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Results for “Double Bind Interaction”

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PubMed1989

Identification of two distinct functional domains on vinculin involved in its association with focal contacts.

We report here on the identification of two distinct functional domains on chicken vinculin molecule, which can, independently, mediate its interaction with focal contacts in living cells. These findings were obtained by immunofluorescent labeling of COS cells transfected with a series of chicken vinculin-specific cDNA constructs derived from clones cVin1 and cVin5 (Bendori, R., D. Salomon, and B. Geiger. 1987. EMBO [Eur. Mol. Biol. Organ.] J. 6:2897-2905). These included a chimeric construct consisting of 5' sequences of cVin1 attached to the complementary 3' region of cVin5, as well as several constructs of either cVin1 or cVin5 from which 3' or 5' sequences were deleted. We show here that the products of both cVin1 and cVin5, and of the cVin1/cVin5 chimera, readily associated with focal contacts in transfected COS cells. Furthermore, 78 and 45 kD NH2-terminal fragments encoded by a deleted cVin1 and the 78-kD COOH-terminal portion of vinculin encoded by cVin5 were capable of binding specifically to focal contact areas. In contrast 3'-deletion mutants prepared from clone cVin5 and a 5'-deletion mutant of cVin1, lacking both NH2- and COOH-terminal sequences, failed to associate with focal contacts in transfected cells. The loss of binding was accompanied by an overall disarray of the microfilament system. These results, together with previous in vitro binding studies, suggest that vinculin contains at least two independent sites for binding to focal contacts; the NH2-terminal domain may contain the talin binding site while the COOH-terminal domain may mediate vinculin-vinculin interaction. Moreover, the disruptive effect of the double-deleted molecule (lacking the two focal-contact binding sites) on the organization of actin suggests that a distinct region involved in the binding of vinculin to the microfilament system is present in the NH2-terminal 45-kD region of the molecule.

Animals↗
PubMed2002

DNA-binding mechanism of O6-alkylguanine-DNA alkyltransferase. Effects of protein and DNA alkylation on complex stability.

The mutagenic and cytotoxic effects of many endogenous and exogenous alkylating agents are mitigated by the actions of O(6)-alkylguanine-DNA alkyltransferase (AGT). In humans this protein protects the integrity of the genome, but it also contributes to the resistance of tumors to DNA-alkylating chemotherapeutic agents. Here we report properties of the interaction between AGT and short DNA oligonucleotides. We show that although AGT sediments as a monomer in the absence of DNA, it binds cooperatively to both single-stranded and double-stranded deoxyribonucleotides. This strong cooperative interaction is only slightly perturbed by active site mutation of AGT or by alkylation of either AGT or DNA. The stoichiometry of complex formation with 16-mer oligonucleotides, assessed by analytical ultracentrifugation and electrophoretic mobility shift assays, is 4:1 on single-stranded and duplex DNA and is unchanged by several active site mutations or by protein or DNA alkylation. These results have significant implications for the mechanisms by which AGT locates and interacts with repairable alkyl lesions to effect DNA repair.

Alkylation↗
PubMed1982

The interactions of androgen receptor with poly(A)-containing RNA and polyribonucleotides.

The androgen receptor from mouse kidney cytosol binds not only to DNA but also to RNA as assayed by competition with DNA-cellulose centrifugation or by direct binding with agarose-polynucleotides. The dihydrotestosterone-receptor complex interacts more strongly to poly(A)-containing mRNA than to native natural DNA; it binds much more tightly to synthetic poly(G) than to natural DNA and other homopolyribonucleotides such as poly(U), poly(A), or poly(C). Poly(U) is moderately effective in competing for the complex, whereas poly(A) and poly(C) re not effective. Competition studies with heteropolyribonucleotides show that polymers containing G are most effective on binding to the androgen receptor, whereas those containing U are moderately effective, and those containing only A and C are least effective. Several analogues of poly(G), such as poly(X), poly(I), and poly(m7G) interact very well with the dihydrotestosterone-receptor with some differences in the bindings. A double-stranded polymer, poly(I) . poly(C) competes poorly for the androgen receptor. The observations that the androgen receptor binds to RNA and interacts with polyribonucleotides selectively suggest that androgen receptor-RNA interaction could play important roles in gene regulation.

