scholarly journals Purification of overexpressed gam gene protein from bacteriophage Mu by denaturation-renaturation techniques and a study of its DNA-binding properties

1990 ◽  
Vol 269 (3) ◽  
pp. 679-684 ◽  
Author(s):  
Z H Abraham ◽  
N Symonds
Keyword(s):  
Inclusion Bodies ◽  
Guanidine Hydrochloride ◽  
Deae Cellulose ◽  
Exonuclease Activity ◽  
Bacteriophage Mu ◽  
Binding Properties ◽  
Pellet Fraction ◽  
High Affinity ◽  
Double Stranded Dna ◽  
Dna Binding Properties

Recombinant Mu gam gene protein (Mu GAM) synthesized in Escherichia coli accumulates in the form of insoluble inclusion bodies which, after cell lysis and low-speed centrifugation, can be recovered in the pellet fraction. This property was utilized in a purification procedure for Mu GAM based on guanidine hydrochloride denaturation-renaturation followed by a single DEAE-cellulose chromatographic step. The purified Mu GAM was shown by nitrocellulose-filter-binding experiments to bind with high affinity to linear double-stranded DNA and more weakly to supercoiled and single-stranded forms. Mu GAM protects linear DNA from degradation by a variety of exonucleases, but only weakly inhibits endonuclease activity. These results are in accord with a model of Mu GAM conferring protection from exonuclease activity by binding to the ends of DNA.

Double-stranded DNA binding properties of Saccharomyces cerevisiae DNA polymerase ɛ and of the Dpb3p-Dpb4p subassembly

2003 ◽  
Vol 8 (11) ◽  
pp. 873-888 ◽  
Author(s):  
Toshiaki Tsubota ◽  
Satoko Maki ◽  
Hajime Kubota ◽  
Akio Sugino ◽  
Hisaji Maki
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Dna Polymerase ◽  
Binding Properties ◽  
Double Stranded Dna ◽  
Dna Binding Properties

scholarly journals DNA-binding activities of the Drosophila melanogaster even-skipped protein are mediated by its homeo domain and influenced by protein context.

1988 ◽  
Vol 8 (11) ◽  
pp. 4598-4607 ◽  
Author(s):  
T Hoey ◽  
R Warrior ◽  
J Manak ◽  
M Levine
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Binding Properties ◽  
Binding Behavior ◽  
High Affinity ◽  
Homeo Box ◽  
Acid Protein ◽  
Dna Binding Properties ◽  
Amino Acid Protein

The homeo box gene even-skipped (eve) encodes a 376-amino-acid protein that binds with high affinity to sequences located near the 5' termini of the eve and en genes. The 5' en sites are A + T rich and contain copies of the 10-base-pair (bp) consensus sequence T-C-A-A-T-T-A-A-A-T. In contrast, the 5' eve sites are G + C rich and contain the 9-bp sequence T-C-A-G-C-A-C-C-G. Among the five different homeo box proteins that have been tested for binding, eve is unique in that it shows virtually equal preference for the A + T-rich 5' en binding sites and the G + C-rich 5' eve sites. Most of the other proteins bind with a relatively higher affinity to the en sites than to the eve sites. In an effort to identify the regions of the eve protein that are responsible for its efficient binding to both classes of recognition sequences, we analyzed the DNA-binding properties of various mutant eve proteins. These studies suggest that the homeo domain of the eve protein is responsible for both binding activities. However, mutations in distant regions of the protein influenced the binding behavior of the eve homeo domain and caused a reduction in binding to the G + C class of recognition sites. We propose that the protein context of the homeo domain can influence its DNA-binding properties.

scholarly journals DNA Binding Proteins of the Filamentous Phages CTXφ and VGJφ of Vibrio cholerae

2009 ◽  
Vol 191 (18) ◽  
pp. 5873-5876 ◽  
Author(s):  
Alina Falero ◽  
Andy Caballero ◽  
Beatriz Ferrán ◽  
Yovanny Izquierdo ◽  
Rafael Fando ◽  
...  
Keyword(s):  
Dna Binding ◽  
Vibrio Cholerae ◽  
Genomic Dna ◽  
Open Reading Frame ◽  
Rolling Circle Replication ◽  
Binding Properties ◽  
Rolling Circle ◽  
Reading Frame ◽  
Double Stranded Dna ◽  
Dna Binding Properties

