DNA-binding domain of RCC1 protein is not essential for coupling mitosis with DNA replication

1992 ◽  
Vol 102 (3) ◽  
pp. 393-400 ◽  
Author(s):  
H. Seino ◽  
N. Hisamoto ◽  
S. Uzawa ◽  
T. Sekiguchi ◽  
T. Nishimoto
Keyword(s):  
Dna Binding ◽  
Nuclear Translocation ◽  
Protein S ◽  
Sucrose Density Gradient ◽  
Binding Activity ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Dna Binding Activity ◽  
Unknown Protein ◽  
Tsbn2 Cells

The RCC1 protein that is required for coupling mitosis with the S phase has a DNA-binding domain in the N-terminal region outside the repeat. We found that RCC1 protein without any DNA-binding activity complemented the tsBN2 mutation with the same efficiency as that of intact RCC1 protein. In ts+ transformants of tsBN2 cells transfected with the RCC1 cDNA lacking the DNA-binding domain, an endogenous RCC1 disappeared at 39.5 degrees C, and the deleted RCC1 protein encoded by the transfected cDNA was found in the cytoplasm, but a significant amount of it was also found in the nuclei. This deleted RCC1 protein was eluted from the nuclei with the same concentration of NaCl and DNase I as was used for the intact RCC1 protein in BHK21 cells. Furthermore, the deleted RCC1 protein co-migrated with the nucleosome fraction on sucrose density gradient analysis. These results indicate that the RCC1 protein binds chromatin with the aid of other unknown protein(s). Thus, the DNA-binding domain of RCC1 protein is not essential for coupling between the S and M phases, but was shown instead to function as a nuclear translocation signal.

scholarly journals A novel DNA-binding motif in the nuclear matrix attachment DNA-binding protein SATB1

1994 ◽  
Vol 14 (3) ◽  
pp. 1852-1860
Author(s):  
K Nakagomi ◽  
Y Kohwi ◽  
L A Dickinson ◽  
T Kohwi-Shigematsu
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Contact ◽  
Binding Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Binding Motif ◽  
Dna Binding Domain ◽  
Sequence Context ◽  
Dna Binding Activity

The nuclear matrix attachment DNA (MAR) binding protein SATB1 is a sequence context-specific binding protein that binds in the minor groove, making virtually no contact with the DNA bases. The SATB1 binding sites consist of a special AT-rich sequence context in which one strand is well-mixed A's, T's, and C's, excluding G's (ATC sequences), which is typically found in clusters within different MARs. To determine the extent of conservation of the SATB1 gene among different species, we cloned a mouse homolog of the human STAB1 cDNA from a cDNA expression library of the mouse thymus, the tissue in which this protein is predominantly expressed. This mouse cDNA encodes a 764-amino-acid protein with a 98% homology in amino acid sequence to the human SATB1 originally cloned from testis. To characterize the DNA binding domain of this novel class of protein, we used the mouse SATB1 cDNA and delineated a 150-amino-acid polypeptide as the binding domain. This region confers full DNA binding activity, recognizes the specific sequence context, and makes direct contact with DNA at the same nucleotides as the whole protein. This DNA binding domain contains a novel DNA binding motif: when no more than 21 amino acids at either the N- or C-terminal end of the binding domain are deleted, the majority of the DNA binding activity is lost. The concomitant presence of both terminal sequences is mandatory for binding. These two terminal regions consist of hydrophilic amino acids and share homologous sequences that are different from those of any known DNA binding motifs. We propose that the DNA binding region of SATB1 extends its two terminal regions toward DNA to make direct contact with DNA.

Characterization of a dominant negativeC. elegansTwist mutant protein with implications for human Saethre-Chotzen syndrome

Development ◽  
2002 ◽  
Vol 129 (11) ◽  
pp. 2761-2772
Author(s):  
Ann K. Corsi ◽  
Thomas M. Brodigan ◽  
Erik M. Jorgensen ◽  
Michael Krause
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Binding Domain ◽  
Dominant Negative ◽  
Mutant Protein ◽  
Dna Binding Domain ◽  
Mesodermal Cell ◽  
C Elegans ◽  
Dna Binding Activity ◽  
A Charge

Twist is a transcription factor that is required for mesodermal cell fates in all animals studied to date. Mutations of this locus in humans have been identified as the cause of the craniofacial disorder Saethre-Chotzen syndrome. The Caenorhabditis elegans Twist homolog is required for the development of a subset of the mesoderm. A semidominant allele of the gene that codes for CeTwist, hlh-8, has defects that occur earlier in the mesodermal lineage than a previously studied null allele of the gene. The semidominant allele has a charge change (E29K) in the basic DNA-binding domain of CeTwist. Surprisingly, the mutant protein retains DNA-binding activity as both a homodimer and a heterodimer with its partner E/Daughterless (CeE/DA). However, the mutant protein blocks the activation of the promoter of a target gene. Therefore, the mutant CeTwist may cause cellular defects as a dominant negative protein by binding to target promoters as a homo- or heterodimer and then blocking transcription. Similar phenotypes as those caused by the E29K mutation were observed when amino acid substitutions in the DNA-binding domain that are associated with the human Saethre-Chotzen syndrome were engineered into the C. elegans protein. These data suggest that Saethre-Chotzen syndrome may be caused, in some cases, by dominant negative proteins, rather than by haploinsufficiency of the locus.

