scholarly journals DNA-Binding and -Bending Activities of SAP30L and SAP30 Are Mediated by a Zinc-Dependent Module and Monophosphoinositides

2008 ◽  
Vol 29 (2) ◽  
pp. 342-356 ◽  
Author(s):  
Keijo M. Viiri ◽  
Janne Jänis ◽  
Trevor Siggers ◽  
Taisto Y. K. Heinonen ◽  
Jarkko Valjakka ◽  
...  
Keyword(s):  
Dna Binding ◽  
Sequence Motif ◽  
Naked Dna ◽  
Corepressor Complex ◽  
Protein Dna Interactions ◽  
Core Histones ◽  
Associated Proteins ◽  
Localization Signal ◽  
Linker Molecules ◽  
Dna Binding And Bending

ABSTRACT Deacetylation of histones is carried out by a corepressor complex in which Sin3A is an essential scaffold protein. Two proteins in this complex, the Sin3A-associated proteins SAP30L and SAP30, have previously been suggested to function as linker molecules between various corepressors. In this report, we demonstrate new functions for human SAP30L and SAP30 by showing that they can associate directly with core histones as well as naked DNA. A zinc-coordinating structure is necessary for DNA binding, one consequence of which is bending of the DNA. We provide evidence that a sequence motif previously shown to be a nuclear localization signal is also a phosphatidylinositol (PI)-binding element and that binding of specific nuclear monophosphoinositides regulates DNA binding and chromatin association of SAP30L. PI binding also decreases the repression activity of SAP30L and affects its translocation from the nucleus to the cytoplasm. Our results suggest that SAP30L and SAP30 play active roles in recruitment of deacetylating enzymes to nucleosomes, and mediate key protein-protein and protein-DNA interactions involved in chromatin remodeling and transcription.

scholarly journals Determinants of HMGB Proteins Required To Promote RAG1/2-Recombination Signal Sequence Complex Assembly and Catalysis during V(D)J Recombination

2005 ◽  
Vol 25 (11) ◽  
pp. 4413-4425 ◽  
Author(s):  
Yan Dai ◽  
Ben Wong ◽  
Yi-Meng Yen ◽  
Marjorie A. Oettinger ◽  
Jongbum Kwon ◽  
...  
Keyword(s):  
Dna Binding ◽  
Signal Sequence ◽  
Recombination Signal Sequence ◽  
Dna Complexes ◽  
Recombination Signal ◽  
Dna Binding Domains ◽  
Naked Dna ◽  
Binding Domains ◽  
Dna Binding And Bending ◽  
Hmgb Proteins

ABSTRACT Efficient assembly of RAG1/2-recombination signal sequence (RSS) DNA complexes that are competent for V(D)J cleavage requires the presence of the nonspecific DNA binding and bending protein HMGB1 or HMGB2. We find that either of the two minimal DNA binding domains of HMGB1 is effective in assembling RAG1/2-RSS complexes on naked DNA and stimulating V(D)J cleavage but that both domains are required for efficient activity when the RSS is incorporated into a nucleosome. The single-domain HMGB protein from Saccharomyces cerevisiae, Nhp6A, efficiently assembles RAG1/2 complexes on naked DNA; however, these complexes are minimally competent for V(D)J cleavage. Nhp6A forms much more stable DNA complexes than HMGB1, and a variety of mutations that destabilize Nhp6A binding to bent microcircular DNA promote increased V(D)J cleavage. One of the two DNA bending wedges on Nhp6A and the analogous phenylalanine wedge at the DNA exit site of HMGB1 domain A were found to be essential for promoting RAG1/2-RSS complex formation. Because the phenylalanine wedge is required for specific recognition of DNA kinks, we propose that HMGB proteins facilitate RAG1/2-RSS interactions by recognizing a distorted DNA structure induced by RAG1/2 binding. The resulting complex must be sufficiently dynamic to enable the series of RAG1/2-mediated chemical reactions on the DNA.

scholarly journals An improved bind-n-seq strategy to determine protein-DNA interactions validated using the bacterial transcriptional regulator YipR

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Shi-qi An ◽  
Miguel A. Valvano ◽  
Yan-hua Yu ◽  
Jeremy S. Webb ◽  
Guillermo Lopez Campos
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Computational Algorithm ◽  
Dna Recombination ◽  
Transcriptional Regulator ◽  
Sequence Motif ◽  
Dependent Manner ◽  
Dna Interactions ◽  
Promoter Regions ◽  
Protein Dna Interactions