Animals↗
PubMed2003

Promyelocytic leukemia protein sensitizes tumor necrosis factor alpha-induced apoptosis by inhibiting the NF-kappaB survival pathway.

The promyelocytic leukemia protein (PML) is a growth/tumor suppressor essential for induction of apoptosis by diverse apoptotic stimuli. The mechanism by which PML regulates cell death remains unclear. In this study we found that ectopic expression of PML potentiates cell death by apoptosis in the tumor necrosis factor alpha (TNFalpha)-resistant cell line U2OS and other cell lines. Treatment with TNFalpha significantly sensitized these cells to apoptosis in a p53-independent manner. PML/TNFalpha-induced cell death is associated with DNA fragmentation, activation of caspase-3, -7, and -8, and degradation of DNA fragmentation factor/inhibitor of CAD. PML/TNFalpha-induced cell death could be blocked by the caspase-8 inhibitors CrmA and c-FLIP but not by Bcl-2. These findings indicate that this cell death event is initiated through the death receptor-dependent apoptosis pathway. PML is a transcriptional repressor of NF-kappaB by interacting with RelA/p65 and prevents its binding to the cognate enhancer through the C terminus. Coimmunoprecipitation and double-color immunofluorescence staining demonstrated that PML physically interacts with RelA/p65 in vivo and the two proteins colocalized at the endogenous levels. Overexpression of NF-kappaB rescued cell death induced by PML/TNFalpha. Furthermore, PML(-/-) mouse embryo fibroblasts are more resistant to TNFalpha-induced apoptosis. Together this study defines a novel mechanism by which PML induces apoptosis through repression of the NF-kappaB survival pathway.

Animals↗
PubMed1986

Identification of the 9-aminoacridine/DNA complex responsible for photodynamic inactivation of P22.

Acridine dyes bound to the condensed DNA within phage particles sensitize them to inactivation by visible light. The mechanism involves absorption of photons by an acridine/DNA complex, generating singlet oxygen, which covalently damages nearby proteins needed for DNA injection [Bryant, J., & King, J. (1985) J. Mol. Biol. 180, 837-863]. Acridines and related dyes interact with double-stranded DNA through a number of binding modes. To determine in condensed phage DNA the binding mode responsible for this inactivation, we have studied the formation of the DNA/acridine target complexes for photoinactivation. Analysis of the kinetics of 9-aminoacridine binding to Salmonella phage P22 particles revealed the formation of two binding species, one of which appeared more rapidly and was apparently an intermediate in the formation of the second. The rapidly forming species represented DNA sites with intercalated acridines, while the more slowly forming species represented the subsequent binding of additional acridine molecules to the DNA backbone of sites already containing intercalated dye. The rates of photoinactivation correlated with the rate of binding of 9-aminoacridine to the DNA backbone. This suggests that the most effective species for sensitizing phage to light-induced damage has acridine molecules stacked alongside the backbone of a region with intercalated molecules.

Aminacrine↗
PubMed2005

Nuclear DNA helicase II (RNA helicase A) interacts with Werner syndrome helicase and stimulates its exonuclease activity.

Nuclear DNA helicase II (NDH II), alternatively named RNA helicase A, is involved in transcription and RNA processing. Here, we report that NDH II interacts with the Werner syndrome helicase WRN, an enzyme associated with premature aging and predisposition to tumorigenesis. NDH II was co-purified with WRN, DNA polymerase delta, and replication protein A (70 kDa) during several steps of conventional column chromatography. Co-immunoprecipitations revealed an association between NDH II, WRN, and polymerase delta. We demonstrate a direct protein-protein interaction between WRN and NDH II that is mediated by the N-terminal double-strand RNA-binding domain II and C-terminal RGG box of NDH II and the N-terminal exonuclease domain of WRN. WRN inhibited the DNA-dependent NTPase and DNA helicase activities of NDH II. On the other hand, the 3' --> 5' exonuclease activity of WRN was increased by the presence of NDH II. NDH II directly stimulated the exonuclease domain of WRN, whereas the exonuclease domain of WRN suppressed the DNA-dependent (but not RNA-dependent) ATPase activity of NDH II. These results suggest that the double-strand RNA-binding domain II and RGG box of NDH II together form a protein-protein interaction surface that contacts the exonuclease domain of WRN. Furthermore, NDH II enhanced the degradation of D-loop DNA by the WRN exonuclease. Taken together, these results suggest that NDH II plays a role in promoting the DNA processing function of WRN, which in turn might be necessary for maintaining genomic stability.