scholarly journals Regulation of the Transcription Factor YY1 in Mitosis through Phosphorylation of Its DNA-binding Domain

2009 ◽  
Vol 20 (22) ◽  
pp. 4766-4776 ◽  
Author(s):  
Raed Rizkallah ◽  
Myra M. Hurt
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Binding Activity ◽  
Binding Domain ◽  
Phosphorylation Sites ◽  
Dna Binding Domain ◽  
Daughter Cells ◽  
Dna Binding Activity ◽  
Yin Yang 1 ◽  
Transcription Factor Yy1

Yin-Yang 1 (YY1) is a ubiquitously expressed zinc finger transcription factor. It regulates a vast array of genes playing critical roles in development, differentiation, and cell cycle. Very little is known about the mechanisms that regulate the functions of YY1. It has long been proposed that YY1 is a phosphoprotein; however, a direct link between phosphorylation and the function of YY1 has never been proven. Investigation of the localization of YY1 during mitosis shows that it is distributed to the cytoplasm during prophase and remains excluded from DNA until early telophase. Immunostaining studies show that YY1 is distributed equally between daughter cells and rapidly associates with decondensing chromosomes in telophase, suggesting a role for YY1 in early marking of active and repressed genes. The exclusion of YY1 from DNA in prometaphase HeLa cells correlated with an increase in the phosphorylation of YY1 and loss of DNA-binding activity that can be reversed by dephosphorylation. We have mapped three phosphorylation sites on YY1 during mitosis and show that phosphorylation of two of these sites can abolish the DNA-binding activity of YY1. These results demonstrate a novel mechanism for the inactivation of YY1 through phosphorylation of its DNA-binding domain.

scholarly journals A novel DNA-binding motif in the nuclear matrix attachment DNA-binding protein SATB1.

1994 ◽  
Vol 14 (3) ◽  
pp. 1852-1860 ◽  
Author(s):  
K Nakagomi ◽  
Y Kohwi ◽  
L A Dickinson ◽  
T Kohwi-Shigematsu
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Contact ◽  
Binding Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Binding Motif ◽  
Dna Binding Domain ◽  
Sequence Context ◽  
Dna Binding Activity

The nuclear matrix attachment DNA (MAR) binding protein SATB1 is a sequence context-specific binding protein that binds in the minor groove, making virtually no contact with the DNA bases. The SATB1 binding sites consist of a special AT-rich sequence context in which one strand is well-mixed A's, T's, and C's, excluding G's (ATC sequences), which is typically found in clusters within different MARs. To determine the extent of conservation of the SATB1 gene among different species, we cloned a mouse homolog of the human STAB1 cDNA from a cDNA expression library of the mouse thymus, the tissue in which this protein is predominantly expressed. This mouse cDNA encodes a 764-amino-acid protein with a 98% homology in amino acid sequence to the human SATB1 originally cloned from testis. To characterize the DNA binding domain of this novel class of protein, we used the mouse SATB1 cDNA and delineated a 150-amino-acid polypeptide as the binding domain. This region confers full DNA binding activity, recognizes the specific sequence context, and makes direct contact with DNA at the same nucleotides as the whole protein. This DNA binding domain contains a novel DNA binding motif: when no more than 21 amino acids at either the N- or C-terminal end of the binding domain are deleted, the majority of the DNA binding activity is lost. The concomitant presence of both terminal sequences is mandatory for binding. These two terminal regions consist of hydrophilic amino acids and share homologous sequences that are different from those of any known DNA binding motifs. We propose that the DNA binding region of SATB1 extends its two terminal regions toward DNA to make direct contact with DNA.

scholarly journals Mutational analysis of the DNA-binding domain of the CYS3 regulatory protein of Neurospora crassa.

1991 ◽  
Vol 11 (9) ◽  
pp. 4356-4362 ◽  
Author(s):  
M N Kanaan ◽  
G A Marzluf
Keyword(s):  
Dna Binding ◽  
Neurospora Crassa ◽  
Mutational Analysis ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Dna Binding Domain ◽  
Dna Binding Activity

cys-3, the major sulfur regulatory gene of Neurospora crassa, activates the expression of a set of unlinked structural genes which encode sulfur catabolic-related enzymes during conditions of sulfur limitation. The cys-3 gene encodes a regulatory protein of 236 amino acid residues with a leucine zipper and an upstream basic region (the b-zip region) which together may constitute a DNA-binding domain. The b-zip region was expressed in Escherichia coli to examine its DNA-binding activity. The b-zip domain protein binds to the promoter region of the cys-3 gene itself and of cys-14, the sulfate permease II structural gene. A series of CYS3 mutant proteins obtained by site-directed mutagenesis were expressed and tested for function, dimer formation, and DNA-binding activity. The results demonstrate that the b-zip region of cys-3 is critical for both its function in vivo and specific DNA-binding in vitro.

