scholarly journals Mutagenesis of the HMGB (high-mobility group B) protein Cmb1 (cytosine-mismatch binding 1) of Schizosaccharomyces pombe: effects on recognition of DNA mismatches and damage

2003 ◽  
Vol 372 (2) ◽  
pp. 651-660 ◽  
Author(s):  
Christophe KUNZ ◽  
Karin ZURBRIGGEN ◽  
Oliver FLECK
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Schizosaccharomyces Pombe ◽  
Excision Repair ◽  
Specific Binding ◽  
High Mobility ◽  
High Mobility Group ◽  
Dna Mismatches ◽  
Terminal Amino ◽  
Group B

Cmb1 (cytosine-mismatch binding 1) is a high-mobility group (HMG) protein of Schizosaccharomyces pombe, which consists of 223 amino acids and has a single HMG domain at the C-terminal end. We have created several mutant and deletion forms of the Cmb1 protein and studied the effects on general DNA binding and specific binding to DNA mismatches and damaged DNA. Cmb1Δ41 (i.e. Cmb1 from which the 41 N-terminal amino acids have been deleted) bound specifically to cytosine-containing mismatches, to the cisplatin-induced intrastrand cross-links cis-GG and cis-AG and to an O6-methylguanine lesion. DNA binding was not affected when the 45 N-terminal amino acids were deleted, but was abolished in the absence of the 50 N-terminal amino acids, and was reduced when Cmb1 was truncated by between five and eleven C-terminal amino acids. Cmb1, both with and without the C-terminal truncations, retained its DNA binding affinity after heating at 95 °C. The cmb1 gene was induced when S. pombe cells were treated with cisplatin. Mitotic mutation rates were increased in a S. pombe cmb1 null mutant and in a cmb1-(1–212) mutant, which encodes a Cmb1 protein lacking the 11 C-terminal amino acids. We conclude that mutation avoidance by Cmb1 is distinct from Msh2-dependent mismatch repair, but related to nucleotide excision repair.

scholarly journals Identification of two Sox17 messenger RNA isoforms, with and without the high mobility group box region, and their differential expression in mouse spermatogenesis.

1996 ◽  
Vol 133 (3) ◽  
pp. 667-681 ◽  
Author(s):  
Y Kanai ◽  
M Kanai-Azuma ◽  
T Noce ◽  
T C Saido ◽  
T Shiroishi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Reporter Gene ◽  
Germ Cells ◽  
Messenger Rna ◽  
High Mobility ◽  
The Other ◽  
High Mobility Group ◽  
Luciferase Reporter ◽  
Mrna Isoforms

The different mRNA isoforms of the mouse Sox17 gene were isolated from adult mouse testis cDNAs. One form (referred to as form Sox17) encodes an Sry-related protein of 419 amino acids containing a single high mobility group box near the NH2-terminus, while the other form (referred to as form t-Sox17) shows a unique mRNA isoform of the Sox17 gene with a partial deletion of the HMG box region. Analysis of genomic DNA revealed that these two isoforms were produced at least by alternative splicing of the exon corresponding to the 5' untranslated region and NH2-terminal 102 amino acids. RNA analyses in the testis revealed that form Sox17 began at the pachytene spermatocyte stage and was highly accumulated in round spermatids. Protein analyses revealed that t-Sox17 isoforms, as well as Sox17 isoforms, were translated into the protein products in the testis, although the amount of t-Sox17 products is lower in comparison to the high accumulation of t-Sox17 mRNA. By the electrophoretic mobility-shift assay and the random selection assay using recombinant Sox17 and t-Sox17 proteins, Sox17 protein is a DNA-binding protein with a similar sequence specificity to Sry and the other members of Sox family proteins, while t-Sox17 shows no apparent DNA-binding activity. Moreover, by a cotransfection experiment using a luciferase reporter gene, Sox17 could stimulate transcription through its binding site, but t-Sox17 had little effect on reporter gene expression. Thus, these findings suggest that Sox17 may function as a transcriptional activator in the premeiotic germ cells, and that a splicing switch into t-Sox17 may lead to the loss of its function in the postmeiotic germ cells.

scholarly journals Efficient Specific DNA Binding by p53 Requires both Its Central and C-Terminal Domains as Revealed by Studies with High-Mobility Group 1 Protein

2002 ◽  
Vol 22 (19) ◽  
pp. 6797-6808 ◽  
Author(s):  
Kristine McKinney ◽  
Carol Prives
Keyword(s):  
Dna Binding ◽  
Specific Binding ◽  
High Mobility ◽  
Dna Structure ◽  
High Mobility Group ◽  
Chromosomal Protein ◽  
Linear Dna ◽  
Binding Domains ◽  
C Terminus ◽  
Group 1

