scholarly journals iASPP mediates p53 selectivity through a modular mechanism fine-tuning DNA recognition

2019 ◽  
Vol 116 (35) ◽  
pp. 17470-17479 ◽  
Author(s):  
Shuo Chen ◽  
Jiale Wu ◽  
Shan Zhong ◽  
Yuntong Li ◽  
Ping Zhang ◽  
...  
Keyword(s):  
Dna Binding ◽  
Tumor Suppressors ◽  
Binding Sites ◽  
Human Cancer ◽  
Binding Domain ◽  
Human Papillomaviruses ◽  
Fine Tuning ◽  
Dna Binding Domain ◽  
Transcription Cofactor ◽  
Dna Binding Site

The most frequently mutated protein in human cancer is p53, a transcription factor (TF) that regulates myriad genes instrumental in diverse cellular outcomes including growth arrest and cell death. Cell context-dependent p53 modulation is critical for this life-or-death balance, yet remains incompletely understood. Here we identify sequence signatures enriched in genomic p53-binding sites modulated by the transcription cofactor iASPP. Moreover, our p53–iASPP crystal structure reveals that iASPP displaces the p53 L1 loop—which mediates sequence-specific interactions with the signature-corresponding base—without perturbing other DNA-recognizing modules of the p53 DNA-binding domain. A TF commonly uses multiple structural modules to recognize its cognate DNA, and thus this mechanism of a cofactor fine-tuning TF–DNA interactions through targeting a particular module is likely widespread. Previously, all tumor suppressors and oncoproteins that associate with the p53 DNA-binding domain—except the oncogenic E6 from human papillomaviruses (HPVs)—structurally cluster at the DNA-binding site of p53, complicating drug design. By contrast, iASPP inhibits p53 through a distinct surface overlapping the E6 footprint, opening prospects for p53-targeting precision medicine to improve cancer therapy.

scholarly journals Dissection of a carboxy-terminal region of the yeast regulatory protein RAP1 with effects on both transcriptional activation and silencing

1992 ◽  
Vol 12 (3) ◽  
pp. 1209-1217
Author(s):  
C F Hardy ◽  
D Balderes ◽  
D Shore
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Binding Protein ◽  
Binding Domain ◽  
Dominant Negative Effect ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Carboxy Terminal

RAP1 is an essential sequence-specific DNA-binding protein in Saccharomyces cerevisiae whose binding sites are found in a large number of promoters, where they function as upstream activation sites, and at the silencer elements of the HMR and HML mating-type loci, where they are important for repression. We have examined the involvement of specific regions of the RAP1 protein in both repression and activation of transcription by studying the properties of a series of hybrid proteins containing RAP1 sequences fused to the DNA-binding domain of the yeast protein GAL4 (amino acids 1 to 147). GAL4 DNA-binding domain/RAP1 hybrids containing only the carboxy-terminal third of the RAP1 protein (which lacks the RAP1 DNA-binding domain) function as transcriptional activators of a reporter gene containing upstream GAL4 binding sites. Expression of some hybrids from the strong ADH1 promoter on multicopy plasmids has a dominant negative effect on silencers, leading to either partial or complete derepression of normally silenced genes. The GAL4/RAP1 hybrids have different effects on wild-type and several mutated but functional silencers. Silencers lacking either an autonomously replicating sequence consensus element or the RAP1 binding site are strongly derepressed, whereas the wild-type silencer or a silencer containing a deletion of the binding site for another silencer-binding protein, ABF1, are only weakly affected by hybrid expression. By examining a series of GAL4 DNA-binding domain/RAP1 hybrids, we have mapped the transcriptional activation and derepression functions to specific parts of the RAP1 carboxy terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Dissection of a carboxy-terminal region of the yeast regulatory protein RAP1 with effects on both transcriptional activation and silencing.

