RIP60, a mammalian origin-binding protein, enhances DNA bending near the dihydrofolate reductase origin of replication

1990 ◽  
Vol 10 (12) ◽  
pp. 6236-6243
Author(s):  
M S Caddle ◽  
L Dailey ◽  
N H Heintz
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Dna Bending ◽  
Chinese Hamster ◽  
Nuclear Extract ◽  
Dna Fragments ◽  
Mobility Shift ◽  
Bent Dna ◽  
Origin Region ◽  
Gel Mobility Shift

Replication of the Chinese hamster dihydrofolate (dhfr) gene initiates near a 281-bp HaeIII fragment of stably bent DNA that binds RIP60, a 60-kDa origin-specific DNA-binding protein that has been purified from HeLa cell nuclear extract (L. Dailey, M. S. Caddle, N. Heintz, and N. H. Heintz, Mol. Cell. Biol. 10:6225-6235, 1990). Circular permutation assays showed that stable DNA bending in the dhfr origin region fragment was due to the presence of five oligo (dA)3-4 tracts, designated bend elements B1 to B5, that are spaced 10 bp apart. DNA bending directed by elements B1 to B5, as assessed by anomolous migration of DNA fragments on polyacrylamide gels, was accentuated at 4 degrees C. Bend element B5, which is in inverse orientation relative to elements B1 to B4, overlaps an ATT-rich motif that comprises the RIP60 protein-binding site. Gel mobility shift assays with circularly permuted bent DNA fragments and purified RIP60 showed that RIP60 markedly enhanced DNA bending of the dhfr origin region sequences. These results suggest that, as in many plasmids, bacteriophages, and eucaryotic viruses, mammalian DNA-binding proteins may enhance DNA bending near origins of replication during initiation of DNA synthesis.

scholarly journals RIP60, a mammalian origin-binding protein, enhances DNA bending near the dihydrofolate reductase origin of replication.

1990 ◽  
Vol 10 (12) ◽  
pp. 6236-6243 ◽  
Author(s):  
M S Caddle ◽  
L Dailey ◽  
N H Heintz
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Dna Bending ◽  
Chinese Hamster ◽  
Nuclear Extract ◽  
Dna Fragments ◽  
Mobility Shift ◽  
Bent Dna ◽  
Origin Region ◽  
Gel Mobility Shift

Replication of the Chinese hamster dihydrofolate (dhfr) gene initiates near a 281-bp HaeIII fragment of stably bent DNA that binds RIP60, a 60-kDa origin-specific DNA-binding protein that has been purified from HeLa cell nuclear extract (L. Dailey, M. S. Caddle, N. Heintz, and N. H. Heintz, Mol. Cell. Biol. 10:6225-6235, 1990). Circular permutation assays showed that stable DNA bending in the dhfr origin region fragment was due to the presence of five oligo (dA)3-4 tracts, designated bend elements B1 to B5, that are spaced 10 bp apart. DNA bending directed by elements B1 to B5, as assessed by anomolous migration of DNA fragments on polyacrylamide gels, was accentuated at 4 degrees C. Bend element B5, which is in inverse orientation relative to elements B1 to B4, overlaps an ATT-rich motif that comprises the RIP60 protein-binding site. Gel mobility shift assays with circularly permuted bent DNA fragments and purified RIP60 showed that RIP60 markedly enhanced DNA bending of the dhfr origin region sequences. These results suggest that, as in many plasmids, bacteriophages, and eucaryotic viruses, mammalian DNA-binding proteins may enhance DNA bending near origins of replication during initiation of DNA synthesis.

scholarly journals Characterization of the maize Mutator transposable element MURA transposase as a DNA-binding protein.

1997 ◽  
Vol 17 (9) ◽  
pp. 5165-5175 ◽  
Author(s):  
M I Benito ◽  
V Walbot
Keyword(s):  
Dna Binding ◽  
Transposable Element ◽  
Binding Protein ◽  
Sequence Similarity ◽  
Functional Characterization ◽  
Dna Binding Protein ◽  
Amino Acid Sequence Similarity ◽  
Mobility Shift ◽  
Gel Mobility Shift

The autonomous MuDR element of the Mutator (Mu) transposable element family of maize encodes at least two proteins, MURA and MURB. Based on amino acid sequence similarity, previous studies have reported that MURA is likely to be a transposase. The functional characterization of MURA has been hindered by the instability of its cDNA, mudrA, in Escherichia coli. In this study, we report the first successful stabilization and expression of MURA in Saccharomyces cerevisiae. Gel mobility shift assays demonstrate that MURA is a DNA-binding protein that specifically binds to sequences within the highly conserved Mu element terminal inverted repeats (TIRs). DNase I and 1,10-phenanthroline-copper footprinting of MURA-Mu1 TIR complexes indicate that MURA binds to a conserved approximately 32-bp region in the TIR of Mu1. In addition, MURA can bind to the same region in the TIRs of all tested actively transposing Mu elements but binds poorly to the diverged Mu TIRs of inactive elements. Previous studies have reported a correlation between Mu transposon inactivation and methylation of the Mu element TIRs. Gel mobility shift assays demonstrate that MURA can interact differentially with unmethylated, hemimethylated, and homomethylated TIR substrates. The significance of MURA's interaction with the TIRs of Mu elements is discussed in the context of what is known about the regulation and mechanisms of Mutator activities in maize.

scholarly journals A novel DNA-binding motif abuts the zinc finger domain of insect nuclear hormone receptor FTZ-F1 and mouse embryonal long terminal repeat-binding protein.

