Detectable DNA-binding Activity Research Articles

The oxidative DNA glycosylases of Mycobacterium tuberculosis exhibit different substrate preferences from their Escherichia coli counterparts

The DNA glycosylases that remove oxidized DNA bases fall into two general families: the Fpg/Nei family and the Nth superfamily. Based on protein sequence alignments, we identified four putative Fpg/Nei family members, as well as a putative Nth protein in Mycobacterium tuberculosis H37Rv. All four Fpg/Nei proteins were successfully overexpressed using a bicistronic vector created in our laboratory. The MtuNth protein was also overexpressed in soluble form. The substrate specificities of the purified enzymes were characterized in vitro with oligodeoxynucleotide substrates containing single lesions. Some were further characterized by gas chromatography/mass spectrometry (GC/MS) analysis of products released from γ-irradiated DNA. MtuFpg1 has substrate specificity similar to that of EcoFpg. Both EcoFpg and MtuFpg1 are more efficient at removing spiroiminodihydantoin (Sp) than 7,8-dihydro-8-oxoguanine (8-oxoG). However, MtuFpg1 shows a substantially increased opposite base discrimination compared to EcoFpg. MtuFpg2 contains only the C-terminal domain of an Fpg protein and has no detectable DNA binding activity or DNA glycosylase/lyase activity and thus appears to be a pseudogene. MtuNei1 recognizes oxidized pyrimidines on both double-stranded and single-stranded DNA and exhibits uracil DNA glycosylase activity. MtuNth recognizes a variety of oxidized bases, including urea, 5,6-dihydrouracil (DHU), 5-hydroxyuracil (5-OHU), 5-hydroxycytosine (5-OHC) and methylhydantoin (MeHyd). Both MtuNei1 and MtuNth excise thymine glycol (Tg); however, MtuNei1 strongly prefers the (5 R) isomers, whereas MtuNth recognizes only the (5 S) isomers. MtuNei2 did not demonstrate activity in vitro as a recombinant protein, but like MtuNei1 when expressed in Escherichia coli, it decreased the spontaneous mutation frequency of both the fpg mutY nei triple and nei nth double mutants, suggesting that MtuNei2 is functionally active in vivo recognizing both guanine and cytosine oxidation products. The kinetic parameters of the MtuFpg1, MtuNei1 and MtuNth proteins on selected substrates were also determined and compared to those of their E. coli homologs.

Structure of the τ60/Δτ91 Subcomplex of Yeast Transcription Factor IIIC: Insights into Preinitiation Complex Assembly

Yeast RNA polymerase III is recruited upon binding of subcomplexes tauA and tauB of transcription factor IIIC (TFIIIC) to the A and B blocks of tRNA gene promoters. The tauB subcomplex consists of subunits tau60, tau91, and tau138. We determined the 3.2 A crystal structure of tau60 bound to a large C-terminal fragment of tau91 (Deltatau91). Deltatau91 protein contains a seven-bladed propeller preceded by an N-terminal extension, whereas tau60 contains a structurally homologous propeller followed by a C-terminal domain with a novel alpha/beta fold. The two propeller domains do not have any detectable DNA binding activity and mediate heterodimer formation that may serve as scaffold for tau138 assembly. We show that the C-terminal tau60 domain interacts with the TATA binding protein (TBP). Recombinant tauB recruits TBP and stimulates TFIIIB-directed transcription on a TATA box containing tRNA gene, implying a combined contribution of tauA and tauB to preinitiation complex formation.

Mutations in the Spacer Region of Drosophila Mitochondrial DNA Polymerase Affect DNA Binding, Processivity, and the Balance between Pol and Exo Function

