scholarly journals An Abundant DNA Binding Protein from the Hyperthermophilic Archaeon Sulfolobus shibatae Affects DNA Supercoiling in a Temperature-Dependent Fashion

2000 ◽  
Vol 182 (14) ◽  
pp. 3929-3933 ◽  
Author(s):  
Hong Xue ◽  
Rong Guo ◽  
Yunfei Wen ◽  
Danxu Liu ◽  
Li Huang
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Dna Supercoiling ◽  
Cellular Protein ◽  
Chromosomal Dna ◽  
Temperature Dependent ◽  
Mobility Shift ◽  
Hyperthermophilic Archaeon ◽  
Sulfolobus Shibatae

ABSTRACT The DNA binding protein Ssh10b, a member of the Sac10b family, has been purified from the hyperthermophilic archaeon Sulfolobus shibatae. Ssh10b constitutes about 4% of the cellular protein. Electrophoretic mobility shift assays showed that Ssh10b first bound a double-stranded DNA fragment with an estimated binding size of ∼∼12 bp, forming distinct shifts, until the DNA was coated with the protein. Binding of more Ssh10b resulted in the formation of smears of lower mobilities. The migration pattern of the smearing Ssh10b-DNA complexes was affected by temperature, whereas that of complexes associated with the distinct shifts was not. Interestingly, Ssh10b was capable of constraining negative DNA supercoils in a temperature-dependent fashion. While the ability of the protein to constrain supercoils was weak at 25°C, it was enhanced substantially at 45°C or higher temperatures (up to 80°C). Taken together, our data suggest that archaeal proteins of the Sac10b family may affect the topology of chromosomal DNA in thermophilic archaea at their growth temperatures.

AND-1, a natural chimeric DNA-binding protein, combines an HMG-box with regulatory WD-repeats

1997 ◽  
Vol 110 (9) ◽  
pp. 1051-1062 ◽  
Author(s):  
A. Kohler ◽  
M.S. Schmidt-Zachmann ◽  
W.W. Franke
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Sequence Motifs ◽  
Mobility Shift ◽  
Hmg Box ◽  
Amino Terminal ◽  
Wd Repeats ◽  
Carboxy Terminal

Using a specific monoclonal antibody (mAb AND-1/23-5-14) we have identified, cDNA-cloned and characterized a novel DNA-binding protein of the clawed toad, Xenopus laevis, that is accumulated in the nucleoplasm of oocytes and various other cells. This protein comprises 1,127 amino acids, with a total molecular mass of 125 kDa and a pI of 5.27. It is encoded by a mRNA of approximately 4 kb and contains, in addition to clusters of acidic amino acids, two hallmark motifs: the amino-terminal part harbours seven consecutive ‘WD-repeats’, which are sequence motifs of about 40 amino acids that are characteristic of a large group of regulatory proteins involved in diverse cellular functions, while the carboxy terminal portion possesses a 63-amino-acid-long ‘HMG-box’, which is typical of a family of DNA-binding proteins involved in regulation of chromatin assembly, transcription and replication. The DNA-binding capability of the protein was demonstrated by DNA affinity chromatography and electrophoretic mobility shift assays using four-way junction DNA. Protein AND-1 (acidic nucleoplasmic DNA-binding protein) appears as an oligomer, probably a homodimer, and has been localized throughout the entire interchromatinic space of the interphase nucleoplasm, whereas during mitosis it is transiently dispersed over the cytoplasm. We also identified a closely related, perhaps orthologous protein in mammals. The unique features of protein AND-1, which is a ‘natural chimera’ combining properties of the WD-repeat and the HMG-box families of proteins, are discussed in relation to its possible nuclear functions.

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.