Animals↗
PubMed2001

Y box-binding protein-1 binds preferentially to single-stranded nucleic acids and exhibits 3'-->5' exonuclease activity.

We have previously shown that Y box-binding protein-1 (YB-1) binds preferentially to cisplatin-modified Y box sequences. Based on structural and biochemical data, we predicted that this protein binds single-stranded nucleic acids. In the present study we confirmed the prediction and also discovered some unexpected functional features of YB-1. We found that the cold shock domain of the protein is necessary but not sufficient for double-stranded DNA binding while the C-tail domain interacts with both single-stranded DNA and RNA independently of the cold shock domain. In an in vitro translation system the C-tail domain of the protein inhibited translation but the cold shock domain did not. Both in vitro pull-down and in vivo co-immunoprecipitation assays revealed that YB-1 can form a homodimer. Deletion analysis mapped the C-tail domain of the protein as the region of homodimerization. We also characterized an intrinsic 3'-->5' DNA exonuclease activity of the protein. The region between residues 51 and 205 of its 324-amino acid extent is required for full exonuclease activity. Our findings suggest that YB-1 functions in regulating DNA/RNA transactions and that these actions involve different domains.

Binding Sites↗
PubMed2000

Yeast glycogen synthase kinase 3 is involved in protein degradation in cooperation with Bul1, Bul2, and Rsp5.

The yeast Saccharomyces cerevisiae has four genes, MCK1, MDS1 (RIM11), MRK1, and YOL128c, that encode glycogen synthase kinase 3 (GSK-3) homologs. The gsk-3 null mutant, in which these four genes are disrupted, shows temperature sensitivity, which is suppressed by the expression of mammalian GSK-3beta and by an osmotic stabilizer. Suppression of temperature sensitivity by an osmotic stabilizer is also observed in the bul1 bul2 double null mutant, and the temperature sensitivity of the bul1 bul2 double null mutant is suppressed by multiple copies of MCK1. We have screened rog mutants (revertants of gsk-3) which suppress the temperature sensitivity of the mck1 mds1 double null mutant and found that two of them, rog1 and rog2, also suppress the temperature sensitivity of the bul1 bul2 double null mutant. Bul1 and Bul2 have been reported to bind to Rsp5, a hect (for homologous to E6-associated-protein carboxyl terminus)-type ubiquitin ligase, but involvement of Bul1 and Bul2 in protein degradation has not been demonstrated. We find that Rog1, but not Rog2, is stabilized in the gsk-3 null and the bul1 bul2 double null mutants. Rog1 binds directly to Rsp5, and their interaction is dependent on GSK-3. Furthermore, Rog1 is stabilized in the npi1 mutant, in which RSP5 expression levels are reduced. These results suggest that yeast GSK-3 regulates the stability of Rog1 in cooperation with Bul1, Bul2, and Rsp5.

Adaptor Proteins, Signal Transducing↗
PubMed1976

[The interaction of pyridine-adenine-dinucleotides containing a hydrazide group with swine muscle lactate dehydrogenase].

Hydrazide group of 4-substituted NAD analogues is shown to interact with functional groups of substrate-binding site in double complexes with pig muscle lactate dehydrogenase (isoenzyme M4). The lactic acid residue, which is structurally incorporated into NAD analogue, improves slightly the binding of dinucleotide, while 2,2,6,6-tetramethylpiperidine-1-oxyl residue considerably decreases the firmless of binding. The comparison of the inhibitory ability of oxamate, incotinic acid hydraxide and their spin-labelled derivatives indicates the restricted and stiff sizes of a substrate-binding site.

Animals↗
PubMed1993

Interaction of the pim1/spi1 mitotic checkpoint with a protein phosphatase.

