Genomic footprinting of the yeast zinc finger protein Rme1p and its roles in repression of the meiotic activator IME1

The zinc finger protein Rme1p is a negative regulator of the meiotic activator IME1 in Saccharomyces cerevisiae . Prior studies have shown that Rme1p binds in vitro to a site near nt -2030 in the IME1 upstream region, but a genomic mutation in that site has little effect on repression of IME1 . To identify Rme1p binding sites in vivo , we have examined the binding of Rme1p to genomic sites through in vivo footprinting. We show that Rme1p binds to two sites in the IME1 upstream region, near nt -1950 and -2030. Mutations in both binding sites abolish repression of chromosomal IME1 by Rme1p, whereas a mutation in either single site causes partial derepression. Therefore, both Rme1p binding sites are essential for repression of IME1 . Prior studies have shown that repression by Rme1p depends upon RGR1 and SIN4 , which specify RNA polymerase II mediator subunits that are required for normal nucleosome density. We find that RGR1 and SIN4 are not simply required for Rme1p to bind to DNA in vivo . These results suggest that Rme1p functions directly as a repressor of IME1 and that Rgr1p and Sin4p are required for DNA-bound Rme1p to exert repression.     

Multiple domains of repressor activator protein 1 contribute to facilitated binding of glycolysis regulatory protein 1

The function of repressor activator protein 1 (Rap1p) at glycolytic enzyme gene upstream activating sequence (UAS) elements in Saccharomyces cerevisiae is to facilitate binding of glycolysis regulatory protein 1 (Gcr1p) at adjacent sites. Rap1p has a modular domain structure. In its amino terminus there is an asymmetric DNA-bending domain, which is distinct from its DNA-binding domain, which resides in the middle of the protein. In the carboxyl terminus of Rap1p lie its silencing and putative activation domains. We carried out a molecular dissection of Rap1p to identify domains contributing to its ability to facilitate binding of Gcr1p. We prepared full-length and three truncated versions of Rap1p and tested their ability to facilitate binding of Gcr1p by gel shift assay. The ability to detect ternary complexes containing Rap1p.DNA. Gcr1p depended on the presence of binding sites for both proteins in the probe DNA. The DNA-binding domain of Rap1p, although competent to bind DNA, was unable to facilitate binding of Gcr1p. Full-length Rap1p and the amino- and carboxyl-truncated versions of Rap1p were each able to facilitate binding of Gcr1p at an appropriately spaced binding site. Under these conditions, Gcr1p displayed an approximately 4-fold greater affinity for Rap1p-bound DNA than for otherwise identical free DNA. When spacing between Rap1p- and Gcr1p-binding sites was altered by insertion of five nucleotides, the ability to form ternary Rap1p.DNA.Gcr1p complexes was inhibited by all but the DNA-binding domain of Rap1p itself; however, the ability of each individual protein to bind the DNA probe was unaffected.     

Ndj1p, a meiotic telomere protein required for normal chromosome synapsis and segregation in yeast

The Saccharomyces cerevisiae gene NDJ1 (nondisjunction) encodes a protein that accumulates at telomeres during meiotic prophase. Deletion of NDJ1 (ndj1Delta) caused nondisjunction, impaired distributive segregation of linear chromosomes, and disordered the distribution of telomeric Rap1p, but it did not affect distributive segregation of circular plasmids. Induction of meiotic recombination and the extent of crossing-over were largely normal in ndj1Delta cells, but formation of axial elements and synapsis were delayed. Thus, Ndj1p may stabilize homologous DNA interactions at telomeres, and possibly at other sites, and it is required for a telomere activity in distributive segregation.     

