scholarly journals Defining the Functionally Important Domain and Amino Acid Residues in Mycobacterium tuberculosis Integration Host Factor for Genome Stability, DNA Binding, and Integrative Recombination

2017 ◽  
Vol 199 (19) ◽  
Author(s):  
Narayanaswamy Sharadamma ◽  
Yadumurthy Harshavardhana ◽  
K. Muniyappa
Keyword(s):  
Dna Damage ◽  
Mycobacterium Tuberculosis ◽  
Amino Acid ◽  
Dna Binding ◽  
Integration Host Factor ◽  
Host Factor ◽  
Content Type ◽  
Amino Acid Region ◽  
N Terminus ◽  
Acid Region

ABSTRACT The integration host factor of Mycobacterium tuberculosis (mIHF) consists of a single polypeptide chain, the product of the ihf gene. We previously revealed that mIHF is a novel member of a new class of nucleoid-associated proteins that have important roles in DNA damage response, nucleoid compaction, and integrative recombination. The mIHF contains a region of 86 amino acids at its N terminus, absent from both α- and β-subunits of Escherichia coli IHF. However, the functional significance of an extra 86-amino-acid region in the full-length protein remains unknown. Here, we report the structure/function relationship of the DNA-binding and integrative recombination-stimulating activity of mIHF. Deletion mutagenesis showed that an extra 86-amino-acid region at the N terminus is dispensable; the C-terminal region possesses the sequences essential for its known biological functions, including the ability to suppress the sensitivity of E. coli ΔihfA and ΔihfB cells to DNA-damaging agents, DNA binding, DNA multimerization-circularization, and stimulation of phage L5 integrase-catalyzed integrative recombination. Single and double alanine substitutions at positions Arg170 and Arg171, located at the mIHF DNA-binding site, abrogated its capacity to suppress the sensitivity of E. coli ΔihfA and ΔihfB cells to DNA-damaging agents. The variants encoded by these mutant alleles failed to bind DNA and stimulate integrative recombination. Interestingly, the DNA-binding activity of the mIHF-R173A variant remained largely unaffected; however, it was unable to stimulate integrative recombination, thus revealing a separation-of-function allele of mIHF. The functional and structural characterization of this separation-of-function allele of mIHF could reveal previously unknown functions of IHF. IMPORTANCE The integration host factor of Mycobacterium tuberculosis is a novel nucleoid-associated protein. mIHF plays a vital role in DNA damage response, nucleoid compaction, and integrative recombination. Intriguingly, mIHF contains an extra 86-amino-acid region at its N terminus, absent from both α- and β-subunits of Escherichia coli IHF, whose functional significance is unknown. Furthermore, a triad of arginine residues located at the mIHF-DNA interface have been implicated in a range of its functions. Here, we reveal the roles of N- and C-terminal regions of mIHF and the individual residues in the Arg triad for their ability to provide protection in vivo against DNA damage, bind DNA, and stimulate integrase-catalyzed site-specific recombination.

scholarly journals The DNA-binding specificity of the hepatocyte nuclear factor 3/forkhead domain is influenced by amino-acid residues adjacent to the recognition helix.

1994 ◽  
Vol 14 (4) ◽  
pp. 2755-2766 ◽  
Author(s):  
D G Overdier ◽  
A Porcella ◽  
R H Costa
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Hepatocyte Nuclear Factor ◽  
Binding Properties ◽  
Winged Helix ◽  
Amino Acid Region ◽  
Dna Binding Specificity ◽  
Recognition Helix ◽  
Dna Binding Properties ◽  
Acid Region