Synthesis of an enzymatically active FLP recombinase in vitro: search for a DNA-binding domain

1989 ◽  
Vol 9 (5) ◽  
pp. 1987-1995
Author(s):  
A A Amin ◽  
P D Sadowski
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
In Vitro Transcription ◽  
Binding Activity ◽  
Binding Domain ◽  
Translation System ◽  
Dna Binding Domain ◽  
Amino Terminal ◽  
Dna Binding Activity

We have used an in vitro transcription and translation system to synthesize an enzymatically active FLP protein. The FLP mRNA synthesized in vitro by SP6 polymerase is translated efficiently in a rabbit reticulocyte lysate to produce enzymatically active FLP. Using this system, we assessed the effect of deletions and tetrapeptide insertions on the ability of the respective variant proteins synthesized in vitro to bind to the FLP recognition target site and to carry out excisive recombination. Deletions of as few as six amino acids from either the carboxy- or amino-terminal region of FLP resulted in loss of binding activity. Likewise, insertions at amino acid positions 79, 203, and 286 abolished DNA-binding activity. On the other hand, a protein with an insertion at amino acid 364 retained significant DNA-binding activity but had no detectable recombination activity. Also, an insertion at amino acid 115 had no measurable effect on DNA binding, but recombination was reduced by 95%. In addition, an insertion at amino acid 411 had no effect on DNA binding and recombination. On the basis of these results, we conclude that this approach fails to define a discrete DNA-binding domain. The possible reasons for this result are discussed.

Cysteine residues in the zinc finger and amino acids adjacent to the finger are necessary for DNA binding by the LAC9 regulatory protein of Kluyveromyces lactis

1988 ◽  
Vol 8 (9) ◽  
pp. 3726-3733
Author(s):  
M M Witte ◽  
R C Dickson
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Zinc Finger ◽  
Kluyveromyces Lactis ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Dna Binding Activity ◽  
Carboxyl Terminal

LAC9 is a positive regulatory protein that controls transcription of the lactose-galactose regulon in Kluyveromyces lactis. LAC9 is homologous to the GAL4 protein of Saccharomyces cerevisiae. Both proteins have a single "zinc finger" which plays a role in DNA binding. We previously hypothesized (L. V. Wray, M. M. Witte, R. C. Dickson, and M. I. Riley, Mol. Cell. Biol. 7:1111-1121, 1987) that the DNA-binding domain of the LAC9 protein consisted of the zinc finger as well as a region of amino acids on the carboxyl-terminal side of the zinc finger. In this study we used oligonucleotide-directed mutagenesis to introduce 13 single-amino-acid changes into the proposed DNA-binding domain of the LAC9 protein. Variant LAC9 proteins carrying an amino acid substitution in any one of the four highly conserved Cys residues of the zinc finger had reduced DNA-binding activity, suggesting that each Cys is necessary for DNA binding. Three of four variant LAC9 proteins with amino acid substitutions located on the carboxyl-terminal side of the zinc finger had reduced DNA-binding activity. These results support our hypothesis that the DNA-binding domain of the LAC9 protein is composed of the zinc finger and the adjacent region on the carboxyl side of the zinc finger, a region that has the potential to form an alpha-helix. Finally, LAC9 proteins containing His residues substituted for the conserved Cys residues also had reduced DNA-binding activity, indicating that His residues are not equivalent to Cys residues, as had been previously thought.

scholarly journals W194XProp1 and S156insTProp1, both of which have intact DNA-binding domain, show a different DNA-binding activity to the Prop1-binding element in human Pit-1 gene

2010 ◽  
Vol 323 (2) ◽  
pp. 167-171 ◽  
Author(s):  
Hiromi Shibahara ◽  
Nobuko Ikeshita ◽  
Yuka Sugiyama ◽  
Keizo Toda ◽  
Daisuke Yamamoto ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Dna Binding Activity

scholarly journals Surface Mutagenesis of the Bovine Papillomavirus E1 DNA Binding Domain Reveals Residues Required for Multiple Functions Related to DNA Replication

2006 ◽  
Vol 80 (15) ◽  
pp. 7491-7499 ◽  
Author(s):  
Stephen Schuck ◽  
Arne Stenlund
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Binding Activity ◽  
Binding Domain ◽  
Bovine Papillomavirus ◽  
Dna Binding Domain ◽  
Multifunctional Protein ◽  
Dna Binding Activity ◽  
E1 Protein ◽  
Complex Functions

ABSTRACT The E1 protein from papillomaviruses is a multifunctional protein with complex functions required for the initiation of viral DNA replication. We have performed a surface mutagenesis of the well-characterized E1 DNA binding domain (DBD). We demonstrate that substitutions of multiple residues on the surface of the E1 DBD are defective for DNA replication without affecting the DNA binding activity of the protein. The defects of individual substitutions include failure to form the double trimer that melts the ori and failure to form the double hexamer that unwinds the ori. These results demonstrate that the DBD plays an essential role in multiple DNA replication-related processes apart from DNA binding.

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