ABSTRACT The native product of open reading frame 112 (orf112) and a recombinant variant of the RstB protein, encoded by Vibrio cholerae pathogen-specific bacteriophages VGJφ and CTXφ, respectively, were purified to more than 90% homogeneity. Orf112 protein was shown to specifically bind single-stranded genomic DNA of VGJφ; however, RstB protein unexpectedly bound double-stranded DNA in addition to the single-stranded genomic DNA. The DNA binding properties of these proteins may explain their requirement for the rolling circle replication of the respective phages and RstB's requirement for single-stranded-DNA chromosomal integration of CTXφ phage dependent on XerCD recombinases.

Synthesis and evaluation of the in vitro DNA-binding properties of chiral cis-dichloro(pyridyloxazoline)platinum(II) complexes

2009 ◽  
Vol 87 (1) ◽  
pp. 321-327 ◽  
Author(s):  
David W Dodd ◽  
Heather E Toews ◽  
Michael J Trevail ◽  
Michael C Jennings ◽  
Robert HE Hudson ◽  
...  
Keyword(s):  
Dna Binding ◽  
Hplc Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Metal Exposure ◽  
Binding Studies ◽  
Binding Properties ◽  
Double Stranded Dna ◽  
Dna Binding Studies ◽  
Dna Binding Properties

A series of chiral cis-dichloro(pyridyloxazoline)platinum(II) and palladium(II) complexes were synthesized and their reactivity towards a defined sequence of single-stranded and double-stranded DNA was investigated in comparison to cisplatin. The compounds differed in the nature and absolute configuration of the substituent at the C4 position of the oxazoline ring. The DNA-binding ability of these compounds was evaluated by HPLC analysis, post metal exposure, of enzymatic digests of an undecamer duplex containing one putative metallation site. Polyacrylamide gel electrophoresis (PAGE) and thermal denaturation confirmed the results of the HPLC analysis, which showed that the stereochemistry and character of the substituent at the C4 position of the oxazoline ring had little effect on DNA binding, possibly due to the formation of monofunctional adducts.Key words: cisplatin, chiral, pyridyloxazoline, DNA-binding studies, platinum, palladium.

DNA-binding activities of the Drosophila melanogaster even-skipped protein are mediated by its homeo domain and influenced by protein context

1988 ◽  
Vol 8 (11) ◽  
pp. 4598-4607
Author(s):  
T Hoey ◽  
R Warrior ◽  
J Manak ◽  
M Levine
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Binding Properties ◽  
Binding Behavior ◽  
High Affinity ◽  
Homeo Box ◽  
Acid Protein ◽  
Dna Binding Properties ◽  
Amino Acid Protein

The homeo box gene even-skipped (eve) encodes a 376-amino-acid protein that binds with high affinity to sequences located near the 5' termini of the eve and en genes. The 5' en sites are A + T rich and contain copies of the 10-base-pair (bp) consensus sequence T-C-A-A-T-T-A-A-A-T. In contrast, the 5' eve sites are G + C rich and contain the 9-bp sequence T-C-A-G-C-A-C-C-G. Among the five different homeo box proteins that have been tested for binding, eve is unique in that it shows virtually equal preference for the A + T-rich 5' en binding sites and the G + C-rich 5' eve sites. Most of the other proteins bind with a relatively higher affinity to the en sites than to the eve sites. In an effort to identify the regions of the eve protein that are responsible for its efficient binding to both classes of recognition sequences, we analyzed the DNA-binding properties of various mutant eve proteins. These studies suggest that the homeo domain of the eve protein is responsible for both binding activities. However, mutations in distant regions of the protein influenced the binding behavior of the eve homeo domain and caused a reduction in binding to the G + C class of recognition sites. We propose that the protein context of the homeo domain can influence its DNA-binding properties.

scholarly journals Identification and Characterization of Saccharomyces cerevisiae Cdc6 DNA-binding Properties

2000 ◽  
Vol 11 (5) ◽  
pp. 1673-1685 ◽  
Author(s):  
Luo Feng ◽  
Bin Wang ◽  
Barbara Driscoll ◽  
Ambrose Jong
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Binding Activity ◽  
Temperature Sensitive ◽  
Sequence Specificity ◽  
Binding Properties ◽  
Dna Binding Activity ◽  
Double Stranded Dna ◽  
Dna Binding Properties ◽  
Mutant Cells