Three distinct hepatocyte nuclear factor 3 (HNF-3) proteins (HNF-3 alpha, -3 beta, and -3 gamma) are known to regulate the transcription of liver-specific genes. The HNF-3 proteins bind to DNA as a monomer through a modified helix-turn-helix, known as the winged helix motif, which is also utilized by a number of developmental regulators, including the Drosophila homeotic forkhead (fkh) protein. We have previously described the isolation, from rodent tissue, of an extensive family of tissue-specific HNF-3/fkh homolog (HFH) genes sharing homology in their winged helix motifs. In this report, we have determined the preferred DNA-binding consensus sequence for the HNF-3 beta protein as well as for two divergent family members, HFH-1 and HFH-2. We show that these HNF-3/fkh proteins bind to distinct DNA sites and that the specificity of protein recognition is dependent on subtle nucleotide alterations in the site. The HNF-3, HFH-1, and HFH-2 consensus binding sequences were also used to search DNA regulatory regions to identify potential target genes. Furthermore, an analysis of the DNA-binding properties of a series of HFH-1/HNF-3 beta protein chimeras has allowed us to identify a 20-amino-acid region, located adjacent to the DNA recognition helix, which contributes to DNA-binding specificity. These sequences are not involved in base-specific contacts and include residues which diverge within the HNF-3/fkh family. Replacement of this 20-amino-acid region in HNF-3 beta with corresponding residues from HFH-1 enabled the HNF-3 beta recognition helix to bind only HFH-1-specific DNA-binding sites. We propose a model in which this 20-amino-acid flanking region influences the DNA-binding properties of the recognition helix.

scholarly journals A Molecular Genetic Dissection of the Evolutionarily Conserved N Terminus of Yeast Rad52

Genetics ◽  
2002 ◽  
Vol 161 (2) ◽  
pp. 549-562
Author(s):  
Uffe H Mortensen ◽  
Naz Erdeniz ◽  
Qi Feng ◽  
Rodney Rothstein
Keyword(s):  
Dna Damage ◽  
Amino Acid ◽  
Dna Binding ◽  
Molecular Genetic ◽  
Mitotic Recombination ◽  
Amino Acid Residues ◽  
Γ Irradiation ◽  
Evolutionarily Conserved ◽  
N Terminus ◽  
Γ Ray

Abstract Rad52 is a DNA-binding protein that stimulates the annealing of complementary single-stranded DNA. Only the N terminus of Rad52 is evolutionarily conserved; it contains the core activity of the protein, including its DNA-binding activity. To identify amino acid residues that are important for Rad52 function(s), we systematically replaced 76 of 165 amino acid residues in the N terminus with alanine. These substitutions were examined for their effects on the repair of γ-ray-induced DNA damage and on both interchromosomal and direct repeat heteroallelic recombination. This analysis identified five regions that are required for efficient γ-ray damage repair or mitotic recombination. Two regions, I and II, also contain the classic mutations, rad52-2 and rad52-1, respectively. Interestingly, four of the five regions contain mutations that impair the ability to repair γ-ray-induced DNA damage yet still allow mitotic recombinants to be produced at rates that are similar to or higher than those obtained with wild-type strains. In addition, a new class of separation-of-function mutation that is only partially deficient in the repair of γ-ray damage, but exhibits decreased mitotic recombination similar to rad52 null strains, was identified. These results suggest that Rad52 protein acts differently on lesions that occur spontaneously during the cell cycle than on those induced by γ-irradiation.

scholarly journals The DNA-binding specificity of the hepatocyte nuclear factor 3/forkhead domain is influenced by amino-acid residues adjacent to the recognition helix

1994 ◽  
Vol 14 (4) ◽  
pp. 2755-2766
Author(s):  
D G Overdier ◽  
A Porcella ◽  
R H Costa
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Hepatocyte Nuclear Factor ◽  
Binding Properties ◽  
Winged Helix ◽  
Amino Acid Region ◽  
Dna Binding Specificity ◽  
Recognition Helix ◽  
Dna Binding Properties ◽  
Acid Region