1992 ◽  
Vol 12 (12) ◽  
pp. 5667-5672 ◽  
Author(s):  
H Ueda ◽  
G C Sun ◽  
T Murata ◽  
S Hirose
Keyword(s):  
Dna Binding ◽  
Long Terminal Repeat ◽  
Zinc Finger ◽  
Hormone Receptor ◽  
Binding Protein ◽  
Nuclear Hormone Receptor ◽  
Zinc Finger Motif ◽  
Binding Region ◽  
Mobility Shift ◽  
Gel Mobility Shift

Fruit fly FTZ-F1, silkworm BmFTZ-F1, and mouse embryonal long terminal repeat-binding protein are members of the nuclear hormone receptor superfamily, which recognizes the same sequence, 5'-PyCAAGGPyCPu-3'. Among these proteins, a 30-amino-acid basic region abutting the C-terminal end of the zinc finger motif, designated the FTZ-F1 box, is conserved. Gel mobility shift competition by various mutant peptides of the DNA-binding region revealed that the FTZ-F1 box as well as the zinc finger motif is involved in the high-affinity binding of FTZ-F1 to its target site. Using a gel mobility shift matrix competition assay, we demonstrated that the FTZ-F1 box governs the recognition of the first three bases, while the zinc finger region recognizes the remaining part of the binding sequence. We also showed that the DNA-binding region of FTZ-F1 recognizes and binds to DNA as a monomer. Occurrence of the FTZ-F1 box sequence in other members of the nuclear hormone receptor superfamily raises the possibility that these receptors constitute a unique subfamily which binds to DNA as a monomer.

A novel DNA-binding motif abuts the zinc finger domain of insect nuclear hormone receptor FTZ-F1 and mouse embryonal long terminal repeat-binding protein

1992 ◽  
Vol 12 (12) ◽  
pp. 5667-5672 ◽  
Author(s):  
H Ueda ◽  
G C Sun ◽  
T Murata ◽  
S Hirose
Keyword(s):  
Dna Binding ◽  
Long Terminal Repeat ◽  
Zinc Finger ◽  
Hormone Receptor ◽  
Binding Protein ◽  
Nuclear Hormone Receptor ◽  
Zinc Finger Motif ◽  
Binding Region ◽  
Mobility Shift ◽  
Gel Mobility Shift

Fruit fly FTZ-F1, silkworm BmFTZ-F1, and mouse embryonal long terminal repeat-binding protein are members of the nuclear hormone receptor superfamily, which recognizes the same sequence, 5'-PyCAAGGPyCPu-3'. Among these proteins, a 30-amino-acid basic region abutting the C-terminal end of the zinc finger motif, designated the FTZ-F1 box, is conserved. Gel mobility shift competition by various mutant peptides of the DNA-binding region revealed that the FTZ-F1 box as well as the zinc finger motif is involved in the high-affinity binding of FTZ-F1 to its target site. Using a gel mobility shift matrix competition assay, we demonstrated that the FTZ-F1 box governs the recognition of the first three bases, while the zinc finger region recognizes the remaining part of the binding sequence. We also showed that the DNA-binding region of FTZ-F1 recognizes and binds to DNA as a monomer. Occurrence of the FTZ-F1 box sequence in other members of the nuclear hormone receptor superfamily raises the possibility that these receptors constitute a unique subfamily which binds to DNA as a monomer.

scholarly journals Identification of the proteins responsible for SAR DNA binding in nuclear matrix of Cucurbita pepo.

1995 ◽  
Vol 42 (2) ◽  
pp. 171-176
Author(s):  
R Rzepecki ◽  
E Markiewicz ◽  
J Szopa
Keyword(s):  
Dna Binding ◽  
Cucurbita Pepo ◽  
Standard Procedure ◽  
Cell Nuclei ◽  
Mobility Shift ◽  
Gel Mobility Shift Assay ◽  
Mobility Shift Assay ◽  
Pre Treatment ◽  
Gel Mobility Shift ◽  
Triton X

The nuclear matrices from White bush (Cucurbita pepo var. patisonina) cell nuclei have been isolated using three methods: I, standard procedure involving extraction of cell nuclei with 2 M NaCl and 1% Triton X-100; II, the same with pre-treatment of cell nuclei with 0.5 mM CuSO4 (stabilisation step); and III, method with extraction by lithium diiodosalicylate (LIS), and compared the polypeptide pattern. The isolated matrices specifically bind SAR DNA derived from human beta-interferon gene in the exogenous SAR binding assay and in the gel mobility shift assay. Using IgG against the 32 kDa endonuclease we have found in the DNA-protein blot assay that this protein is one of the proteins binding SAR DNA. We have identified three proteins with molecular mass of 65 kDa, 60 kDa and 32 kDa which are responsible for SAR DNA binding in the gel mobility shift assay experiments.

