scholarly journals Control of Recombination Directionality by the Listeria Phage A118 Protein Gp44 and the Coiled-Coil Motif of Its Serine Integrase

2017 ◽  
Vol 199 (11) ◽  
Author(s):  
Sridhar Mandali ◽  
Kushol Gupta ◽  
Anthony R. Dawson ◽  
Gregory D. Van Duyne ◽  
Reid C. Johnson
Keyword(s):  
Listeria Monocytogenes ◽  
Dna Binding ◽  
Coiled Coil ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Content Type ◽  
Bacterial Chromosomes ◽  
Carboxyl Terminal ◽  
Recombination Reactions ◽  
Serine Integrase

ABSTRACT The serine integrase of phage A118 catalyzes integrative recombination between attP on the phage and a specific attB locus on the chromosome of Listeria monocytogenes, but it is unable to promote excisive recombination between the hybrid attL and attR sites found on the integrated prophage without assistance by a recombination directionality factor (RDF). We have identified and characterized the phage-encoded RDF Gp44, which activates the A118 integrase for excision and inhibits integration. Gp44 binds to the C-terminal DNA binding domain of integrase, and we have localized the primary binding site to be within the mobile coiled-coil (CC) motif but distinct from the distal tip of the CC that is required for recombination. This interaction is sufficient to inhibit integration, but a second interaction involving the N-terminal end of Gp44 is also required to activate excision. We provide evidence that these two contacts modulate the trajectory of the CC motifs as they extend out from the integrase core in a manner dependent upon the identities of the four att sites. Our results support a model whereby Gp44 shapes the Int-bound complexes to control which att sites can synapse and recombine. IMPORTANCE Serine integrases mediate directional recombination between bacteriophage and bacterial chromosomes. These highly regulated site-specific recombination reactions are integral to the life cycle of temperate phage and, in the case of Listeria monocytogenes lysogenized by A118 family phage, are an essential virulence determinant. Serine integrases are also utilized as tools for genetic engineering and synthetic biology because of their exquisite unidirectional control of the DNA exchange reaction. Here, we identify and characterize the recombination directionality factor (RDF) that activates excision and inhibits integration reactions by the phage A118 integrase. We provide evidence that the A118 RDF binds to and modulates the trajectory of the long coiled-coil motif that extends from the large carboxyl-terminal DNA binding domain and is postulated to control the early steps of recombination site synapsis.

Conserved DNA binding and self-association domains of the Drosophila zeste protein

1992 ◽  
Vol 12 (2) ◽  
pp. 598-608
Author(s):  
J D Chen ◽  
C S Chan ◽  
V Pirrotta
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Amino Acid Sequence ◽  
Coiled Coil ◽  
Helical Structure ◽  
Binding Activity ◽  
Binding Domain ◽  
Drosophila Virilis ◽  
Predicted Amino Acid Sequence ◽  
Dna Binding Domain

The zeste gene product is involved in two types of genetic effects dependent on chromosome pairing: transvection and the zeste-white interaction. Comparison of the predicted amino acid sequence with that of the Drosophila virilis gene shows that several blocks of amino acid sequence have been very highly conserved. One of these regions corresponds to the DNA binding domain. Site-directed mutations in this region indicate that a sequence resembling that of the homeodomain DNA recognition helix is essential for DNA binding activity. The integrity of an amphipathic helical region is also essential for binding activity and is likely to be responsible for dimerization of the DNA binding domain. Another very strongly conserved domain of zeste is the C-terminal region, predicted to form a long helical structure with two sets of heptad repeats that constitute two long hydrophobic ridges at opposite ends and on opposite faces of the helix. We show that this domain is responsible for the extensive aggregation properties of zeste that are required for its role in transvection phenomena. A model is proposed according to which the hydrophobic ridges induce the formation of open-ended coiled-coil structures holding together many hundreds of zeste molecules and possibly anchoring these complexes to other nuclear structures.

scholarly journals Conserved DNA binding and self-association domains of the Drosophila zeste protein.