Mouse beta-globin DNA-binding protein B1 is identical to a proto-oncogene, the transcription factor Spi-1/PU.1, and is restricted in expression to hematopoietic cells and the testis

1993 ◽  
Vol 13 (5) ◽  
pp. 2929-2941
Author(s):  
D L Galson ◽  
J O Hensold ◽  
T R Bishop ◽  
M Schalling ◽  
A D D'Andrea ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Consensus Sequence ◽  
Cell Types ◽  
Dna Binding Protein ◽  
Globin Genes ◽  
Beta Globin ◽  
Chromosome 11 ◽  
Mobility Shift ◽  
Murine Erythroleukemia

The hematopoietic-specific DNA-binding protein B1 binds to the DNA consensus sequence AAAGRGGAARYG located twice in intervening sequence 2 of both of the mouse beta-globin genes (D. L. Galson and D.E. Housman, Mol. Cell. Biol. 8:381-392, 1988). B1 was cloned by expression of a murine erythroleukemia (MEL) cell cDNA library in transfected COS cells and screening by electrophoretic mobility shift analysis. B1 is identical to the proto-oncogene Spi-1/PU.1 (Spi-1), an ets family member. Protein-DNA contacts are shown to resemble those of the helix-turn-helix homeodomain proteins. By Northern (RNA) analysis, we found that Spi-1 mRNA is present at low levels during murine CFU-E maturation and is at least 20-fold higher in uninduced MEL, a transformed proerythroblast-like cell line which contains an activating/transforming insertion of spleen focus-forming virus at the Spi-1 locus. Dimethyl sulfoxide-induced MEL cell differentiation decreases Spi-1 mRNA to approximately 20% of the uninduced level before commitment occurs. In addition to erythroid cells, Spi-1 mRNA is present in B cells, myelomonocytes, and mast cells but not in T cells and nonhematopoietic cell types. In situ hybridization demonstrated Spi-1 mRNA expression in bone marrow, spleen, interstitial nonhepatocytes of the liver, and interstitial nontubular cells of the testis. The Spi-1 locus was mapped on human chromosome 11 to the same interval as ACP2 (lysosomal acid phosphatase), between the anonymous DNA markers D11S33 and D11S14. This region has not yet been found to be associated with a human malignancy.

scholarly journals Adenovirus DNA Binding Protein Interacts with the SNF2-Related CBP Activator Protein (SrCap) and Inhibits SrCap-Mediated Transcription

2001 ◽  
Vol 75 (21) ◽  
pp. 10033-10040 ◽  
Author(s):  
Xiequn Xu ◽  
Isaac Chackalaparampil ◽  
M. Alexandra Monroy ◽  
Maria T. Cannella ◽  
Elizabeth Pesek ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Protein A ◽  
Dna Binding Protein ◽  
Cellular Protein ◽  
Protein Family ◽  
Dependent Manner ◽  
Transcription Activity ◽  
Activator Protein

ABSTRACT The SNF2-related CBP activator protein, SrCap (pronounced “sir cap”), shares homology with the SNF2/SWI2 protein family. SrCap was cloned through its ability to bind CBP. SrCap can function as a CBP coactivator and can activate transcription in a reporter assay when expressed as a Gal-SrCap fusion protein. A monoclonal antibody raised against the carboxyl terminus of SrCap coimmunoprecipitates CBP/p300, supporting the model that SrCap is a CBP binding protein and that these proteins can be found together in a cellular protein complex. In addition, several cellular proteins are coimmunoprecipitated by the SrCap-specific antibody. Since adenovirus E1A proteins interact with CBP/p300 proteins, we examined what proteins could be copurified in a SrCap-specific coimmunoprecipitation assay from lysates of adenovirus-infected cells. While E1A proteins were not detected in this complex, to our surprise, we observed the presence of an infected-cell-specific band of 72 kDa, which we suspected might be the adenovirus DNA binding protein, DBP. The adenovirus DBP is a multifunctional protein involved in several aspects of the adenovirus life cycle, including an ability to modulate transcription. The identity of DBP was confirmed by DBP-specific Western blot analysis and by reimmunoprecipitating DBP from denatured SrCap-specific protein complexes. Using in vitro-translated DBP and SrCap proteins, we demonstrated that these proteins interact. To determine whether this interaction could affect SrCap-mediated transcription, we tested whether increasing amounts of DBP could modulate the Gal-SrCap transcription activity. We observed that DBP inhibited Gal-SrCap transcription activity in a dose-dependent manner. These data suggest a novel mechanism of adenovirus host cell control by which DBP binds to and inactivates SrCap, a member of the SNF2 chromatin-remodeling protein family.

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