DNA-binding properties of the yeast transcriptional activator, Gcr1p

In Saccharomyces cerevisiae the GCRI gene product is required for high-level expression of genes encoding glycolytic enzymes. In this communication, we extend our analysis of the DNA binding properties of Gcr1p. The DNA-binding domain of Gcr1p binds DNA with high affinity. The apparent dissociation constant of the Gcr1p DNA-binding domain for one of its specific binding sites (TTTCAGCTTCCTCTAT) is 2.9 x 10(-10) M. However, competition experiments showed that Gcr1p binds this site in vitro with a low degree of specificity. We measured a 33-fold difference between the ability of specific competitor and DNA of random sequence to inhibit the formation of nucleoprotein complexes between Gcr1p and a radiolabeled DNA probe containing its binding site. DNA band-shift experiments, utilizing probes of constant length in which the positions of Gcr1p-binding sites are varied relative to the ends, indicated that Gcr1p-DNA nucleoprotein complexes contain bent DNA. The implications of these findings in terms of the combinatorial interactions that occur at the upstream activating sequence elements of genes encoding glycolytic enzymes are discussed.     

Cloning, expression, and characterization of a DNA binding domain of gpNu1, a phage lambda DNA packaging protein

Terminase is an enzyme from bacteriophage lambda that is required for insertion of the viral genome into an empty pro-capsid. This enzyme is composed of the viral proteins gpNu1 (20.4 kDa) and gpA (73.3 kDa) in a holoenzyme complex. Current models for terminase assembly onto DNA suggest that gpNu1 binds to three repeating elements within a region of the lambda genome known as cosB which, in turn, stimulates the assembly of a gpA dimer at the cosN subsite. This prenicking complex is the first of several stable nucleoprotein intermediates required for DNA packaging. We have noted a hydrophobic region within the primary amino acid sequence of the terminase gpNu1 subunit and hypothesized that this region constitutes a protein-protein interaction domain required for cooperative assembly at cosB and that is also responsible for the observed aggregation behavior of the isolated protein. We therefore constructed a mutant of gpNu1 in which this hydrophobic "domain" has been deleted in order to test these hypotheses. The deletion mutant protein, gpNu1DeltaK, is fully soluble and, unlike full-length protein, shows no tendency toward aggregation; However, the protein is a dimer under all experimental conditions examined as determined by gel permeation and sedimentation equilibrium analysis. The truncated protein is folded with evidence of secondary and tertiary structural elements by circular dichroism and NMR spectroscopy. While physical and biological assays demonstrate that gpNu1DeltaK does not interact with the terminase gpA subunit, the deletion mutant binds with specificity to cos-containing DNA. We have thus constructed a deletion mutant of the phage lambda terminase gpNu1 subunit which constitutes a highly soluble DNA binding domain of the protein. We further propose that the hydrophobic amino acids found between Lys100 and Pro141 define a self-association domain that is required for the assembly of stable nucleoprotein packaging complexes and that the C-terminal tail of the protein defines a distinct gpA-binding site that is responsible for terminase holoenzyme formation.     

DNA binding properties of a chemically synthesized DNA binding domain of hRFX1

The RFX DNA binding domain (DBD) is a novel highly conserved motif belonging to a large number of dimeric DNA binding proteins which have diverse regulatory functions in eukaryotic organisms, ranging from yeasts to human. To characterize this novel motif, solid phase synthesis of a 76mer polypeptide corresponding to the DBD of human hRFX1 (hRFX1/DBD), a prototypical member of the RFX family, has been optimized to yield large quantities (approximately 90 mg) of pure compound. Preliminary two-dimensional1H NMR experiments suggested the presence of helical regions in this sequence in agreement with previously reported secondary structure predictions. In gel mobility shift assays, this synthetic peptide was shown to bind in a cooperative manner the 23mer duplex oligodeoxynucleotide corresponding to the binding site of hRFX1, with a 2:1 stoichoimetry due to an inverse repeat present in the 23mer. The stoichiometry of this complex was reduced to 1:1 by decreasing the length of the DNA sequence to a 13mer oligonucleotide containing a single half-site. Surface plasmon resonance measurements were achieved using this 5'-biotylinated 13mer oligonucleotide immobilized on an avidin-coated sensor chip. Using this method an association constant (K a = 4 x 10(5)/M/s), a dissociation constant (K d = 6 x 10(-2)/s) and an equilibrium dissociation constant (K D = 153 nM) were determined for binding of hRFX1/DBD to the double-stranded 13mer oligonucleotide. In the presence of hRFX1/DBD the melting temperature of the 13mer DNA was increased by 16 degreesC, illustrating stabilization of the double-stranded conformation induced by the peptide.     

