RecA protein mediates homologous recognition via non-Watson-Crick bonds in base triplets

1995 ◽  
Vol 347 (1319) ◽  
pp. 5-12 ◽  
Keyword(s):  
Biological Significance ◽  
Reca Protein ◽  
Triplex Dna ◽  
Duplex Dna ◽  
The Novel ◽  
Hydrophobic Core ◽  
Dna Interactions ◽  
Homologous Proteins ◽  
Widespread Distribution ◽  
E Coli

E. coli RecA protein, the prototype of a class, forms a helical nucleoprotein filament on single-stranded DNA that recognizes homology in duplex DNA, and initiates the exchange of strands in homologous recombination. The discovery of this reaction some years ago posed a quandary on how a third strand recognizes homology in duplex DNA, whose Watson-Crick bonds face inward in a hydrophobic core of stacked bases. Recent studies have shown that RecA protein promotes homologous recognition via nonW atson-Crick bonds in base triplets. The intermediates in the RecA reaction differ distinctly from triplex DNA that forms non-enzymically. The biological significance of the novel set of DNA interactions by which RecA protein effects homologous recognition is indicated by the importance of this protein in recombination, and the widespread distribution of homologous proteins in prokaryotes and eukaryotes.

scholarly journals Evidence for conservative (two-progeny) DNA double-strand break repair.

Genetics ◽  
1995 ◽  
Vol 139 (1) ◽  
pp. 5-17 ◽  
Author(s):  
T Yokochi ◽  
K Kusano ◽  
I Kobayashi
Keyword(s):  
Reca Protein ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Duplex Dna ◽  
Experimental Conditions ◽  
E Coli ◽  
Dna Double Strand Break ◽  
Break Repair ◽  
Set Up

Abstract The double-strand break repair models for homologous recombination propose that a double-strand break in a duplex DNA segment is repaired by gene conversion copying a homologous DNA segment. This is a type of conservative recombination, or two-progeny recombination, which generates two duplex DNA segments from two duplex DNA segments. Transformation with a plasmid carrying a double-strand gap and an intact homologous DNA segment resulted in products expected from such conservative (two-progeny) repair in Escherichia coli cells with active E. coli RecE pathway (recBC sbcA) or with active bacteriophage lambda Red pathway. Apparently conservative double-strand break repair, however, might result from successive events of nonconservative recombination, or one-progeny recombination, which generates only one recombinant duplex DNA segment from two segments, involving multiple plasmid molecules. Contribution of such intermolecular recombination was evaluated by transformation with a mixture of two isogenic parental plasmids marked with a restriction site polymorphism. Most of the gap repair products were from intramolecular and, therefore, conservative (two-progeny) reaction under the conditions chosen. Most were conservative even in the absence of RecA protein. The double-strand gap repair reaction was not affected by inversion of the unidirectional replication origin on the plasmid. These results demonstrate the presence of the conservative (two-progeny) double-strand break repair mechanism. These experiments do not rule out the occurrence of nonconservative (one-progeny) recombination since we set up experimental conditions that should favor detection of conservative (two-progeny) recombination.

Electron Microscopy and image analysis of nucleo-protein complexes

1994 ◽  
Vol 52 ◽  
pp. 88-89
Author(s):  
E. H. Egelman ◽  
X. Yu
Keyword(s):  
Electron Microscopy ◽  
Image Analysis ◽  
Normal Cell ◽  
Genetic Recombination ◽  
Protein Complexes ◽  
Chromosomal Rearrangements ◽  
Reca Protein ◽  
E Coli ◽  
Helical Polymer ◽  
Specific Protease

The RecA protein of E. coli has been shown to mediate genetic recombination, regulate its own synthesis, control the expression of other genes, act as a specific protease, form a helical polymer and have an ATPase activity, among other observed properties. The unusual filament formed by the RecA protein on DNA has not previously been shown to exist outside of bacteria. Within this filament, the 36 Å pitch of B-form DNA is extended to about 95 Å, the pitch of the RecA helix. We have now establishedthat similar nucleo-protein complexes are formed by bacteriophage and yeast proteins, and availableevidence suggests that this structure is universal across all of biology, including humans. Thus, understanding the function of the RecA protein will reveal basic mechanisms, in existence inall organisms, that are at the foundation of general genetic recombination and repair.Recombination at this moment is assuming an importance far greater than just pure biology. The association between chromosomal rearrangements and neoplasms has become stronger and stronger, and these rearrangements are most likely products of the recombinatory apparatus of the normal cell. Further, damage to DNA appears to be a major cause of cancer.