1992 ◽  
Vol 12 (3) ◽  
pp. 1209-1217 ◽  
Author(s):  
C F Hardy ◽  
D Balderes ◽  
D Shore
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Binding Protein ◽  
Binding Domain ◽  
Dominant Negative Effect ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Carboxy Terminal

RAP1 is an essential sequence-specific DNA-binding protein in Saccharomyces cerevisiae whose binding sites are found in a large number of promoters, where they function as upstream activation sites, and at the silencer elements of the HMR and HML mating-type loci, where they are important for repression. We have examined the involvement of specific regions of the RAP1 protein in both repression and activation of transcription by studying the properties of a series of hybrid proteins containing RAP1 sequences fused to the DNA-binding domain of the yeast protein GAL4 (amino acids 1 to 147). GAL4 DNA-binding domain/RAP1 hybrids containing only the carboxy-terminal third of the RAP1 protein (which lacks the RAP1 DNA-binding domain) function as transcriptional activators of a reporter gene containing upstream GAL4 binding sites. Expression of some hybrids from the strong ADH1 promoter on multicopy plasmids has a dominant negative effect on silencers, leading to either partial or complete derepression of normally silenced genes. The GAL4/RAP1 hybrids have different effects on wild-type and several mutated but functional silencers. Silencers lacking either an autonomously replicating sequence consensus element or the RAP1 binding site are strongly derepressed, whereas the wild-type silencer or a silencer containing a deletion of the binding site for another silencer-binding protein, ABF1, are only weakly affected by hybrid expression. By examining a series of GAL4 DNA-binding domain/RAP1 hybrids, we have mapped the transcriptional activation and derepression functions to specific parts of the RAP1 carboxy terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystallization and Preliminary X-ray Analysis of the DNA Binding Domain of the Hin Recombinase with Its DNA Binding Site

1993 ◽  
Vol 232 (3) ◽  
pp. 982-986
Author(s):  
Jin-An Feng ◽  
Melvin Simon ◽  
David P. Mack ◽  
Peter B. Dervan ◽  
Reid C. Johnson ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
X Ray ◽  
Dna Binding Site ◽  
Hin Recombinase

scholarly journals The DNA Binding Domain of a Papillomavirus E2 Protein Programs a Chimeric Nuclease To Cleave Integrated Human Papillomavirus DNA in HeLa Cervical Carcinoma Cells

2007 ◽  
Vol 81 (12) ◽  
pp. 6254-6264 ◽  
Author(s):  
Stacy M. Horner ◽  
Daniel DiMaio
Keyword(s):  
Human Papillomavirus ◽  
Dna Binding ◽  
Binding Site ◽  
Cancer Cells ◽  
Binding Sites ◽  
Binding Domain ◽  
Carcinoma Cells ◽  
Dna Binding Domain ◽  
Viral Dna ◽  
Infected Cells

ABSTRACT Viral DNA binding proteins that direct nucleases or other protein domains to viral DNA in lytically or latently infected cells may provide a novel approach to modulate viral gene expression or replication. Cervical carcinogenesis is initiated by high-risk human papillomavirus (HPV) infection, and viral DNA persists in the cancer cells. To test whether a DNA binding domain of a papillomavirus protein can direct a nuclease domain to cleave HPV DNA in cervical cancer cells, we fused the DNA binding domain of the bovine papillomavirus type 1 (BPV1) E2 protein to the catalytic domain of the FokI restriction endonuclease, generating a BPV1 E2-FokI chimeric nuclease (BEF). BEF introduced DNA double-strand breaks on both sides of an E2 binding site in vitro, whereas DNA binding or catalytic mutants of BEF did not. After expression of BEF in HeLa cervical carcinoma cells, we detected cleavage at E2 binding sites in the integrated HPV18 DNA in these cells and also at an E2 binding site in cellular DNA. BEF-expressing cells underwent senescence, which required the DNA binding activity of BEF, but not its nuclease activity. These results demonstrate that DNA binding domains of viral proteins can target effector molecules to cognate binding sites in virally infected cells.

scholarly journals Crystal Structure of the DNA-Binding Domain of Human Herpesvirus 6A Immediate Early Protein 2

2017 ◽  
Vol 91 (21) ◽  
Author(s):  
Mitsuhiro Nishimura ◽  
Junjie Wang ◽  
Aika Wakata ◽  
Kento Sakamoto ◽  
Yasuko Mori
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Site ◽  
Human Herpesvirus ◽  
Binding Domain ◽  
Immediate Early ◽  
Dna Binding Domain ◽  
Factor Binding ◽  
Early Protein ◽  
Dna Binding Site