Recent studies have shown that Cdc6 is an essential regulator in the formation of DNA replication complexes. However, the biochemical nature of the Cdc6 molecule is still largely unknown. In this report, we present evidence that the Saccharomyces cerevisiaeCdc6 protein is a double-stranded DNA-binding protein. First, we have demonstrated that the purified yeast Cdc6 can bind to double-stranded DNA (dissociation constant ∼ 1 × 10−7 M), not to single-stranded DNA, and that the Cdc6 molecule is a homodimer in its native form. Second, we show that GST-Cdc6 fusion proteins expressed in Escherichia coli bind DNA in an electrophoretic mobility shift assay. Cdc6 antibodies and GST antibodies, but not preimmune serum, induce supershifts of GST-Cdc6 and DNA complexes in these assays, which also showed that GST-Cdc6 binds to various DNA probes without apparent sequence specificity. Third, the minimal requirement for the binding of Cdc6 to DNA has been mapped within its N-terminal 47-amino acid sequence (the NP6 region). This minimal binding domain shows identical DNA-binding properties to those possessed by full-length Cdc6. Fourth, the GST-NP6 protein competes for DNA binding with distamycin A, an antibiotic that chelates DNA within the minor groove of the A+T-rich region. Finally, site-direct mutagenesis studies revealed that the29KRKK region of Cdc6 is essential for Cdc6 DNA-binding activity. To further elucidate the function of Cdc6 DNA binding in vivo, we demonstrated that a binding mutant of Cdc6 fails to complement either cdc6-1 temperature-sensitive mutant cells orΔcdc6 null mutant cells at the nonpermissive temperature. The mutant gene also conferred growth impairments and increased the plasmid loss in its host, indicative of defects in DNA synthesis. Because the mutant defective in DNA binding also fails to stimulate Abf1 ARS1 DNA-binding activity, our results suggest that Cdc6 DNA-binding activity may play a pivotal role in the initiation of DNA replication.

scholarly journals An Arginine Switch in the Species B Adenovirus Knob Determines High-Affinity Engagement of Cellular Receptor CD46

2008 ◽  
Vol 83 (2) ◽  
pp. 673-686 ◽  
Author(s):  
B. David Persson ◽  
Steffen Müller ◽  
Dirk M. Reiter ◽  
Benedikt B. T. Schmitt ◽  
Marko Marttila ◽  
...  
Keyword(s):  
Respiratory Infections ◽  
Hemorrhagic Cystitis ◽  
Cellular Receptor ◽  
Cell Tropism ◽  
Healthy Individuals ◽  
Binding Properties ◽  
High Affinity ◽  
Double Stranded Dna ◽  
Kinetics Of ◽  
Very High

ABSTRACT Adenoviruses (Ads) are icosahedral, nonenveloped viruses with a double-stranded DNA genome. The 51 known Ad serotypes exhibit profound variations in cell tropism and disease types. The number of observed Ad infections is steadily increasing, sometimes leading to fatal outcomes even in healthy individuals. Species B Ads can cause kidney infections, hemorrhagic cystitis, and severe respiratory infections, and most of them use the membrane cofactor protein CD46 as a cellular receptor. The crystal structure of the human Ad type 11 (Ad11) knob complexed with CD46 is known; however, the determinants of CD46 binding in related species B Ads remain unclear. We report here a structural and functional analysis of the Ad11 knob, as well as the Ad7 and Ad14 knobs, which are closely related in sequence to the Ad11 knob but have altered CD46-binding properties. The comparison of the structures of the three knobs, which we determined at very high resolution, provides a platform for understanding these differences and allows us to propose a mechanism for productive high-affinity engagement of CD46. At the center of this mechanism is an Ad knob arginine that needs to switch its orientation in order to engage CD46 with high affinity. Quantum chemical calculations showed that the CD46-binding affinity of Ad11 is significantly higher than that of Ad7. Thus, while Ad7 and Ad14 also bind CD46, the affinity and kinetics of these interactions suggest that these Ads are unlikely to use CD46 productively. The proposed mechanism is likely to determine the receptor usage of all CD46-binding Ads.

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