1992 ◽  
Vol 12 (2) ◽  
pp. 598-608 ◽  
Author(s):  
J D Chen ◽  
C S Chan ◽  
V Pirrotta
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Amino Acid Sequence ◽  
Coiled Coil ◽  
Helical Structure ◽  
Binding Activity ◽  
Binding Domain ◽  
Drosophila Virilis ◽  
Predicted Amino Acid Sequence ◽  
Dna Binding Domain

The zeste gene product is involved in two types of genetic effects dependent on chromosome pairing: transvection and the zeste-white interaction. Comparison of the predicted amino acid sequence with that of the Drosophila virilis gene shows that several blocks of amino acid sequence have been very highly conserved. One of these regions corresponds to the DNA binding domain. Site-directed mutations in this region indicate that a sequence resembling that of the homeodomain DNA recognition helix is essential for DNA binding activity. The integrity of an amphipathic helical region is also essential for binding activity and is likely to be responsible for dimerization of the DNA binding domain. Another very strongly conserved domain of zeste is the C-terminal region, predicted to form a long helical structure with two sets of heptad repeats that constitute two long hydrophobic ridges at opposite ends and on opposite faces of the helix. We show that this domain is responsible for the extensive aggregation properties of zeste that are required for its role in transvection phenomena. A model is proposed according to which the hydrophobic ridges induce the formation of open-ended coiled-coil structures holding together many hundreds of zeste molecules and possibly anchoring these complexes to other nuclear structures.

scholarly journals DNA-binding domain of the RepE initiator protein of mini-F plasmid: involvement of the carboxyl-terminal region.

1995 ◽  
Vol 177 (8) ◽  
pp. 1994-2001 ◽  
Author(s):  
F Matsunaga ◽  
Y Kawasaki ◽  
M Ishiai ◽  
K Nishikawa ◽  
T Yura ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Domain ◽  
Terminal Region ◽  
Dna Binding Domain ◽  
F Plasmid ◽  
Initiator Protein ◽  
Carboxyl Terminal

The Carboxyl-Terminal Region Common to Lamins A and C Contains a DNA Binding Domain†

Biochemistry ◽  
2003 ◽  
Vol 42 (17) ◽  
pp. 4819-4828 ◽  
Author(s):  
Vérène Stierlé ◽  
Joël Couprie ◽  
Cecilia Östlund ◽  
Isabelle Krimm ◽  
Sophie Zinn-Justin ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Domain ◽  
Terminal Region ◽  
Dna Binding Domain ◽  
Carboxyl Terminal

Cysteine residues in the zinc finger and amino acids adjacent to the finger are necessary for DNA binding by the LAC9 regulatory protein of Kluyveromyces lactis

1988 ◽  
Vol 8 (9) ◽  
pp. 3726-3733
Author(s):  
M M Witte ◽  
R C Dickson
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Zinc Finger ◽  
Kluyveromyces Lactis ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Dna Binding Activity ◽  
Carboxyl Terminal

LAC9 is a positive regulatory protein that controls transcription of the lactose-galactose regulon in Kluyveromyces lactis. LAC9 is homologous to the GAL4 protein of Saccharomyces cerevisiae. Both proteins have a single "zinc finger" which plays a role in DNA binding. We previously hypothesized (L. V. Wray, M. M. Witte, R. C. Dickson, and M. I. Riley, Mol. Cell. Biol. 7:1111-1121, 1987) that the DNA-binding domain of the LAC9 protein consisted of the zinc finger as well as a region of amino acids on the carboxyl-terminal side of the zinc finger. In this study we used oligonucleotide-directed mutagenesis to introduce 13 single-amino-acid changes into the proposed DNA-binding domain of the LAC9 protein. Variant LAC9 proteins carrying an amino acid substitution in any one of the four highly conserved Cys residues of the zinc finger had reduced DNA-binding activity, suggesting that each Cys is necessary for DNA binding. Three of four variant LAC9 proteins with amino acid substitutions located on the carboxyl-terminal side of the zinc finger had reduced DNA-binding activity. These results support our hypothesis that the DNA-binding domain of the LAC9 protein is composed of the zinc finger and the adjacent region on the carboxyl side of the zinc finger, a region that has the potential to form an alpha-helix. Finally, LAC9 proteins containing His residues substituted for the conserved Cys residues also had reduced DNA-binding activity, indicating that His residues are not equivalent to Cys residues, as had been previously thought.

Gain-of-Phosphorylation Mutations in Coiled-Coil and DNA-Binding Domain of STAT1 Identified in Japanese Patients with Chronic Mucocutaneous Candidiasis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4831-4831
Author(s):  
Yoko Mizoguchi ◽  
Satoshi Okada ◽  
Miyuki Tsumura ◽  
Osamu Hirata ◽  
Jean-Laurent Casanova ◽  
...  
Keyword(s):  
Dna Binding ◽  
Coiled Coil ◽  
Binding Domain ◽  
Specific Gene ◽  
Chronic Mucocutaneous Candidiasis ◽  
Dna Binding Domain ◽  
Mean Flow ◽  
Transcription Activity ◽  
Mucocutaneous Candidiasis ◽  
Ifn Γ