Characterization of a novel DNA binding domain within the amino-terminal region of the RAG-1 protein

Rag-1 and Rag-2 are the critical components of the V-(D)-J recombinase required for site-specific recombination of the antigen receptor genes. In this study, we have examined the ability of recombinant (r) Rag-1 and Rag-2 to bind the recombination signal sequences (RSS) and have determined that rRag-1, but not rRag-2, is able to directly bind DNA. rRAG-1 DNA binding activity was found to reside within a novel amino-terminal arginine-rich (RR) domain with partial homology to a variety of nucleic acid binding domains. Although the RR-domain did not demonstrate RSS-specificity, this DNA binding domain may stabilize the interaction of RAG-1 with, or increase the affinity for, the V-(D)-J recombination signals.     

Hus1p, a conserved fission yeast checkpoint protein, interacts with Rad1p and is phosphorylated in response to DNA damage

The hus1+ gene is one of six fission yeast genes, termed the checkpoint rad genes, which are essential for both the S-M and DNA damage checkpoints. Classical genetics suggests that these genes are required for activation of the PI-3 kinase-related (PIK-R) protein, Rad3p. Using a dominant negative allele of hus1+, we have demonstrated a genetic interaction between hus1+ and another checkpoint rad gene, rad1+. Hus1p and Rad1p form a stable complex in wild-type fission yeast, and the formation of this complex is dependent on a third checkpoint rad gene, rad9+, suggesting that these three proteins may exist in a discrete complex in the absence of checkpoint activation. Hus1p is phosphorylated in response to DNA damage, and this requires rad3+ and each of the other checkpoint rad genes. Although there is no gene related to hus1+ in the Saccharomyces cerevisiae genome, we have identified closely related mouse and human genes, suggesting that aspects of the checkpoint control mechanism are conserved between fission yeast and higher eukaryotes.     

Localization of vitronectin binding domain in plasminogen activator inhibitor-1

Plasminogen activator inhibitor type 1 (PAI-1) is the rapid physiologic inhibitor of tissue-type plasminogen activator and urokinase-type plasminogen activator (uPA). In plasma and the extracellular matrix, PAI-1 is associated with the adhesive glycoprotein vitronectin. In order to characterize the PAI-1 structural domain responsible for binding to vitronectin, the segment of the PAI-1 cDNA encoding amino acids 13-147 (nucleotides 248-650) was randomly mutagenized and subcloned into a bacterial expression vector containing the mature PAI-1 coding sequence. Recombinant PAI-1 mutants were expressed in Escherichia coli and bacterial lysates assayed in duplicate for uPA inhibitory activity and vitronectin binding. Of 190 clones screened, six consistently demonstrated decreased vitronectin binding relative to uPA inhibitory activity. DNA sequence analysis of four of these clones identified 10 unique missense mutations, all located between base pairs 298 and 641, with each clone containing between one and four substitutions. Each substitution was expressed independently by site-directed mutagenesis and again analyzed for uPA inhibitory activity and vitronectin binding. Five point mutations that selectively disrupt vitronectin binding were identified. All 5 residues are located on the exterior of the PAI-1 structure. These findings appear to define a complex binding surface that bridges alpha-helices C and E to beta-strand 1A and includes amino acids 55, 109, 110, 116, and 123. These results suggest that vitronectin binding may stabilize the active conformation of PAI-1 by restricting the movement of beta-sheet A and thereby preventing insertion of the reactive center loop.     

