scholarly journals DNA-binding Activity of the ERp57 C-terminal Domain Is Related to a Redox-dependent Conformational Change

2007 ◽  
Vol 282 (14) ◽  
pp. 10299-10310 ◽  
Author(s):  
Caterina Grillo ◽  
Chiara D'Ambrosio ◽  
Valerio Consalvi ◽  
Roberta Chiaraluce ◽  
Andrea Scaloni ◽  
...  
Keyword(s):  
Dna Binding ◽  
Conformational Change ◽  
Binding Activity ◽  
Terminal Domain ◽  
Dna Binding Activity

Characterization of DNA-binding activity in the N-terminal domain of the DNA methyltransferase Dnmt3a

2011 ◽  
Vol 437 (1) ◽  
pp. 141-148 ◽  
Author(s):  
Isao Suetake ◽  
Yuichi Mishima ◽  
Hironobu Kimura ◽  
Young-Ho Lee ◽  
Yuji Goto ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Amino Acid ◽  
Dna Binding ◽  
Dna Methyltransferase ◽  
Basic Amino Acid ◽  
Binding Activity ◽  
Amino Acid Residues ◽  
Terminal Sequence ◽  
Terminal Domain ◽  
Dna Binding Activity

The Dnmt3a gene, which encodes de novo-type DNA methyltransferase, encodes two isoforms, full-length Dnmt3a and Dnmt3a2, which lacks the N-terminal 219 amino acid residues. We found that Dnmt3a showed higher DNA-binding and DNA-methylation activities than Dnmt3a2. The N-terminal sequence from residues 1 to 211 was able to bind to DNA, but could not distinguish methylated and unmethylated CpG. Its binding to DNA was inhibited by a major groove binder. Four basic amino acid residues, Lys51, Lys53, Arg177 and Arg179, in the N-terminal region were crucial for the DNA-binding activity. The ectopically expressed N-terminal sequence (residues 1–211) was localized in nuclei, whereas that harbouring mutations at the four basic amino acid residues was also detected in the cytoplasm. The DNA-methylation activity of Dnmt3a with the mutations was suppressed under physiological salt conditions, which is similar that of Dnmt3a2. In addition, ectopically expressed Dnmt3a with mutations, as well as Dnmt3a2, could not be retained efficiently in nuclei on salt extraction. We conclude that the DNA-binding activity of the N-terminal domain contributes to the DNA-methyltransferase activity via anchoring of the whole molecule to DNA under physiological salt conditions.

scholarly journals Domain organization of I kappa B alpha and sites of interaction with NF-kappa B p65.

1995 ◽  
Vol 15 (4) ◽  
pp. 2166-2172 ◽  
Author(s):  
E Jaffray ◽  
K M Wood ◽  
R T Hay
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Ankyrin Repeat ◽  
Inhibitor Protein ◽  
Nf Kappa B ◽  
I Kappa B Alpha ◽  
Terminal Domain ◽  
Dna Binding Activity ◽  
Protease Cleavage ◽  
Flexible Region

The DNA-binding activity and cellular distribution of the transcription factor NF-kappa B are regulated by the inhibitor protein I kappa B alpha. I kappa B alpha belongs to a family of proteins that contain multiple repeats of a 30- to 35-amino-acid sequence that was initially recognized in the erythrocyte protein ankyrin. Partial proteolysis has been used to study the domain structure of I kappa B alpha and to determine the sites at which it interacts with NF-kappa B. The data reveal a tripartite structure for I kappa B alpha in which a central, protease-resistant domain composed of five ankyrin repeats is flanked by an unstructured N-terminal extension and a compact, highly acidic C-terminal domain that is connected to the core of the protein by a flexible linker. Functional analysis of V8 cleavage products indicates that I kappa B alpha molecules lacking the N-terminal region can interact with and inhibit the DNA-binding activity of the p65 subunit of NF-kappa B, whereas I kappa B alpha molecules which lack both the N- and C-terminal regions are incapable of doing so. Protease cleavage of the N terminus of I kappa B alpha was unaffected by the presence of the p65 subunit of NF-kappa B, whereas bound p65 blocked cleavage of the flexible linker connecting the C-terminal domain to the ankyrin repeat-containing core of the protein. This linker region is highly conserved within the human, rat, pig, and chicken homologs of I kappa B alpha, and while it has been suggested that it represents a sixth ankyrin repeat, it is also likely that this is a flexible region of the protein that interacts with NF-kappa B.

scholarly journals DNA binding activity of Helicobacter pylori DnaB helicase: the role of the N-terminal domain in modulating DNA binding activities

FEBS Journal ◽  
2011 ◽  
Vol 279 (2) ◽  
pp. 234-250 ◽  
Author(s):  
Ram G. Nitharwal ◽  
Vijay Verma ◽  
Naidu Subbarao ◽  
Santanu Dasgupta ◽  
Nirupam R. Choudhury ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Dna Binding ◽  
Binding Activity ◽  
Terminal Domain ◽  
Dna Binding Activity ◽  
Dnab Helicase

The DNA-binding activity of mouse DNA methyltransferase 1 is regulated by phosphorylation with casein kinase 1δ/ε

2010 ◽  
Vol 427 (3) ◽  
pp. 489-497 ◽  
Author(s):  
Yasunori Sugiyama ◽  
Naoya Hatano ◽  
Noriyuki Sueyoshi ◽  
Isao Suetake ◽  
Shoji Tajima ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dna Binding ◽  
Dna Methyltransferase ◽  
Gel Filtration ◽  
Binding Activity ◽  
Casein Kinase ◽  
Protein Kinase Activity ◽  
Terminal Domain ◽  
Dna Binding Activity ◽  
Methylation Patterns