Abstract Abstract 4831 Chronic mucocutaneous candidiasis (CMCD) is a rare congenital disorder characterized by persistent or recurrent skin, nails and mucosal membranes infections caused by Candida albicans. Several studies suggest that impairment of development in Th17 lineage and/or IL-17 signaling could be responsible for development of CMCD and seven responsible genes, CARD9, STAT3, IL12B, IL12RB1, IL17RA, IL17F, and AIRE have been identified. Recently, heterozygous mutations in coiled-coil domain (CCD) and DNA-binding domain (DBD) of STAT1 are identified in approximately 40% of patients with CMCD. Signal transducer and activation of transcription 1 (STAT1) is a DNA-binding factor which regulates specific gene transcription. IFN-γ stimulation results in phosphorylation of STAT1 at Tyr701 (pSTAT1) to induce the homodimerization, a gamma-activated factor (GAF), through the conformational change and the nuclear import. The GAF binds to the gamma-activated sequence (GAS) to induce the transcriptional activities. STAT1 mutations identified in patients with CMCD are gain-of-phosphorylation, gain-of-GAF DNA binding and gain-of-GAS transcription activity in response to IFN-γ, IFN-α and IL-27. Based on the results of transient gene experiments, impairment in dephosphorylation of STAT1 has been considered to be a molecular pathogenesis underlying the increased pSTAT1. Here we report six heterozygous missense mutations in CCD and DBD of STAT1, including two novel heterozygous STAT1 mutation, 1060C>A (L354M) and 986C>T (P329L), in two sporadic and four multiplex cases with CMCD in Japan. We investigated functional significance of these mutations by transient gene expression experiments using U3C STAT1 null fibrosarcoma cells. Similar to the previous studies, all mutant proteins showed increased pSTAT1 in response to IFN-α and IFN-γ. Increased GAF-DNA binding and GAS transcription activity were observed in mutant expressed cells. Thus, these mutations are gain-of-function mutations against GAF mediated transcription activity. We then studied dephosphorylation of STAT1 using peripheral blood mononuclear cells (PBMCs) from the patients. The PBMCs were incubated with Staurosporine, tyrosine kinase inhibitor, followed by IFN-γ stimulation and analyzed by flowcytometry and immunoblot. In both experiments, PBMCs from the patients showed increased pSTAT1 after IFN-γ stimulation. Furthermore, we observed persistent pSTAT1 after Staurosporine treatment. These findings suggest that excess pSTAT1 is caused by an impairment of dephosphorylation. The flowcytometry analysis showed no overlap in mean flow intensity of pSTAT1 between 6 patients and 11 healthy controls after Staurosporine treatment. Therefore, this method can be useful for rapid test of STAT1 function in patients with CMCD. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Independent and Interchangeable Multimerization Domains of the AbrB, Abh, and SpoVT Global Regulatory Proteins

2005 ◽  
Vol 187 (18) ◽  
pp. 6354-6362 ◽  
Author(s):  
Fude Yao ◽  
Mark A. Strauch
Keyword(s):  
Dna Binding ◽  
Binding Domain ◽  
Chimeric Proteins ◽  
Dna Binding Domain ◽  
Global Regulators ◽  
Amino Terminal ◽  
Sequence Homologies ◽  
Carboxyl Terminal ◽  
Paralogous Proteins

ABSTRACT The global regulators AbrB, Abh, and SpoVT are paralogous proteins showing their most extensive sequence homologies in the DNA-binding amino-terminal regions (about 50 residues). The carboxyl-terminal portion of AbrB has been hypothesized to be a multimerization domain with little if any role in DNA-binding recognition or specificity. To investigate the multimerization potentials of the carboxyl-terminal portions of AbrB, Abh, and SpoVT we utilized an in vivo multimerization assay system based upon fusion of the domains to the DNA binding domain of the λ cI repressor protein. The results indicate that the N and C domains of all three paralogues are independent dimerization modules and that the intact Abh and SpoVT proteins are most probably tetramers. Chimeric proteins consisting of the AbrB N-terminal DNA-binding domain fused to the C domain of either Abh or SpoVT are indistinguishable from wild-type AbrB in their ability to regulate an AbrB target promoter in vivo.

scholarly journals The ChiS-Family DNA-Binding Domain Contains a Cryptic Helix-Turn-Helix Variant

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Catherine A. Klancher ◽  
George Minasov ◽  
Ram Podicheti ◽  
Douglas B. Rusch ◽  
Triana N. Dalia ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Vibrio Cholerae ◽  
Binding Domain ◽  
Structural Basis ◽  
Striking Difference ◽  
Dna Binding Domain ◽  
Structural Domains ◽  
Content Type ◽  
Domains Of Life