The catalytic subunit (alpha) of mitochondrial DNA polymerase (pol gamma) shares conserved DNA polymerase and 3'-5' exonuclease active site motifs with Escherichia coli DNA polymerase I and bacteriophage T7 DNA polymerase. A major difference between the prokaryotic and mitochondrial proteins is the size and sequence of the region between the exonuclease and DNA polymerase domains, referred to as the spacer in pol gamma-alpha. Four gamma-specific conserved sequence elements are located within the spacer region of the catalytic subunit in eukaryotic species from yeast to humans. To elucidate the functional roles of the spacer region, we pursued deletion and site-directed mutagenesis of Drosophila pol gamma. Mutant proteins were expressed from baculovirus constructs in insect cells, purified to near homogeneity, and analyzed biochemically. We find that mutations in three of the four conserved sequence elements within the spacer alter enzyme activity, processivity, and/or DNA binding affinity. In addition, several mutations affect differentially DNA polymerase and exonuclease activity and/or functional interactions with mitochondrial single-stranded DNA-binding protein. Based on these results and crystallographic evidence showing that the template-primer binds in a cleft between the exonuclease and DNA polymerase domains in family A DNA polymerases, we propose that conserved sequences within the spacer of pol gamma may position the substrate with respect to the enzyme catalytic domains.

Transfection of keratinocytes abrogates detectable DNA-binding activity of CCAAT displacement protein.

Transfection of keratinocytes with plasmid DNA leads to the loss of detectable DNA-binding activity of CCAAT displacement protein but not of Yin Yang 1, as monitored by electrophoretic mobility shift assay. This phenomenon was found to be attributable to the presence of plasmid DNA in the nuclear extracts prepared from transfected cells. Treatment of these nuclear extracts with DNase I restored the ability to monitor DNA-binding activity of CDP. This report documents a new pitfall associated with transfection.

Cloning and functional characterization of an NAD(+)-dependent DNA ligase from Staphylococcus aureus.

A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus ligA gene. Orthologues of the putative S. aureus NAD(+)-dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli, and the enzyme was purified to near homogeneity. NAD(+)-dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of (32)P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD(+)-dependent DNA ligase from B. stearothermophilus, two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.

Dominant Negative Mutants Implicate STAT5 in Myeloid Cell Proliferation and Neutrophil Differentiation

STAT5 is a member of the signal transducers and activation of transcription (STAT) family of latent transcription factors activated in a variety of cytokine signaling pathways. We introduced alanine substitution mutations in highly conserved regions of murine STAT5A and studied the mutants for dimerization, DNA binding, transactivation, and dominant negative effects on erythropoietin-induced STAT5-dependent transcriptional activation. The mutations included two near the amino-terminus (W255KR→AAA and R290QQ→AAA), two in the DNA-binding domain (E437E→AA and V466VV→AAA), and a carboxy-terminal truncation of STAT5A (STAT5A/▵53C) analogous to a naturally occurring isoform of rat STAT5B. All of the STAT mutant proteins were tyrosine phosphorylated by JAK2 and heterodimerized with STAT5B except for the WKR mutant, suggesting an important role for this region in STAT5 for stabilizing dimerization. The WKR, EE, and VVV mutants had no detectable DNA-binding activity, and the WKR and VVV mutants, but not EE, were defective in transcriptional induction. The VVV mutant had a moderate dominant negative effect on erythropoietin-induced STAT5 transcriptional activation, which was likely due to the formation of heterodimers that are defective in DNA binding. Interestingly, the WKR mutant had a potent dominant negative effect, comparable to the transactivation domain deletion mutant, ▵53C. Stable expression of either the WKR or ▵53C STAT5 mutants in the murine myeloid cytokine-dependent cell line 32D inhibited both interleukin-3–dependent proliferation and granulocyte colony-stimulating factor (G-CSF)–dependent differentiation, without induction of apoptosis. Expression of these mutants in primary murine bone marrow inhibited G-CSF–dependent granulocyte colony formation in vitro. These results demonstrate that mutations in distinct regions of STAT5 exert dominant negative effects on cytokine signaling, likely through different mechanisms, and suggest a role for STAT5 in proliferation and differentiation of myeloid cells.