ABSTRACT Immediate early proteins of human herpesvirus 6A (HHV-6A) are expressed at the outset of lytic infection and thereby regulate viral gene expression. Immediate early protein 2 (IE2) of HHV-6A is a transactivator that drives a variety of promoters. The C-terminal region of HHV-6A IE2 is shared among IE2 homologs in betaherpesviruses and is involved in dimerization, DNA binding, and transcription factor binding. In this study, the structure of the IE2 C-terminal domain (IE2-CTD) was determined by X-ray crystallography at a resolution of 2.5 Å. IE2-CTD forms a homodimer stabilized by a β-barrel core with two interchanging long loops. Unexpectedly, the core structure resembles those of the gammaherpesvirus factors EBNA1 of Epstein-Barr virus and LANA of Kaposi sarcoma-associated herpesvirus, but the interchanging loops are longer in IE2-CTD and form helix-turn-helix (HTH)-like motifs at their tips. The HTH and surrounding α-helices form a structural feature specific to the IE2 group. The apparent DNA-binding site (based on structural similarity with EBNA1 and LANA) resides on the opposite side of the HTH-like motifs, surrounded by positive electrostatic potential. Mapping analysis of conserved residues on the three-dimensional structure delineated a potential factor-binding site adjacent to the expected DNA-binding site. The predicted bi- or tripartite functional sites indicate a role for IE2-CTD as an adapter connecting the promoter and transcriptional factors that drive gene expression. IMPORTANCE Human herpesvirus 6A (HHV-6A) and HHV-6B belong to betaherpesvirus subfamily. Both viruses establish lifelong latency after primary infection, and their reactivation poses a significant risk to immunocompromised patients. Immediate early protein 2 (IE2) of HHV-6A and HHV-6B is a transactivator that triggers viral replication and contains a DNA-binding domain shared with other betaherpesviruses such as human herpesvirus 7 and human cytomegalovirus. In this study, an atomic structure of the DNA-binding domain of HHV-6A IE2 was determined and analyzed, enabling a structure-based understanding of the functions of IE2, specifically DNA recognition and interaction with transcription factors. Unexpectedly, the dimeric core resembles the DNA-binding domain of transcription regulators from gammaherpesviruses, showing structural conservation as a DNA-binding domain but with its own unique structural features. These findings facilitate further characterization of this key viral transactivator.

scholarly journals Xbp1, a stress-induced transcriptional repressor of the Saccharomyces cerevisiae Swi4/Mbp1 family.

1997 ◽  
Vol 17 (11) ◽  
pp. 6491-6501 ◽  
Author(s):  
B Mai ◽  
L Breeden
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Binding ◽  
Binding Site ◽  
Binding Sites ◽  
Binding Protein ◽  
Transcriptional Repressor ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
G1 Cyclin

We have identified Xbp1 (XhoI site-binding protein 1) as a new DNA-binding protein with homology to the DNA-binding domain of the Saccharomyces cerevisiae cell cycle regulating transcription factors Swi4 and Mbp1. The DNA recognition sequence was determined by random oligonucleotide selection and confirmed by gel retardation and footprint analyses. The consensus binding site of Xbp1, GcCTCGA(G/A)G(C/A)g(a/g), is a palindromic sequence, with an XhoI restriction enzyme recognition site at its center. This Xbpl binding site is similar to Swi4/Swi6 and Mbp1/Swi6 binding sites but shows a clear difference from these elements in one of the central core bases. There are binding sites for Xbp1 in the G1 cyclin promoter (CLN1), but they are distinct from the Swi4/Swi6 binding sites in CLN1, and Xbp1 will not bind to Swi4/Swi6 or Mbp1/Swi6 binding sites. The XBP1 promoter contains several stress-regulated elements, and its expression is induced by heat shock, high osmolarity, oxidative stress, DNA damage, and glucose starvation. When fused to the LexA DNA-binding domain, Xbp1 acts as transcriptional repressor, defining it as the first repressor in the Swi4/Mbp1 family and the first potential negative regulator of transcription induced by stress. Overexpression of XBP1 results in a slow-growth phenotype, lengthening of G1, an increase in cell volume, and a repression of G1 cyclin expression. These observations suggest that Xbp1 may contribute to the repression of specific transcripts and cause a transient cell cycle delay under stress conditions.

scholarly journals Mapping protein binding stability on nucleosomal DNA by single-molecule approach

2014 ◽  
Vol 70 (a1) ◽  
pp. C111-C111
Author(s):  
Jianshi Jin ◽  
Teng-fei Lian ◽  
Xiaoliang Xie ◽  
Xiao-Dong Su
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Conformational Changes ◽  
Protein Interaction ◽  
Binding Sites ◽  
Binding Domain ◽  
Shielding Effect ◽  
Dna Binding Domain ◽  
Nucleosomal Dna ◽  
Dna Protein Interaction