Three distinct hepatocyte nuclear factor 3 (HNF-3) proteins (HNF-3 alpha, -3 beta, and -3 gamma) are known to regulate the transcription of liver-specific genes. The HNF-3 proteins bind to DNA as a monomer through a modified helix-turn-helix, known as the winged helix motif, which is also utilized by a number of developmental regulators, including the Drosophila homeotic forkhead (fkh) protein. We have previously described the isolation, from rodent tissue, of an extensive family of tissue-specific HNF-3/fkh homolog (HFH) genes sharing homology in their winged helix motifs. In this report, we have determined the preferred DNA-binding consensus sequence for the HNF-3 beta protein as well as for two divergent family members, HFH-1 and HFH-2. We show that these HNF-3/fkh proteins bind to distinct DNA sites and that the specificity of protein recognition is dependent on subtle nucleotide alterations in the site. The HNF-3, HFH-1, and HFH-2 consensus binding sequences were also used to search DNA regulatory regions to identify potential target genes. Furthermore, an analysis of the DNA-binding properties of a series of HFH-1/HNF-3 beta protein chimeras has allowed us to identify a 20-amino-acid region, located adjacent to the DNA recognition helix, which contributes to DNA-binding specificity. These sequences are not involved in base-specific contacts and include residues which diverge within the HNF-3/fkh family. Replacement of this 20-amino-acid region in HNF-3 beta with corresponding residues from HFH-1 enabled the HNF-3 beta recognition helix to bind only HFH-1-specific DNA-binding sites. We propose a model in which this 20-amino-acid flanking region influences the DNA-binding properties of the recognition helix.

The Oct-2 glutamine-rich and proline-rich activation domains can synergize with each other or duplicates of themselves to activate transcription

1994 ◽  
Vol 14 (9) ◽  
pp. 6046-6055
Author(s):  
M Tanaka ◽  
W M Clouston ◽  
W Herr
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Activation Domain ◽  
Activation Domains ◽  
Amino Acid Region ◽  
Pou Domain ◽  
Octamer Motif ◽  
P Domain ◽  
Acid Region

The B-cell POU homeodomain protein Oct-2 contains two transcriptional activation domains, one N terminal and the other C terminal of the central DNA-binding POU domain. The synergistic action of these two activation domains makes Oct-2 a more potent activator of mRNA promoters than the related broadly expressed octamer motif-binding protein Oct-1, which contains an N-terminal but not a C-terminal Oct-2-like activation domain. Both Oct-2 mRNA promoter activation domains were delineated by truncation analysis: the N-terminal Q domain is a 66-amino-acid region rich in glutamines, and the C-terminal P domain is a 42-amino-acid region rich in prolines. The Q and P domains synergized with each other or duplicates of themselves, independently of their N-terminal or C-terminal position relative to the POU domain. The C-terminal P domain, which differentiates Oct-2 from Oct-1, also activated transcription in conjunction with the heterologous GAL4 DNA-binding domain. Oct-2 thus contains three modular functional units, the DNA-binding POU domain and the two P and Q activation domains. An electrophoretic mobility shift assay with a variety of these Oct-2 activators revealed a distinct complex called QA that was dependent on the presence of an active glutamine-rich activation domain and migrated more slowly than the Oct-2-DNA complexes. Formation of the QA complex is consistent with interaction of the glutamine-rich activation domains with a regulatory protein important for the process of transcriptional activation.

scholarly journals The Oct-2 glutamine-rich and proline-rich activation domains can synergize with each other or duplicates of themselves to activate transcription.

1994 ◽  
Vol 14 (9) ◽  
pp. 6046-6055 ◽  
Author(s):  
M Tanaka ◽  
W M Clouston ◽  
W Herr
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Activation Domain ◽  
Activation Domains ◽  
Amino Acid Region ◽  
Pou Domain ◽  
Octamer Motif ◽  
P Domain ◽  
Acid Region

The B-cell POU homeodomain protein Oct-2 contains two transcriptional activation domains, one N terminal and the other C terminal of the central DNA-binding POU domain. The synergistic action of these two activation domains makes Oct-2 a more potent activator of mRNA promoters than the related broadly expressed octamer motif-binding protein Oct-1, which contains an N-terminal but not a C-terminal Oct-2-like activation domain. Both Oct-2 mRNA promoter activation domains were delineated by truncation analysis: the N-terminal Q domain is a 66-amino-acid region rich in glutamines, and the C-terminal P domain is a 42-amino-acid region rich in prolines. The Q and P domains synergized with each other or duplicates of themselves, independently of their N-terminal or C-terminal position relative to the POU domain. The C-terminal P domain, which differentiates Oct-2 from Oct-1, also activated transcription in conjunction with the heterologous GAL4 DNA-binding domain. Oct-2 thus contains three modular functional units, the DNA-binding POU domain and the two P and Q activation domains. An electrophoretic mobility shift assay with a variety of these Oct-2 activators revealed a distinct complex called QA that was dependent on the presence of an active glutamine-rich activation domain and migrated more slowly than the Oct-2-DNA complexes. Formation of the QA complex is consistent with interaction of the glutamine-rich activation domains with a regulatory protein important for the process of transcriptional activation.