Loss of p58pim1, a homolog of human RCC1, results in uncoupling of mitosis from the completion of DNA replication in fission yeast. An extragenic suppressor of a mutant allele of pim1, esp1, has been isolated and characterized. esp1 encodes a predicted product of 305 amino acid residues, which shares 71% identity with budding yeast SIT4, a type2A related protein phosphatase. p58pim1 binds p25spi1, a 25-kd ras-related GTPase previously isolated as a high dosage suppressor of pim1. The complex dissociates in the presence of guanine nucleotides and Mg2+. The mutant p58pim1 is defective in its ability to bind p25spi1, suggesting that the physical interaction is essential for the maintenance of the interdependency of cell cycle event. In the esp1 pim1 double mutant, the mutant p58pim1 protein is still defective in its ability to bind to p25spi1. However, pmi1 induced premature mitosis is completely suppressed, suggesting that esp1 may act downstream of the p58pim1/p25spi1 physical interaction but upstream of the activation of the M-phase specific histone H1 kinase.

Amino Acid Sequence↗
PubMed1991

Actinomycin D and 7-aminoactinomycin D binding to single-stranded DNA.

The potent RNA polymerase inhibitors actinomycin D and 7-aminoactinomycin D are shown to bind to single-stranded DNAs. The binding occurs with particular DNA sequences containing guanine residues and is characterized by hypochromic UV absorption changes similar to those observed in interactions of the drugs with double-stranded duplex DNAs. The most striking feature of the binding is the dramatic (ca. 37-fold) enhancement in fluorescence that occurs when the 7-aminoactinomycin is bound to certain single-stranded DNAs. This fluorescence of the complex is also characterized by a 40-nm hypsochromic shift in the emission spectrum of the drug and an increase in the emission anisotropy relative to the free drug or the drug bound to calf thymus DNA. The fluorescence lifetimes change in the presence of the single-stranded DNA in a manner compatible with the intensity difference. Thus, there is an increase in the fraction of the emission corresponding to a 2-ns lifetime component compared to the predominant approximately 0.5-ns lifetime of the free drug. The 7-aminoactinomycin D comigrates in polyacrylamide gels with the single-stranded DNAs, and the fluorescence of the bound drug can be visualized by excitation with 540-nm light. The binding interactions are characterized by association constants of 2.0 x 10(6) to 1.1 x 10(7) M-1.

Base Sequence↗
PubMed1996

Construction of a dimeric DNA-binding peptide model by peptide-anthraquinone conjugation.

A peptide-anthraquinone conjugate was designed and synthesized containing linked peptide chains composed of Asp-Pro-Ala-Ala-Leu-Lys-Arg-Ala-Arg-Asn-Thr-Glu-Ala- Ala-Arg-Arg-Ser-Arg-Ala-Arg-Lys-Leu-Gln-Arg-Met, representing the basic region of GCN4. The two peptides were joined with anthraquinone at its 1- and 8-positions in a two-fold symmetric fashion, mimicking dimeric DNA-binding proteins. Experimental data indicated both an interaction of the anthraquinone moiety with the DNA double strand and an increase in the alpha-helicity of the peptide moieties of the ligand when it binds to DNA.

Amino Acid Sequence↗
PubMed2006

Molecular mechanism of interaction between oligopeptides and double-stranded DNA.

Formation of Ala-Glu-Asp-Gly peptide complex with duplex DNA [poly(dA-dT):poly(dA-dT)] at neutral pH and ambient temperature was studied experimentally. Peptide binding to duplex DNA was associated with hyperchromic effect indicating local separation of the double helix. Energies of paired interactions between amino acid residues and nucleotide bases were compared with the energy of bond between two bases in the nucleotide pair (dA-dT). A new concept of interaction between two types of information-carrying molecules is put forward. This interaction underlies triggering of protein synthesis mechanism and can explain the emergence of life on the Earth.

Chromatin↗
PubMed1997

Profile of the DNA recognition site of the archaeal homing endonuclease I-DmoI.