Abstract Background Interactions between transcription factors and DNA lie at the centre of many biological processes including DNA recombination, replication, repair and transcription. Most bacteria encode diverse proteins that act as transcription factors to regulate various traits. Several technologies for identifying protein–DNA interactions at the genomic level have been developed. Bind-n-seq is a high-throughput in vitro method first deployed to analyse DNA interactions associated with eukaryotic zinc-finger proteins. The method has three steps (i) binding protein to a randomised oligonucleotide DNA target library, (ii) deep sequencing of bound oligonucleotides, and (iii) a computational algorithm to define motifs among the sequences. The classical Bind-n-seq strategy suffers from several limitations including a lengthy wet laboratory protocol and a computational algorithm that is difficult to use. We introduce here an improved, rapid, and simplified Bind-n-seq protocol coupled with a user-friendly downstream data analysis and handling algorithm, which has been optimized for bacterial target proteins. We validate this new protocol by showing the successful characterisation of the DNA-binding specificities of YipR (YajQ interacting protein regulator), a well-known transcriptional regulator of virulence genes in the bacterial phytopathogen Xanthomonas campestris pv. campestris (Xcc). Results The improved Bind-n-seq approach identified several DNA binding motif sequences for YipR, in particular the CCCTCTC motif, which were located in the promoter regions of 1320 Xcc genes. Informatics analysis revealed that many of these genes regulate functions associated with virulence, motility, and biofilm formation and included genes previously found involved in virulence. Additionally, electromobility shift assays show that YipR binds to the promoter region of XC_2633 in a CCCTCTC motif-dependent manner. Conclusion We present a new and rapid Bind-n-seq protocol that should be useful to investigate DNA-binding proteins in bacteria. The analysis of YipR DNA binding using this protocol identifies a novel DNA sequence motif in the promoter regions of target genes that define the YipR regulon.

scholarly journals Facilitated Dissociation of Nucleoid Associated Proteins from DNA in the Bacterial Confinement

2021 ◽  
Author(s):  
Zafer Koşar ◽  
A. Göktuĝ Attar ◽  
Aykut Erbaş
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Experimental Studies ◽  
Dual Purpose ◽  
E Coli ◽  
Protein Dna Interactions ◽  
Factor For Inversion Stimulation ◽  
Associated Proteins ◽  
Order Of Magnitude ◽  
Single Molecule Studies

Transcription machinery ultimately depends on the temporal formation of protein-DNA complexes. Recent experimental studies demonstrate that residence time (i.e., inverse off-rate) of a transcription factor protein can be a contributor to the functional diversity of the protein. In the meantime, single-molecule experiments showed that the off-rates of a wide array of DNA-binding proteins accelerate as the bulk concentration of the protein increases via a concentration-dependent mechanism (i.e., facilitated dissociation, FD). In this study, inspired by the previous single-molecule studies on the factor for inversion stimulation (Fis) protein of E. coli, which is a dual-purpose protein with a diverse functionality, we model the unbinding of Fis from specific bindings sites along a high-molecular-weight circular DNA in a cylindrical structure mimicking the cellular confinement of chromosome. Our simulations show that FD of Fis can well occur in confinement at physiological concentrations. Particularly, when nutrient-rich conditions are emulated with Fis concentrations around micromolar levels, the off-rates increase one order of magnitude compared to the lower Fis levels. However, Fis significantly changes the chromosome structure at higher concentrations by forming dense protein clusters bridging specific sites and juxtaposing remote DNA segments. As a result, at the physiologically observed maximum levels of Fis, the off-rates significantly slow down. Overall, our results indicate that cellular-concentration levels of a structural DNA-binding protein is intermingled with the genome architecture and DNA residence times, thereby providing a basis for understanding the complex effects of dynamic protein-DNA interactions on gene regulation.

scholarly journals Genomic analysis of DNA binding and gene regulation by homologous nucleoid-associated proteins IHF and HU in Escherichia coli K12

2011 ◽  
Vol 40 (8) ◽  
pp. 3524-3537 ◽  
Author(s):  
Ana I. Prieto ◽  
Christina Kahramanoglou ◽  
Ruhi M. Ali ◽  
Gillian M. Fraser ◽  
Aswin S. N. Seshasayee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Regulation ◽  
Dna Binding ◽  
Genomic Analysis ◽  
Escherichia Coli K12 ◽  
Associated Proteins

Fluorescence Study of DNA Binding and Bending byEcoRI DNA Methyltransferase

2006 ◽  
Vol 110 (39) ◽  
pp. 19647-19651
Author(s):  
Baocheng Ma ◽  
Jun Wang ◽  
Xiaohong Fang
Keyword(s):  
Dna Binding ◽  
Dna Methyltransferase ◽  
Fluorescence Study ◽  
Dna Binding And Bending

Identification of single-stranded-DNA-binding proteins that interact with muscle gene elements

1991 ◽  
Vol 11 (4) ◽  
pp. 1944-1953
Author(s):  
I M Santoro ◽  
T M Yi ◽  
K Walsh
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Regulatory Element ◽  
Dna Binding Proteins ◽  
Binding Activity ◽  
Sequence Motif ◽  
Mobility Shift ◽  
Specific Sequence ◽  
Single Stranded Dna ◽  
Muscle Gene