Development of 96 Multiple Injection-GC-MS Technique and Its Application in Protein Engineering of Natural and Non-Natural Enzymatic Reactions

2019 ◽  
Author(s):  
Anja Knorrscheidt ◽  
Pascal Püllmann ◽  
Eugen Schell ◽  
Dominik Homann ◽  
Erik Freier ◽  
...  
Keyword(s):  
Microtiter Plate ◽  
Axial Ligand ◽  
Cell System ◽  
Enzymatic Reactions ◽  
The Novel ◽  
Internal Standards ◽  
E Coli ◽  
Enzyme Screening ◽  
Plate Analysis ◽  
Microtiter Plate Analysis

Directed evolution requires the screening of enzyme libraries in biological matrices. Available assays are mostly substrate or enzyme specific. Chromatographic techniques like LC and GC overcome this limitation, but require long analysis times. The herein developed multiple injections in a single experimental run (MISER) using GC coupled to MS allows the injection of samples every 33 s resulting in 96-well microtiter plate analysis within 50 min. This technique is implementable in any GC-MS system with autosampling. Since the GC-MS is far less prone to ion suppression than LCMS, no chromatographic separation is required. This allows the utilisation of an internal standards and the detection of main and side-product. To prove the feasibility of the system in enzyme screening, two libraries were assessed: i) YfeX library in an E. coli whole cell system for the carbene-transfer reaction on indole revealing the novel axial ligand tryptophan, ii) a library of 616 chimeras of fungal unspecific peroxygenase (UPO) in S. cerevisiae supernatant for hydroxylation of tetralin resulting in novel constructs. The data quality and representation are automatically assessed by a new R-script.

Mixed ligand-metal Complexes of 2-(butan-2-ylidene) hydrazinecarbothioamide- Synthesis, Characterization, Computer Aided Drug Character Evaluation and in vitro Biological Activity Assessment

2019 ◽  
Vol 15 ◽  
Author(s):  
Tahmeena Khan ◽  
Rumana Ahmad ◽  
Iqbal Azad ◽  
Saman Raza ◽  
Seema Joshi ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Metal Complexes ◽  
Condensation Reaction ◽  
Dose Range ◽  
Biological Significance ◽  
Mixed Ligand ◽  
Binding Energies ◽  
Mixed Ligand Complexes ◽  
E Coli ◽  
Topo Ii

Background: Mixed ligand-metal complexes are efficient chelating agents because of flexible donor ability. Mixed ligand complexes containing hetero atoms sulphur, nitrogen and oxygen have been probed for their biological significance. Objective: Nine mixed ligand-metal complexes of 2-(butan-2-ylidene) hydrazinecarbothioamide (2-butanone thiosemicarbazone) and pyridine, bipyridine or 2-picoline as co-ligands were synthesized with Cu, Fe and Zn. The complexes were tested against MDA-MB231 (MDA) and A549 cell lines. Antibacterial activity was tested against S. aureus and E. coli. The drug character of the complexes was evaluated on several parameters viz. physicochemical properties, bioactivity scores, toxicity assessment and absorption, distribution, metabolism, excretion and toxicity (ADMET) profile assessment using various automated softwares. Molecular docking of the complexes was also performed with two target proteins. Method and Results: The mixed ligand-metal complexes were synthesized by condensation reaction for 4-5 h. The characterization was done by elemental analysis, 1H-NMR, FT-IR, molar conductance and UV spectroscopies. Molecular docking was performed against ribonucleotide reductase (RR) and topoisomerase II (topo II). [Cu(C5H11N3S)(py)2(CH3COO)2], [Zn(C5H11N3S)(bpy)(SO4)] and [Zn(C5H11N3S)(2-pic)2(SO4)] displayed the lowest binding energies with respect to RR. Against topo II [Cu(C5H11N3S)(py)2(CH3COO)2], [Cu(C5H11N3S)(bpy)(CH3COO)2] and [Zn(C5H11N3S)(2-pic)2(SO4)] had the lowest energies. The druglikness assessment was done using Leadlikeness and Lipinski’s rules. Against topo II [Cu(C5H11N3S)(py)2(CH3COO)2], [Cu(C5H11N3S)(bpy)(CH3COO)2] and [Zn(C5H11N3S)(2-pic)2(SO4)] had the lowest energies. Not more than two violations were obtained in case of each filtering rule showing drug like character of the mixed ligand complexes. Several of the complexes exhibited positive bioactivity scores and almost all the complexes were predicted to be safe with no hazardous effects. All the complexes were predicted to have no mutagenic character as shown by the Ames test [Zn(C5H11N3S)(py)2(SO4)] showed potential activity against MDA. [Co(C5H11N3S(bpy)(Cl)2] was also active against MDA. [Cu(C5H11N3S)(2-pic)2(CH3COO)2] also showed 27.6% cell viability at 100 µM against MDA. Against A549 [Co(C5H11N3S)(py)2(Cl)2], [Cu(C5H11N3S)(py)2(CH3COO)2] and [Co(C5H11N3S(bpy)(Cl)2] were active. [Co(C5H11N3S)(bpy)(Cl)2] and [Cu(C5H11N3S)(2-pic)2(CH3COO)2] were active against S. aureus. [Co(C5H11N3S)(2-pic)2(Cl)2] and [Zn(C5H11N3S)(2-pic)2(SO4)] were active at lower concentrations against S.aureus. Against E. coli, [Zn(C5H11N3S)(2-pic)2(SO4)] showed activity at 18-20mg dose range.