I- Dmo I is a homing enzyme of the LAGLI-DADG type that recognizes up to 20 bp of DNA and is encoded by an archaeal intron of the hyperthermophilic archaeon Desulfurococcus mobilis . A combined mutational and DNA footprinting approach was employed to investigate the specificity of the I- Dmo I-substrate interaction. The results indicate that the enzyme binds primarily to short base paired regions that border the sites of DNA cleavage and intron insertion. The minimal substrate spans no more than 15 bp and while sequence degeneracy is tolerated in the DNA binding regions, the sequence and size of the cleavage region is highly conserved. The enzyme has a slow turnover rate and cuts the coding strand with a slight preference over the non-coding strand. Complex formation produces some distortion of the DNA double helix within the cleavage region. The data are compatible with the two DNA-binding domains of I- Dmo I bridging the minor groove, where cleavage occurs, and interacting within the major groove on either side, thereby stabilizing a distorted DNA double helix. This may provide a general mode of DNA interaction at least for the LAGLIDADG-type homing enzymes.

Amino Acid Sequence↗
PubMed1996

Protein-protein and protein-DNA interaction regions within the DNA end-binding protein Ku70-Ku86.

DNA ends are generated during double-strand-break repair and recombination. A p70-p86 heterodimer, Ku, accounts for the DNA end binding activity in eukaryotic cell extracts. When one or both subunits of Ku are missing, mammalian cells are deficient in double-strand-break repair and in specialized recombination, such as V(D)J recombination. Little is known of which regions of Ku70 and Ku86 bind to each other to form the heterodimeric complex or of which regions are important for DNA end binding. We have done genetic and biochemical studies to examine the domains within the two subunits important for protein assembly and for DNA end binding. We found that the C-terminal 20-kDa region of Ku70 and the C-terminal 32-kDa region of Ku86 are important for subunit-subunit interaction. For DNA binding, full-length individual subunits are inactive, indicating that heterodimer assembly precedes DNA binding. DNA end binding activity by the heterodimer requires the C-terminal 40-kDa region of Ku70 and the C-terminal 45-kDa region of Ku86. Leucine zipper-like motifs in both subunits that have been suggested as the Ku70-Ku86 interaction domains do not appear to be the sites of such interaction because these are dispensable for both assembly and DNA end binding. On the basis of these studies, we have organized Ku70 into nine sequence regions conserved between Saccharomyces cerevisiae, Drosophila melanogaster, mice, and humans; only the C-terminal three regions are essential for assembly (amino acids [aa] 439 to 609), and the C-terminal four regions appear to be essential for DNA end binding (aa 254 to 609). Within the minimal active fragment of Ku86 necessary for subunit interaction (aa 449 to 732) and DNA binding (aa 334 to 732), a proline-rich region is the only defined motif.

Amino Acid Sequence↗
PubMed1990

Characterization of the DNA binding activity of stable RecA-DNA complexes. Interaction between the two DNA binding sites within RecA helical filaments.

The DNA-binding, annealing and recombinational activities of purified RecA-DNA complexes stabilized by ATP gamma S (a slowly hydrolysable analog of ATP) are described. Electrophoretic analysis, DNase protection experiments and observations by electron microscopy suggest that saturated RecA complexes formed with single- or double-stranded DNA are able to accommodate an additional single strand of DNA with a stoichiometry of about one nucleotide of added single-stranded DNA per nucleotide or base-pair, respectively, of DNA resident in the complex. This strand uptake is independent of complementarity or homology between the added and resident DNA molecules. In the complex, the incoming and resident single-stranded DNA molecules are in close proximity as the two strands can anneal in case of their complementarity. Stable RecA complexes formed with single-stranded DNA bind double-stranded DNA efficiently when the added DNA is homologous to the complexed strand and then initiate a strand exchange reaction between the partner DNA molecules. Electron microscopy of the RecA-single-stranded DNA complexes associated with homologous double-stranded DNA suggests that a portion of duplex DNA is taken into the complex and placed in register with the resident single strand. Our experiments indicate that both DNA binding sites within RecA helical filaments can be occupied by either single- or double-stranded DNA. Presumably, the same first DNA binding site is used by RecA during its polymerization on single- or double-stranded DNA and the second DNA binding site becomes available for subsequent interaction of the protein-saturated complexes with naked DNA. The way by which additional DNA is taken into RecA-DNA complexes shows co-operative character and this helps to explain how topological problems are avoided during RecA-mediated homologous recombination.

Adenosine Triphosphate↗