scholarly journals Deletion of a 19-Amino-Acid Region inClostridioides difficileTcdB2 Results in Spontaneous Autoprocessing and Reduced Cell Binding and Provides a Nontoxic Immunogen for Vaccination

2019 ◽  
Vol 87 (8) ◽  
Author(s):  
Sarah J. Bland ◽  
Jason L. Larabee ◽  
Tyler M. Shadid ◽  
Mark L. Lang ◽  
Jimmy D. Ballard
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Conformational Changes ◽  
Small Gtpases ◽  
Cell Entry ◽  
Bacterial Toxin ◽  
Target Cells ◽  
Content Type ◽  
Amino Acid Region ◽  
Acid Region

ABSTRACTClostridioides difficiletoxin B (TcdB) is an intracellular toxin responsible for many of the pathologies ofC. difficileinfection. The two variant forms of TcdB (TcdB1 and TcdB2) share 92% sequence identity but have reported differences in rates of cell entry, autoprocessing, and overall toxicity. This 2,366-amino-acid, multidomain bacterial toxin glucosylates and inactivates small GTPases in the cytosol of target cells, ultimately leading to cell death. Successful cell entry and intoxication by TcdB are known to involve various conformational changes in the protein, including a proteolytic autoprocessing event. Previous studies found that amino acids 1753 to 1852 influence the conformational states of the proximal carboxy-terminal domain of TcdB and could contribute to differences between TcdB1 and TcdB2. In the current study, a combination of approaches was used to identify sequences within the region from amino acids 1753 to 1852 that influence the conformational integrity and cytotoxicity of TcdB2. Four deletion mutants with reduced cytotoxicity were identified, while one mutant, TcdB2Δ1769–1787, exhibited no detectable cytotoxicity. TcdB2Δ1769–1787underwent spontaneous autoprocessing and was unable to interact with CHO-K1 or HeLa cells, suggesting a potential change in the conformation of the mutant protein. Despite the putative alteration in structural stability, vaccination with TcdB2Δ1769–1787induced a TcdB2-neutralizing antibody response and protected againstC. difficiledisease in a mouse model. These findings indicate that the 19-amino-acid region spanning residues 1769 to 1787 in TcdB2 is crucial to cytotoxicity and the structural regulation of autoprocessing and that TcdB2Δ1769–1787is a promising candidate for vaccination.

scholarly journals Amino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry

mSphere ◽  
2017 ◽  
Vol 2 (4) ◽  
Author(s):  
Jonathan J. Hunt ◽  
Jason L. Larabee ◽  
Jimmy D. Ballard
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Clostridium Difficile ◽  
Cell Entry ◽  
Cell Binding ◽  
Content Type ◽  
Amino Acid Region ◽  
Antibiotic Associated Diarrhea ◽  
Insight Into ◽  
Acid Region