Formation, translocation and resolution of Holliday junctions during homologous genetic recombination

1995 ◽  
Vol 347 (1319) ◽  
pp. 21-25 ◽  
Keyword(s):  
Molecular Mechanisms ◽  
Genetic Recombination ◽  
Holliday Junctions ◽  
The Novel ◽  
E Coli ◽  
The Past ◽  
Endonucleolytic Cleavage ◽  
Insight Into ◽  
Made In

Over the past three or four years, great strides have been made in our understanding of the proteins involved in recombination and the mechanisms by which recombinant molecules are formed. This review summarizes our current understanding of the process by focusing on recent studies of proteins involved in the later steps of recombination in bacteria. In particular, biochemical investigation of the in vitro properties of the E. coli RuvA, RuvB and RuvC proteins have provided our first insight into the novel molecular mechanisms by which Holliday junctions are moved along DNA and then resolved by endonucleolytic cleavage.

Mechanism of E. coli RecA protein directed strand exchanges in post-replication repair of DNA

Nature ◽  
1981 ◽  
Vol 294 (5842) ◽  
pp. 659-662 ◽  
Author(s):  
Stephen C. West ◽  
Era Cassuto ◽  
Paul Howard-Flanders
Keyword(s):  
Reca Protein ◽  
E Coli ◽  
Post Replication Repair

Characterization of novel ybjG and dacC variants in Escherichia coli

2013 ◽  
Vol 62 (11) ◽  
pp. 1728-1734 ◽  
Author(s):  
Dongguo Wang ◽  
Enping Hu ◽  
Jiayu Chen ◽  
Xiulin Tao ◽  
Katelyn Gutierrez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Multiple Drug Resistance ◽  
Multiple Drug ◽  
The Novel ◽  
Conventional Pcr ◽  
E Coli ◽  
Novel Variants ◽  
Restriction Sites ◽  
Genetic Structures

A total of 69 strains of Escherichia coli from patients in the Taizhou Municipal Hospital, China, were isolated, and 11 strains were identified that were resistant to bacitracin, chloramphenicol, tetracycline and erythromycin. These strains were PCR positive for at least two out of three genes, ybjG, dacC and mdfA, by gene mapping with conventional PCR detection. Conjugation experiments demonstrated that these genes existed in plasmids that conferred resistance. Novel ybjG and dacC variants were isolated from E. coli strains EC2163 and EC2347, which were obtained from the sputum of intensive care unit patients. Genetic mapping showed that the genes were located on 8200 kb plasmid regions flanked by EcoRI restriction sites. Three distinct genetic structures were identified among the 11 PCR-positive strains of E. coli, and two contained the novel ybjG and dacC variants. The putative amino acid differences in the ybjG and dacC gene variants were characterized. These results provide evidence for novel variants of ybjG and dacC, and suggest that multiple drug resistance in hospital strains of E. coli depends on the synergistic function of ybjG, dacC and mdfA within three distinct genetic structures in conjugative plasmids.

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