Dominant Negative Mutants Implicate STAT5 in Myeloid Cell Proliferation and Neutrophil Differentiation

AbstractSTAT5 is a member of the signal transducers and activation of transcription (STAT) family of latent transcription factors activated in a variety of cytokine signaling pathways. We introduced alanine substitution mutations in highly conserved regions of murine STAT5A and studied the mutants for dimerization, DNA binding, transactivation, and dominant negative effects on erythropoietin-induced STAT5-dependent transcriptional activation. The mutations included two near the amino-terminus (W255KR→AAA and R290QQ→AAA), two in the DNA-binding domain (E437E→AA and V466VV→AAA), and a carboxy-terminal truncation of STAT5A (STAT5A/▵53C) analogous to a naturally occurring isoform of rat STAT5B. All of the STAT mutant proteins were tyrosine phosphorylated by JAK2 and heterodimerized with STAT5B except for the WKR mutant, suggesting an important role for this region in STAT5 for stabilizing dimerization. The WKR, EE, and VVV mutants had no detectable DNA-binding activity, and the WKR and VVV mutants, but not EE, were defective in transcriptional induction. The VVV mutant had a moderate dominant negative effect on erythropoietin-induced STAT5 transcriptional activation, which was likely due to the formation of heterodimers that are defective in DNA binding. Interestingly, the WKR mutant had a potent dominant negative effect, comparable to the transactivation domain deletion mutant, ▵53C. Stable expression of either the WKR or ▵53C STAT5 mutants in the murine myeloid cytokine-dependent cell line 32D inhibited both interleukin-3–dependent proliferation and granulocyte colony-stimulating factor (G-CSF)–dependent differentiation, without induction of apoptosis. Expression of these mutants in primary murine bone marrow inhibited G-CSF–dependent granulocyte colony formation in vitro. These results demonstrate that mutations in distinct regions of STAT5 exert dominant negative effects on cytokine signaling, likely through different mechanisms, and suggest a role for STAT5 in proliferation and differentiation of myeloid cells.

The Zn(II) Binding Motifs ofE. coliDNA Topoisomerase I Is Part of a High-Affinity DNA Binding Domain

Escherichia coliDNA topoisomerase I binds three Zn(II) with three tetracysteine motifs. Three subclones containing these tetracysteine motifs were expressed and purified. Subclone ZD1 contained the minimal tetracysteine motifs sequence. A larger subclone ZD2 corresponded to a region bordered by two protease sensitive sites. Subclone ZD3 also included the 14-kDa C-terminal domain that has been shown to bind DNA. Subclones ZD1 and ZD2 were found to bind one and two Zn(II), respectively, and neither had detectable DNA binding activity. ZD3 could bind three Zn(II) and had higher DNA binding affinity than the 14-kDa C-terminal domain. The complex formed between ZD3 and a single-stranded 31mer could be detected by the gel shift assay while the complex formed by the 14-kDa C-terminal domain was not stable under gel electrophoresis conditions. The three Zn(II) binding motifs appeared to be part of a high-affinity DNA binding domain.

Molecular characterization of the DNA-binding and dimerization domains of the bZIP transcription factor, EmBP-1.

The wheat basic-leucine zipper (bZIP) DNA-binding protein EmBP-1 has been implicated in the mechanisms of abscisic acid (ABA) mediated gene regulation. Sequence and structural homology to the yeast bZIP protein GCN4 has been used to predict the location of the functional domains of EmBP-1. In order to test these predictions, the presumptive DNA-binding and dimerization domains of EmBP-1 were mapped by producing a series of truncated protein fragments and functionally testing them in vitro. Deletion of 5 amino acids of the predicted basic domain resulted in a loss of all DNA-binding activity. A fragment containing all six leucine repeat elements showed strong DNA-binding activity. Sequential deletion of the leucine repeat elements resulted in first an increase in DNA-binding activity (-L6 and -L5) followed by a reduction in binding activity (-L4) and eventually complete elimination of all detectable DNA-binding activity (-L3 and -L2). This demonstrates the importance of an intact leucine zipper domain of at least 4 repeat elements for efficient DNA-binding. The smallest polypeptide that retained DNA-binding activity is a fragment spanning amino acid residues 248-308 (ca. 8.4 kDa) consisting of minimal basic and leucine zipper domains. Dimerization of EmBP-1 was demonstrated by co-translation of fragments of differing molecular weights and identification of a DNA-protein complex with intermediate mobility to that produced by each fragment alone. A unique leucine-proline repeat element found N-terminal to the DNA-binding domain of EmBP-1 does not appear to play a role in DNA-binding or dimerization. These results confirm the locations of the functional domains of EmBP-1 predicted by similarity to GCN4. The high degree of functional conservation of the bZIP proteins spanning organisms from plants to fungi highlights the ancient origin of this class of transcription factors and of the mechanisms of gene regulation in which they participate.