Dnmt1 (DNA methyltansferase 1) is an enzyme that recognizes and methylates hemimethylated DNA during DNA replication to maintain methylation patterns. The N-terminal region of Dnmt1 is known to form an independent domain structure that interacts with various regulatory proteins and DNA. In the present study, we investigated protein kinases in the mouse brain that could bind and phosphorylate the N-terminal regulatory domain of Dnmt1. A protein fraction containing protein kinase activity for phosphorylation of Dnmt1(1–290) was prepared using Dnmt1(1–290)-affinity, DNA–cellulose and gel-filtration columns. When the proteins in this fraction were analysed by LC-MS/MS (liquid chromatography tandem MS), CK1δ/ε (casein kinase 1δ/ε) was the only protein kinase identified. Recombinant CK1δ/ε was found to bind to the N-terminal domain of Dnmt1 and significantly phosphorylated this domain, especially in the presence of DNA. Phosphorylation analyses using various truncation and point mutants of Dnmt1 revealed that the major priming site phosphorylated by CK1δ/ε was Ser146, and that subsequent phosphorylation at other sites may occur after phosphorylation of the priming site. When the DNA-binding activity of phosphorylated Dnmt1 was compared with that of the non-phosphorylated form, phosphorylation of Dnmt1 was found to decrease the affinity for DNA. These results suggest that CK1δ/ε binds to and phosphorylates the N-terminal domain of Dnmt1 and regulates Dnmt1 function by reducing the DNA-binding activity.

scholarly journals Identification of residues in the human DNA repair enzyme HAP1 (Ref-1) that are essential for redox regulation of Jun DNA binding.

1993 ◽  
Vol 13 (9) ◽  
pp. 5370-5376 ◽  
Author(s):  
L J Walker ◽  
C N Robson ◽  
E Black ◽  
D Gillespie ◽  
I D Hickson
Keyword(s):  
Dna Repair ◽  
Dna Binding ◽  
Active Site ◽  
Cysteine Residue ◽  
Binding Activity ◽  
Repair Enzyme ◽  
Exonuclease Iii ◽  
Terminal Domain ◽  
Dna Binding Activity ◽  
Redox Active

The DNA binding activity of the c-jun proto-oncogene product is inhibited by oxidation of a specific cysteine residue (Cys-252) in the DNA binding domain. Jun protein inactivated by oxidation of this residue can be efficiently reactivated by a factor from human cell nuclei, recently identified as a DNA repair enzyme (termed HAP1 or Ref-1). The HAP1 protein consists of a core domain, which is highly conserved in a family of prokaryotic and eukaryotic DNA repair enzymes, and a 61-amino-acid N-terminal domain absent from bacterial homologs such as Escherichia coli exonuclease III. The eukaryote-specific N-terminal domain was dispensable for the DNA repair functions of the HAP1 protein but was essential for reactivation of the DNA binding activity of oxidized Jun protein. Consistent with this finding, exonuclease III protein could not reactive Jun. A minimal 26-residue region of the N-terminal domain proximal to the core of the HAP1 enzyme was required for redox activity. By site-directed mutagenesis, cysteine 65 was identified as the redox active site in the HAP1 enzyme. In addition, it is proposed that cysteine 93 interacts with the redox active site, probably via disulfide bridge formation. It is concluded that the HAP1 protein has evolved a novel redox activation domain capable of regulating the DNA binding activity of a proto-oncogene product which is not essential for its DNA repair functions. Identification of a putative active site cysteine residue should facilitate analysis of the mechanism by which the HAP1 protein may alter the redox state of a wide range of transcription factors.

Identification of residues in the human DNA repair enzyme HAP1 (Ref-1) that are essential for redox regulation of Jun DNA binding

1993 ◽  
Vol 13 (9) ◽  
pp. 5370-5376
Author(s):  
L J Walker ◽  
C N Robson ◽  
E Black ◽  
D Gillespie ◽  
I D Hickson
Keyword(s):  
Dna Repair ◽  
Dna Binding ◽  
Active Site ◽  
Cysteine Residue ◽  
Binding Activity ◽  
Repair Enzyme ◽  
Exonuclease Iii ◽  
Terminal Domain ◽  
Dna Binding Activity ◽  
Redox Active

The DNA binding activity of the c-jun proto-oncogene product is inhibited by oxidation of a specific cysteine residue (Cys-252) in the DNA binding domain. Jun protein inactivated by oxidation of this residue can be efficiently reactivated by a factor from human cell nuclei, recently identified as a DNA repair enzyme (termed HAP1 or Ref-1). The HAP1 protein consists of a core domain, which is highly conserved in a family of prokaryotic and eukaryotic DNA repair enzymes, and a 61-amino-acid N-terminal domain absent from bacterial homologs such as Escherichia coli exonuclease III. The eukaryote-specific N-terminal domain was dispensable for the DNA repair functions of the HAP1 protein but was essential for reactivation of the DNA binding activity of oxidized Jun protein. Consistent with this finding, exonuclease III protein could not reactive Jun. A minimal 26-residue region of the N-terminal domain proximal to the core of the HAP1 enzyme was required for redox activity. By site-directed mutagenesis, cysteine 65 was identified as the redox active site in the HAP1 enzyme. In addition, it is proposed that cysteine 93 interacts with the redox active site, probably via disulfide bridge formation. It is concluded that the HAP1 protein has evolved a novel redox activation domain capable of regulating the DNA binding activity of a proto-oncogene product which is not essential for its DNA repair functions. Identification of a putative active site cysteine residue should facilitate analysis of the mechanism by which the HAP1 protein may alter the redox state of a wide range of transcription factors.

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