A Gel Mobility Shift Assay for Probing the Effect of Drug–DNA Adducts on DNA-Binding Proteins

2003 ◽  
pp. 95-106 ◽  
Author(s):  
Suzanne M. Cutts ◽  
Andrew Masta ◽  
Con Panousis ◽  
Peter G. Parsons ◽  
Richard A. Sturm ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Adducts ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Mobility Shift ◽  
Gel Mobility Shift Assay ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift

DNA-binding factors of B lymphoid cells are susceptible to limited proteolytic cleavage during nuclear extract preparation

1988 ◽  
Vol 8 (4) ◽  
pp. 1812-1815
Author(s):  
E L Mather
Keyword(s):  
Dna Binding ◽  
Nuclear Extract ◽  
Lymphoid Cells ◽  
Chain Gene ◽  
Immunoglobulin Heavy Chain Gene ◽  
Mobility Shift ◽  
Lymphoid Lines ◽  
Dna Binding Factors ◽  
Mobility Shift Assay ◽  
B Lymphoid

DNA-binding proteins that interact with the 3' end of the mouse mu immunoglobulin heavy chain gene were identified by the electrophoretic mobility shift assay. Complexes of distinctly different mobilities were formed by extracts prepared from B lymphoid lines representing different stages of maturation. The apparent stage-specific differences are shown to be due to proteolytic events that occurred during extract preparation.

The suppression of testis-brain RNA binding protein and kinesin heavy chain disrupts mRNA sorting in dendrites

1999 ◽  
Vol 112 (21) ◽  
pp. 3691-3702 ◽  
Author(s):  
W.L. Severt ◽  
T.U. Biber ◽  
X. Wu ◽  
N.B. Hecht ◽  
R.J. DeLorenzo ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Antisense Oligonucleotides ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Neuronal Remodeling ◽  
Kinesin Heavy Chain ◽  
Gel Mobility Shift

Ribonucleoprotein particles (RNPs) are thought to be key players in somato-dendritic sorting of mRNAs in CNS neurons and are implicated in activity-directed neuronal remodeling. Here, we use reporter constructs and gel mobility shift assays to show that the testis brain RNA-binding protein (TB-RBP) associates with mRNPs in a sequence (Y element) dependent manner. Using antisense oligonucleotides (anti-ODN), we demonstrate that blocking the TB-RBP Y element binding site disrupts and mis-localizes mRNPs containing (alpha)-calmodulin dependent kinase II (alpha)-CAMKII) and ligatin mRNAs. In addition, we show that suppression of kinesin heavy chain motor protein alters only the localization of (alpha)-CAMKII mRNA. Thus, differential sorting of mRNAs involves multiple mRNPs and selective motor proteins permitting localized mRNAs to utilize common mechanisms for shared steps.

USF-1 and USF-2 trans-repress IL-1β-induced iNOS transcription in mesangial cells

2002 ◽  
Vol 283 (4) ◽  
pp. C1065-C1072 ◽  
Author(s):  
Ashish K. Gupta ◽  
Bruce C. Kone
Keyword(s):  
Dna Binding ◽  
Promoter Activity ◽  
Mesangial Cells ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Inos Gene ◽  
E Box ◽  
Inos Promoter ◽  
Gel Mobility Shift

Transcriptional activation of the inducible nitric oxide synthase (iNOS) gene requires multiple interactions of cis elements and trans-acting factors. Previous in vivo footprinting studies (Goldring CE, Reveneau S, Algarte M, and Jeannin JF. Nucleic Acids Res 24: 1682–1687, 1996) of the murine iNOS gene demonstrated lipopolysaccharide-inducible protection of guanines in the region −904/−883, which includes an E-box motif. In this report, by using site-directed mutagenesis of the −893/−888 E-box and correlating functional assays of the mutated iNOS promoter with upstream stimulatory factor (USF) DNA-binding activities, we demonstrate that the −893/−888 E-box motif is functionally required for iNOS regulation in murine mesangial cells and that USFs are in vivo components of the iNOS transcriptional response complex. Mutation of the E-box sequence augmented the iNOS response to interleukin-1β (IL-1β) in transiently transfected mesangial cells. Gel mobility shift assays demonstrated that USFs cannot bind to the −893/−888 E-box promoter region when the E-box is mutated. Cotransfection of USF-1 and USF-2 expression vectors with iNOS promoter-luciferase reporter constructs suppressed IL-1β-simulated iNOS promoter activity. Cotransfection of dominant-negative USF-2 mutants lacking the DNA binding domain or cis-element decoys containing concatamers of the −904/−883 region augmented IL-1β stimulation of iNOS promoter activity. Gel mobility shift assays showed that only USF-1 and USF-2 supershifted the USF protein-DNA complexes. These results demonstrated that USF binding to the E-box at −893/−888 serves to trans-repress basal expression and IL-1β induction of the iNOS promoter.

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