Characterization of baculovirus recombinant wild-type p53. Dimerization of p53 is required for high-affinity DNA binding and cysteine oxidation inhibits p53 DNA binding.

A high-yield, rapid and non-denaturing purification protocol for baculovirus recombinant wild-type p53 is described. Gel-filtration chromatography and chemical cross-linking experiments indicated that purified p53 assembles into multimeric forms ranging from tetramer to higher oligomers. A gel-mobility-shift assay and protein-DNA cross-linking studies demonstrated that purified baculovirus recombinant p53 binds to consensus DNA target as a dimer but that additional p53 molecules may then associate with the preformed p53-dimer-DNA complexes to form larger p53 DNA complexes. These observations suggest that the p53 tetramers and higher oligomers that form the minimal p53 association in solution dissociate upon DNA binding to form p53 dimer-DNA complexes. Binding of the mAB PAb 421 to the oligomerization-promoting domain on p53 stimulated sequentially formation of both p53-dimer-DNA and larger p53-DNA complexes. This observation suggests that factors may exist in vivo that could participate in the formation and the stabilization of the various p53-DNA complexes. Further characterization of the purified p53 revealed that the protein possesses highly reactive cysteine residues. We show that intrachain disulfide bonds form within the purified p53 molecules during storage in the absence of reducing agent. Zn2+ binding to p53 protect sulfhydryl groups from oxidation. Cysteine oxidation by intramolecular disulfide-bond formation did not modify the wild-type immunoreactive phenotype of the p53 protein but totally inhibited its DNA-binding activities. The oxidation of the p53 cysteine residues was also observed for nuclear p53 in baculovirus-infected insect cells. The redox status of the nuclear p53 regulates its DNA-binding activity in vitro confirming the essential role of the reduced state of cysteine residues in p53 for detectable DNA-binding activity.

Functional analysis of the herpes simplex virus UL42 protein

The herpes simplex virus UL42 gene encodes a multifunctional polypeptide (UL42) that is essential for virus DNA replication. To further understand the relationship between the structure of UL42 and the role that it plays during virus replication, we analyzed an extensive set of mutant UL42 proteins for the ability to perform the three major biochemical functions ascribed to the protein:binding to DNA, stably associating with the virus DNA polymerase (Pol), and acting to increase the length of DNA chains synthesized by Pol. Selected mutants were also assayed for their ability to complement the replication of a UL42 null virus. The results indicated that the N-terminal 340 amino acids of UL42 were sufficient for all three biochemical activities and could also support virus replication. Progressive C-terminal truncation resulted in the loss of detectable DNA-binding activity before Pol binding, while several mutations near the N terminus of the polypeptide resulted in an altered interaction with DNA but had no apparent affect on Pol binding. More dramatically, an insertion mutation at residue 160 destroyed the ability to bind Pol but had no effect on DNA binding. This altered polypeptide also failed to increase the length of DNA product synthesized by Pol, and the mutant gene could not complement the growth of a UL42 null virus, indicating that the specific interaction between Pol and UL42 is necessary for full Pol function and for virus replication. This study confirms the validity of the Pol-UL42 interaction as a target for the design of novel therapeutic agents.

Synergistic activation of transcription is mediated by the N-terminal domain of Drosophila fushi tarazu homeoprotein and can occur without DNA binding by the protein.

Synergistic activation of transcription by Drosophila segmentation genes in tissue culture cells provides a model with which to study combinatorial regulation. We examined the synergistic activation of an engrailed-derived promoter by the pair-rule proteins paired (PRD) and fushi tarazu (FTZ). Synergistic activation by PRD requires regions of the homeodomain or adjacent sequences, and that by FTZ requires the first 171 residues. Surprisingly, deletion of the FTZ homeodomain does not reduce the capacity of the protein for synergistic activation, although this mutation abolishes any detectable DNA-binding activity. This finding suggests that FTZ can function through protein-protein interactions with PRD or other components of the homeoprotein transcription complex, adding a new layer of mechanisms that could underlie the functional specificities and combinatorial regulation of homeoproteins.