ABSTRACT TcdB is a major virulence factor produced by Clostridium difficile, a leading cause of antibiotic-associated diarrhea. Hypervirulent strains of C. difficile encode a variant of TcdB (TcdB2) that is more toxic than toxin derived from historical strains (TcdB1). Though TcdB1 and TcdB2 exhibit 92% overall identity, a 99-amino-acid region previously associated with cell entry and spanning amino acids 1753 to 1851 has only 77% sequence identity. Results from the present study indicate that the substantial sequence variation in this region could contribute to the differences in cell entry between TcdB1 and TcdB2 and possibly explain TcdB2’s heightened toxicity. Finally, during the course of these studies, an unusual aspect of TcdB cell entry was discovered wherein cell binding appeared to depend on endocytosis. These findings provide insight into TcdB’s variant forms and their mechanisms of cell entry. Clostridium difficile TcdB2 enters cells with a higher efficiency than TcdB1 and exhibits an overall higher level of toxicity. However, the TcdB2-specific sequences that account for more efficient cell entry have not been reported. In this study, we examined the contribution of carboxy-terminal sequence differences to TcdB activity by comparing the binding, uptake, and endosomal localization of TcdB1 and TcdB2 or selected recombinant fragments of these proteins. Our findings suggest that sequence differences in the amino acid 1753 to 1851 region proximal to the combined repetitive oligopeptide domain (CROP) support enhanced uptake of TcdB2 and localization of toxin in acidified endosomes. In the absence of this region, the CROP domains of both forms of the toxin exhibited similar levels of cell interaction, while the addition of amino acids 1753 to 1851 greatly increased toxin binding by only TcdB2. Moreover, the amino acid 1753 to 2366 fragment of TcdB2, but not TcdB1, accumulated to detectable levels in acidified endosomes. Unexpectedly, we discovered an unusual relationship between endocytosis and the efficiency of cell binding for TcdB1 and TcdB2 wherein inhibition of endocytosis by a chemical inhibitor or incubation at a low temperature resulted in a dramatic reduction in cell binding. These findings provide information on sequence variations that may contribute to differences in TcdB1 and TcdB2 toxicity and reveal a heretofore unknown connection between endocytosis and cell binding for this toxin. IMPORTANCE TcdB is a major virulence factor produced by Clostridium difficile, a leading cause of antibiotic-associated diarrhea. Hypervirulent strains of C. difficile encode a variant of TcdB (TcdB2) that is more toxic than toxin derived from historical strains (TcdB1). Though TcdB1 and TcdB2 exhibit 92% overall identity, a 99-amino-acid region previously associated with cell entry and spanning amino acids 1753 to 1851 has only 77% sequence identity. Results from the present study indicate that the substantial sequence variation in this region could contribute to the differences in cell entry between TcdB1 and TcdB2 and possibly explain TcdB2’s heightened toxicity. Finally, during the course of these studies, an unusual aspect of TcdB cell entry was discovered wherein cell binding appeared to depend on endocytosis. These findings provide insight into TcdB’s variant forms and their mechanisms of cell entry.

scholarly journals A Unique 45-Amino-Acid Region in the Toprim Domain of Plasmodium falciparum Gyrase B Is Essential for Its Activity

2009 ◽  
Vol 8 (11) ◽  
pp. 1759-1769 ◽  
Author(s):  
Ashraf Dar ◽  
Dhaneswar Prusty ◽  
Neelima Mondal ◽  
Suman K. Dhar
Keyword(s):  
Plasmodium Falciparum ◽  
Amino Acid ◽  
Dna Binding ◽  
Atpase Activity ◽  
Dna Cleavage ◽  
Atp Hydrolysis ◽  
Dna Gyrase ◽  
Binding Region ◽  
Amino Acid Region ◽  
Acid Region

ABSTRACT DNA gyrase is the only topoisomerase that can introduce negative supercoils into the DNA at the cost of ATP hydrolysis. Some but not all the steps of the topoisomerization reaction are understood clearly for both eukaryotic topoII and DNA gyrase. This study is an attempt to understand whether the B subunit of DNA gyrase binds to DNA directly, which may be central to the stimulation of its ATPase activity essential for gyrase function. We have dissected the Plasmodium falciparum gyrase B (PfGyrB) subunit to identify a 45-amino-acid region in the toprim domain that is responsible for its intrinsic DNA binding activity, DNA-stimulated ATPase activity, and DNA cleavage. We find that DNA has to enter through the ATP-operated clamp of PfGyrB to gain access to the DNA binding region. Furthermore, the rate of ATP hydrolysis of PfGyrB increases significantly with increasing DNA length, suggesting a possible communication between the ATPase domain and the DNA binding region that can account for its optimal ATPase activity. These results not only highlight the mechanism of GyrB action in the deadly human parasite P. falciparum but also provide meaningful insights into the current mechanistic model of DNA transport by gyrase during the topoisomerization reaction.

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