ABSTRACT Sequence-specific DNA-binding domains (DBDs) are conserved in all domains of life. These proteins carry out a variety of cellular functions, and there are a number of distinct structural domains already described that allow for sequence-specific DNA binding, including the ubiquitous helix-turn-helix (HTH) domain. In the facultative pathogen Vibrio cholerae, the chitin sensor ChiS is a transcriptional regulator that is critical for the survival of this organism in its marine reservoir. We recently showed that ChiS contains a cryptic DBD in its C terminus. This domain is not homologous to any known DBD, but it is a conserved domain present in other bacterial proteins. Here, we present the crystal structure of the ChiS DBD at a resolution of 1.28 Å. We find that the ChiS DBD contains an HTH domain that is structurally similar to those found in other DNA-binding proteins, like the LacI repressor. However, one striking difference observed in the ChiS DBD is that the canonical tight turn of the HTH is replaced with an insertion containing a β-sheet, a variant which we term the helix-sheet-helix. Through systematic mutagenesis of all positively charged residues within the ChiS DBD, we show that residues within and proximal to the ChiS helix-sheet-helix are critical for DNA binding. Finally, through phylogenetic analyses we show that the ChiS DBD is found in diverse proteobacterial proteins that exhibit distinct domain architectures. Together, these results suggest that the structure described here represents the prototypical member of the ChiS-family of DBDs. IMPORTANCE Regulating gene expression is essential in all domains of life. This process is commonly facilitated by the activity of DNA-binding transcription factors. There are diverse structural domains that allow proteins to bind to specific DNA sequences. The structural basis underlying how some proteins bind to DNA, however, remains unclear. Previously, we showed that in the major human pathogen Vibrio cholerae, the transcription factor ChiS directly regulates gene expression through a cryptic DNA-binding domain. This domain lacked homology to any known DNA-binding protein. In the current study, we determined the structure of the ChiS DNA-binding domain (DBD) and found that the ChiS-family DBD is a cryptic variant of the ubiquitous helix-turn-helix (HTH) domain. We further demonstrate that this domain is conserved in diverse proteins that may represent a novel group of transcriptional regulators.

scholarly journals Interaction of ArmZ with the DNA-Binding Domain of MexZ Induces Expression of mexXY Multidrug Efflux Pump Genes and Antimicrobial Resistance in Pseudomonas aeruginosa

2019 ◽  
Vol 63 (12) ◽  
Author(s):  
Adam Kawalek ◽  
Magdalena Modrzejewska ◽  
Bartlomiej Zieniuk ◽  
Aneta Agnieszka Bartosik ◽  
Grazyna Jagura-Burdzy
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antimicrobial Resistance ◽  
Dna Binding ◽  
Molecular Mechanism ◽  
Bacterial Species ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Multidrug Efflux ◽  
Intrinsic Resistance ◽  
Content Type

ABSTRACT Multidrug efflux pumps play an important role in antibiotic resistance in bacteria. In Pseudomonas aeruginosa, the MexXY pump provides intrinsic resistance to many antimicrobials, including aminoglycosides. The expression of the mexXY operon is negatively regulated by the MexZ repressor. This repression is alleviated in response to antibiotic-induced ribosome stress, which results in increased synthesis of the antirepressor ArmZ, interacting with MexZ. The molecular mechanism of MexZ inactivation by ArmZ is not known. Here, we show that the N-terminal part of MexZ, encompassing the DNA-binding domain, is required for the interaction with ArmZ. Using bacterial two-hybrid system-based mutant screening and pulldown analyses, we identified substitutions in MexZ that diminished (R3S, K6E, and R13H) or completely impaired (K53E) the interaction with ArmZ without blocking MexZ activity as a transcriptional repressor. The introduction of the corresponding mexZ missense mutations into the P. aeruginosa PAO1161 chromosome impaired (mexZK6E and mexZR13H) or blocked (mexZK53E) tetracycline-mediated induction of mexY expression. Concomitantly, the PAO1161 mexZK53E strain was more susceptible to aminoglycosides. The identified residues are highly conserved in MexZ-like transcriptional regulators found in bacterial genomes encoding both MexX/MexY/MexZ and ArmZ/PA5470 orthologs, suggesting that a similar mechanism may contribute to the induction of efflux-mediated resistance in other bacterial species. Overall, our data shed light on the molecular mechanism of ArmZ-mediated induction of intrinsic antimicrobial resistance in P. aeruginosa.

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