A sequence-specific DNA-binding protein from skeletal-muscle extracts that binds to probes of three muscle gene DNA elements is identified. This protein, referred to as muscle factor 3, forms the predominant nucleoprotein complex with the MCAT gene sequence motif in an electrophoretic mobility shift assay. This protein also binds to the skeletal actin muscle regulatory element, which contains the conserved CArG motif, and to a creatine kinase enhancer probe, which contains the E-box motif, a MyoD-binding site. Muscle factor 3 has a potent sequence-specific, single-stranded-DNA-binding activity. The specificity of this interaction was demonstrated by sequence-specific competition and by mutations that diminished or eliminated detectable complex formation. MyoD, a myogenic determination factor that is distinct from muscle factor 3, also bound to single-stranded-DNA probes in a sequence-specific manner, but other transcription factors did not. Multiple copies of the MCAT motif activated the expression of a heterologous promoter, and a mutation that eliminated expression was correlated with diminished factor binding. Muscle factor 3 and MyoD may be members of a class of DNA-binding proteins that modulate gene expression by their abilities to recognize DNA with unusual secondary structure in addition to specific sequence.

N-terminal DNA-binding domains contribute to differential DNA-binding specificities of NF-kappa B p50 and p65

1993 ◽  
Vol 13 (2) ◽  
pp. 852-860
Author(s):  
M B Toledano ◽  
D Ghosh ◽  
F Trinh ◽  
W J Leonard
Keyword(s):  
Dna Binding ◽  
Point Mutations ◽  
Terminal Region ◽  
Sequence Motif ◽  
Structural Determinants ◽  
Nf Kappa B ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Dna Binding Specificity

We previously reported that either oxidation or alkylation of NF-kappa B in vitro abrogates DNA binding. We used this phenomenon to help elucidate structural determinants of NF-kappa B binding. We now demonstrate that Cys-62 of NF-kappa B p50 mediates the redox effect and lies within an N-terminal region required for DNA binding but not for dimerization. Several point mutations in this region confer a transdominant negative binding phenotype to p50. The region is highly conserved in all Rel family proteins, and we have determined that it is also critical for DNA binding of NF-kappa B p65. Replacement of the N-terminal region of p65 with the corresponding region from p50 changes its DNA-binding specificity towards that of p50. These data suggest that the N-terminal regions of p50 and p65 are critical for DNA binding and help determine the DNA-binding specificities of p50 and p65. We have defined within the N-terminal region a sequence motif, R(F/G)(R/K)YXCE, which is present in Rel family proteins and also in zinc finger proteins capable of binding to kappa B sites. The potential significance of this finding is discussed.

scholarly journals Identification of single-stranded-DNA-binding proteins that interact with muscle gene elements.

1991 ◽  
Vol 11 (4) ◽  
pp. 1944-1953 ◽  
Author(s):  
I M Santoro ◽  
T M Yi ◽  
K Walsh
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Regulatory Element ◽  
Dna Binding Proteins ◽  
Binding Activity ◽  
Sequence Motif ◽  
Mobility Shift ◽  
Specific Sequence ◽  
Single Stranded Dna ◽  
Muscle Gene

A sequence-specific DNA-binding protein from skeletal-muscle extracts that binds to probes of three muscle gene DNA elements is identified. This protein, referred to as muscle factor 3, forms the predominant nucleoprotein complex with the MCAT gene sequence motif in an electrophoretic mobility shift assay. This protein also binds to the skeletal actin muscle regulatory element, which contains the conserved CArG motif, and to a creatine kinase enhancer probe, which contains the E-box motif, a MyoD-binding site. Muscle factor 3 has a potent sequence-specific, single-stranded-DNA-binding activity. The specificity of this interaction was demonstrated by sequence-specific competition and by mutations that diminished or eliminated detectable complex formation. MyoD, a myogenic determination factor that is distinct from muscle factor 3, also bound to single-stranded-DNA probes in a sequence-specific manner, but other transcription factors did not. Multiple copies of the MCAT motif activated the expression of a heterologous promoter, and a mutation that eliminated expression was correlated with diminished factor binding. Muscle factor 3 and MyoD may be members of a class of DNA-binding proteins that modulate gene expression by their abilities to recognize DNA with unusual secondary structure in addition to specific sequence.

scholarly journals DNA Binding and Bending by the Human Papillomavirus Type 16 E2 Protein

1997 ◽  
Vol 272 (13) ◽  
pp. 8236-8242 ◽  
Author(s):  
Alison Thain ◽  
Kenneth Webster ◽  
Dave Emery ◽  
Anthony R. Clarke ◽  
Kevin Gaston
Keyword(s):  
Human Papillomavirus ◽  
Dna Binding ◽  
E2 Protein ◽  
Human Papillomavirus Type 16 ◽  
Dna Binding And Bending
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