it is imposed without being in breach of a vital constitutional mandate. Further, it is the constitutional duty of the courts, when called upon, to declare such act or conduct an infringement of the Constitution.

The lacI Gene as a Target for Mutation in Transgenic Rodents and Escherichia coli

Genetics ◽  
1998 ◽  
Vol 148 (4) ◽  
pp. 1441-1451
Author(s):  
Johan G de Boer ◽  
Barry W Glickman
Keyword(s):  
Escherichia Coli ◽  
Dna Repair ◽  
Dna Binding ◽  
Transgenic Animals ◽  
Single Copy ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Large Database ◽  
Deletion Mutations ◽  
Laci Gene

Abstract The lacI gene has been used extensively for the recovery and analysis of mutations in bacteria with various DNA repair backgrounds and after exposure to a wide variety of mutagens. This has resulted in a large database of information on mutational mechanisms and specificity of many mutagens, as well as the effect of DNA repair background on mutagenicity. Most importantly, knowledge about the mutational sensitivity of the lacI gene is now available, yielding information about mutable nucleotides. This popularity and available knowledge resulted in the use of the lacI gene in transgenic rodents for the study of mutagenesis in mammals, where it resides in ~40 repeated copies. As the number of sequenced mutations recovered from these animals increases, we are able to analyze the sites at which mutations have been recovered in great detail and to compare the recovered sites between bacteria and transgenic animals. The nucleotides that code for the DNA-binding domain are nearly saturated with base substitutions. Even after determining the sequences of ~10,000 mutations recovered from the animals, however, new sites and new changes are still being recovered. In addition, we compare the nature of deletion mutations between bacteria and animals. Based on the nature of deletions in the animals, we conclude that each deletion occurs in a single copy of the gene.

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