The conformation of nucleosomal DNA is significantly different from that of a canonical B-form double stranded DNA (dsDNA), and is generally regarded to be less flexible and less accessible than free dsDNA due to the tight association of histone cores. Previous studies have demonstrated that the key mechanism involved in nucleosomal DNA-protein interaction is the protein accessibility to the DNA binding site. In this work, we used single molecule assays to measure the stability of two transcriptional factors (glucocorticoid receptor DNA binding domain (GRDBD) and estrogen receptor DNA-binding domain (ERDBD)) bound to their binding sites on different positions of the nucleosomal DNA. Interestingly, the results demonstrated that the nucleosomal DNA-GRDBD binding is not always consistent with the histone shielding effect, but adjusted by additional structural changes. Furthermore, the changes of these DNA-GRDBD interaction profiles were confirmed using molecular modeling and docking approaches based on their crystal structures. Very differently, ERDBD essentially is unable to bind to the nucleosomal DNA anywhere including the unblocked positions. We thus have concluded that the nucleosomal DNA-protein interaction is regulated not only by the histone shielding of the DNA binding sites, but also by the conformational changes of the nucleosomal DNA.

scholarly journals High-resolution footprints of the DNA-binding domain of Epstein-Barr virus nuclear antigen 1.

1989 ◽  
Vol 9 (6) ◽  
pp. 2738-2742 ◽  
Author(s):  
A S Kimball ◽  
G Milman ◽  
T D Tullius
Keyword(s):  
Dna Binding ◽  
Epstein Barr Virus ◽  
Binding Domain ◽  
Nuclear Antigen ◽  
Dna Binding Domain ◽  
Major Groove ◽  
Barr Virus ◽  
Dna Binding Site ◽  
Epstein Barr ◽  
Hydroxyl Radical Footprinting

The DNA-binding domain of Epstein-Barr virus nuclear antigen 1 was found by hydroxyl radical footprinting to protect backbone positions on one side of its DNA-binding site. The guanines contacted in the major groove by the DNA-binding domain of Epstein-Barr virus nuclear antigen 1 were identified by methylation protection. No difference was found in the interaction of the DNA-binding domain of Epstein-Barr virus nuclear antigen 1 with tandemly repeated and overlapping binding sites.

scholarly journals Functionally distinct roles for T and Tbx6 during mouse development

Biology Open ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. bio054692
Author(s):  
Amy K. Wehn ◽  
Deborah R. Farkas ◽  
Carly E. Sedlock ◽  
Dibya Subedi ◽  
Deborah L. Chapman
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Transcriptional Activity ◽  
Es Cells ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Mouse Development ◽  
T Box

ABSTRACTThe mouse T-box transcription factors T and Tbx6 are co-expressed in the primitive streak and have unique domains of expression; T is expressed in the notochord, while Tbx6 is expressed in the presomitic mesoderm. T-box factors are related through a shared DNA binding domain, the T-domain, and can therefore bind to similar DNA sequences at least in vitro. We investigated the functional similarities and differences of T and Tbx6 DNA binding and transcriptional activity in vitro and their interaction genetically in vivo. We show that at one target, Dll1, the T-domains of T and Tbx6 have different affinities for the binding sites present in the mesoderm enhancer. We further show using in vitro assays that T and Tbx6 differentially affect transcription with Tbx6 activating expression tenfold higher than T, that T and Tbx6 can compete at target gene enhancers, and that this competition requires a functional DNA binding domain. Next, we addressed whether T and Tbx6 can compete in vivo. First, we generated embryos that express Tbx6 at greater than wild-type levels embryos and show that these embryos have short tails, resembling the T heterozygous phenotype. Next, using the dominant-negative TWis allele, we show that Tbx6+/− TWis/+ embryos share similarities with embryos homozygous for the Tbx6 hypomorphic allele rib-vertebrae, specifically fusions of several ribs and malformation of some vertebrae. Finally, we tested whether Tbx6 can functionally replace T using a knockin approach, which resulted in severe T null-like phenotypes in chimeric embryos generated with ES cells heterozygous for a Tbx6 knockin at the T locus. Altogether, our results of differences in affinity for DNA binding sites and transcriptional activity for T and Tbx6 provide a potential mechanism for the failure of Tbx6 to functionally replace T and possible competition phenotypes in vivo.

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