ABSTRACT The nonhistone chromosomal protein high-mobility group 1 protein (HMG-1/HMGB1) can serve as an activator of p53 sequence-specific DNA binding (L. Jayaraman, N. C. Moorthy, K. G. Murthy, J. L. Manley, M. Bustin, and C. Prives, Genes Dev. 12:462-472, 1998). HMGB1 is capable of interacting with DNA in a non-sequence-specific manner and causes a significant bend in the DNA helix. Since p53 requires a significant bend in the target site, we examined whether DNA bending by HMGB1 may be involved in its enhancement of p53 sequence-specific binding. Accordingly, a 66-bp oligonucleonucleotide containing a p53 binding site was locked in a bent conformation by ligating its ends to form a microcircle. Indeed, p53 had a dramatically greater affinity for the microcircle than for the linear 66-bp DNA. Moreover, HMGB1 augmented binding to the linear DNA but not to the microcircle, suggesting that HMGB1 works by providing prebent DNA to p53. p53 contains a central core sequence-specific DNA binding region and a C-terminal region that recognizes various forms of DNA non-sequence specifically. The p53 C terminus has also been shown to serve as an autoinhibitor of core-DNA interactions. Remarkably, although the p53 C terminus inhibited p53 binding to the linear DNA, it was required for the increased affinity of p53 for the microcircle. Thus, depending on the DNA structure, the p53 C terminus can serve as a negative or a positive regulator of p53 binding to the same sequence and length of DNA. We propose that both DNA binding domains of p53 cooperate to recognize sequence and structure in genomic DNA and that HMGB1 can help to provide the optimal DNA structure for p53.

scholarly journals Epitope Mapping of Monoclonal Antibodies to Calreticulin Reveals That Charged Amino Acids Are Essential for Antibody Binding

Antibodies ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 31
Author(s):  
Ann Christina Bergmann ◽  
Cecilie Kyllesbech ◽  
Rimantas Slibinskas ◽  
Evaldas Ciplys ◽  
Peter Højrup ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Epitope Mapping ◽  
Biological Function ◽  
Specific Binding ◽  
Enzyme Linked Immunosorbent Assay ◽  
Potential Therapeutic Target ◽  
Charged Amino Acids ◽  
Chaperone Protein ◽  
Terminal Amino

Calreticulin is a chaperone protein, which is associated with myeloproliferative diseases. In this study, we used resin-bound peptides to characterize two monoclonal antibodies (mAbs) directed to calreticulin, mAb FMC 75 and mAb 16, which both have significantly contributed to understanding the biological function of calreticulin. The antigenicity of the resin-bound peptides was determined by modified enzyme-linked immunosorbent assay. Specific binding was determined to an 8-mer epitope located in the N-terminal (amino acids 34–41) and to a 12-mer peptide located in the C-terminal (amino acids 362–373). Using truncated peptides, the epitopes were identified as TSRWIESK and DEEQRLKEEED for mAb FMC 75 and mAb 16, respectively, where, especially the charged amino acids, were found to have a central role for a stable binding. Further studies indicated that the epitope of mAb FMC 75 is assessable in the oligomeric structure of calreticulin, making this epitope a potential therapeutic target.

Comparative analysis of the influence of the high-mobility group box 1 protein on DNA binding and transcriptional activation by the androgen, glucocorticoid, progesterone and mineralocorticoid receptors

2001 ◽  
Vol 361 (1) ◽  
pp. 97-103 ◽  
Author(s):  
Guy VERRIJDT ◽  
Annemie HAELENS ◽  
Erik SCHOENMAKERS ◽  
Wilfried ROMBAUTS ◽  
Frank CLAESSENS
Keyword(s):  
Androgen Receptor ◽  
Comparative Analysis ◽  
Dna Binding ◽  
Mineralocorticoid Receptor ◽  
Hormone Receptors ◽  
Steroid Hormone ◽  
High Mobility ◽  
Steroid Hormone Receptors ◽  
High Mobility Group ◽  
Mineralocorticoid Receptors

We performed a comparative analysis of the effect of high-mobility group box protein 1 (HMGB1) on DNA binding by the DNA-binding domains (DBDs) of the androgen, glucocorticoid, progesterone and mineralocorticoid receptors. The affinity of the DBDs of the different receptors for the tyrosine aminotransferase glucocorticoid response element, a classical high-affinity binding element, was augmented up to 7-fold by HMGB1. We found no major differences in the effects of HMGB1 on DNA binding between the different steroid hormone receptors. In transient transfection assays, however, HMGB1 significantly enhances the activity of the glucocorticoid and progesterone receptors but not the androgen or mineralocorticoid receptor. We also investigated the effect of HMGB1 on the binding of the androgen receptor DBD to a subclass of directly repeated response elements that is recognized exclusively by the androgen receptor and not by the glucocorticoid, progesterone or mineralocorticoid receptor. Surprisingly, a deletion of 26 amino acid residues from the C-terminal extension of the androgen receptor DBD does not influence DNA binding but destroys its sensitivity to HMGB1. Deletion of the corresponding fragment in the DBDs of the glucocorticoid, progesterone and mineralocorticoid receptor destroyed their DNA binding. This 26-residue fragment is therefore essential for the influence of HMGB1 on DNA recognition by all steroid hormone receptors that were tested. However, it is dispensable for DNA binding by the androgen receptor.

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