Synergistic Activation of Transcription Is Mediated by the N-Terminal Domain of Drosophila fushi tarazu Homeoprotein and Can Occur without DNA Binding by the Protein

Synergistic activation of transcription by Drosophila segmentation genes in tissue culture cells provides a model with which to study combinatorial regulation. We examined the synergistic activation of an engrailed-derived promoter by the pair-rule proteins paired (PRD) and fushi tarazu (FTZ). Synergistic activation by PRD requires regions of the homeodomain or adjacent sequences, and that by FTZ requires the first 171 residues. Surprisingly, deletion of the FTZ homeodomain does not reduce the capacity of the protein for synergistic activation, although this mutation abolishes any detectable DNA-binding activity. This finding suggests that FTZ can function through protein-protein interactions with PRD or other components of the homeoprotein transcription complex, adding a new layer of mechanisms that could underlie the functional specificities and combinatorial regulation of homeoproteins.

Myogenin resides in the nucleus and acquires high affinity for a conserved enhancer element on heterodimerization.

Myogenin is a member of a family of muscle-specific factors that can activate the muscle differentiation program in nonmyogenin cells. Using antibodies directed against unique domains of myogenin, we show in the present study that myogenin resides in the nucleus of differentiated muscle cells. Myogenin translated in vitro does not exhibit detectable DNA binding activity; however, when dimerized with the ubiquitous enhancer-binding factor E12, it acquires high affinity for an element in the core of the muscle creatine kinase (MCK) enhancer that is conserved among many muscle-specific genes. Antibody disruption experiments show that myogenin, synthesized during differentiation of the BC3H1 and C2 muscle cell lines, is part of a complex that binds to the same site in the MCK enhancer as myogenin-E12 translated in vitro. Mutagenesis of the myogenin-E12-binding site in the MCK enhancer abolishes binding of the heterodimer and prevents trans-activation of the enhancer by myogenin. The properties of myogenin suggest that its functions as a sequence-specific DNA-binding factor that interacts directly with muscle-specific genes during myogenesis. The dependence of myogenin on E12 for high-affinity DNA binding activity also suggests that the susceptibility of various cell types to the actions of myogenin may be influenced by the cellular factors with which it may interact.

Trypsin cleavage of chick 1,25-dihydroxyvitamin D3 receptors. Generation of discrete polypeptides which retain hormone but are unreactive to DNA and monoclonal antibody.

Intestinal cytosol receptors for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) were subjected to limited trypsin digestion, and the properties of the resulting discrete polypeptide fragments were identified and contrasted with the native 1,25(OH)2D3 receptor. Physical characterization was achieved through sedimentation analysis, gel filtration chromatography, and DEAE anion exchange high performance liquid chromatography. Intactness of functional ligand-binding domains was evaluated by assessing macromolecular retention of 1,25(OH)2D3 as well as by determining reactivity to DNA and monoclonal antibody. While two differentially trypsin-sensitive effects on the 1,25(OH)2D3 receptor were noted, both produced a major polypeptide fragment which retained 1,25(OH)2D3. Action within region I (1 microgram of trypsin/A280-A310) had no effect on net charge but significantly decreased the Stokes radius of the 1,25(OH)2D3 receptor from 3.6 nm (60,000 daltons) to 3.2 nm, concomitant with a significant reduction in receptor aggregational capacity. This large hormone-bound fragment did not elicit detectable DNA-binding activity, and only a portion displayed reactivity to monoclonal antibody. Activity within region II (25 micrograms of trypsin/A280-A310) resulted in a less charged, more globular macromolecule with a Stokes radius of 2.9 nm which was completely unreactive to monoclonal antibody. Immunoblot methodology confirmed the protease-dependent loss of immunologic reactivity of the 60,000-dalton 1,25(OH)2D3 receptor and correspondingly identified receptor fragments of 50,000 and 20,000 daltons displaying positive immunologic reactivity. These studies provide the first evidence for the distinct nature of the molecular domains for 1,25(OH)2D3 and DNA on 1,25(OH)2D3 receptors while confirming the close spatial relationship between interactive sites for DNA and monoclonal antibody.