Mec1p is essential for phosphorylation of the yeast DNA damage checkpoint protein Ddc1p, which physically interacts with Mec3p

Checkpoints prevent DNA replication or nuclear division when chromosomes are damaged. The Saccharomyces cerevisiae DDC1 gene belongs to the RAD17, MEC3 and RAD24 epistasis group which, together with RAD9, is proposed to act at the beginning of the DNA damage checkpoint pathway. Ddc1p is periodically phosphorylated during unperturbed cell cycle and hyperphosphorylated in response to DNA damage. We demonstrate that Ddc1p interacts physically in vivo with Mec3p, and this interaction requires Rad17p. We also show that phosphorylation of Ddc1p depends on the key checkpoint protein Mec1p and also on Rad24p, Rad17p and Mec3p. This suggests that Mec1p might act together with the Rad24 group of proteins at an early step of the DNA damage checkpoint response. On the other hand, Ddc1p phosphorylation is independent of Rad53p and Rad9p. Moreover, while Ddc1p is required for Rad53p phosphorylation, it does not play any major role in the phosphorylation of the anaphase inhibitor Pds1p, which requires RAD9 and MEC1. We suggest that Rad9p and Ddc1p might function in separated branches of the DNA damage checkpoint pathway, playing different roles in determining Mec1p activity and/or substrate specificity.     

DNA binding by the KP repressor protein inhibits P-element transposase activity in vitro

P elements are a family of mobile DNA elements found in Drosophila. P-element transposition is tightly regulated, and P-element-encoded repressor proteins are responsible for inhibiting transposition in vivo. To investigate the molecular mechanisms by which one of these repressors, the KP protein, inhibits transposition, a variety of mutant KP proteins were prepared and tested for their biochemical activities. The repressor activities of the wild-type and mutant KP proteins were tested in vitro using several different assays for P-element transposase activity. These studies indicate that the site-specific DNA-binding activity of the KP protein is essential for repressing transposase activity. The DNA-binding domain of the KP repressor protein is also shared with the transposase protein and resides in the N-terminal 88 amino acids. Within this region, there is a C2HC putative metal-binding motif that is required for site-specific DNA binding. In vitro the KP protein inhibits transposition by competing with the transposase enzyme for DNA-binding sites near the P-element termini.     

DNA bending is essential for the site-specific recognition of DNA response elements by the DNA binding domain of the tumor suppressor protein p53

We have used circular permutation assays to determine the extent and location of the DNA bend induced by the DNA binding domain of human wild type p53 (p53DBD) upon binding to several naturally occurring DNA response elements. We have found that p53DBD binding induces axial bending in all of the response elements investigated. In particular, response elements having a d(CATG) sequence at the junction of two consensus pentamers in each half-site favor highly bent complexes (bending angle is approximately 50 degrees ), whereas response elements having d(CTTG) bases at this position are less bent (bending angles from approximately 37 to approximately 25 degrees ). Quantitative electrophoretic mobility shift assays of different complexes show a direct correlation between the DNA bending angle and the binding affinity of the p53DBD with the response elements, i.e. the greater the stability of the complex, the more the DNA is bent by p53DBD binding. The study provides evidence that the energetics of DNA bending, as determined by the presence or absence of flexible sites in the response elements, may contribute significantly to the overall binding affinity of the p53DBD for different sequences. The results therefore suggest that both the structure and the stability of the p53-DNA complex may vary with different response elements. This variability may be correlated with variability in p53 function.     

Four hydrophobic amino acid residues in the C-terminal effector domain of the yeast Mig1p repressor are important for its in vivo activity

The Mig1 repressor is a zinc finger protein that mediates glucose repression in yeast. Previous work in Saccharomyces cerevisiae has shown that two domains in Miglp are required for repression: the N-terminal zinc finger region and a C-terminal effector domain. Both domains are also conserved in Miglp homologs from the distantly related yeasts Kluyveromyces lactis and K. marxianus, and these Mig1 proteins can fully replace the endogenous Mig1p in S. cerevisiae. We have now made a detailed analysis of the conserved C-terminal effector domain in Mig1p from K. marxianus, using expression in S. cerevisiae to monitor its function. First, a series of small deletions were made within the effector domain. Second, an alanine scan mutagenesis was carried out across the effector domain. Third, double, triple and quadruple mutants were made that affect certain residues within the effector domain. Our results show that four conserved residues within the effector domain, three leucines and one isoleucine, are particularly important for its function in vivo. The analysis further revealed that while the C-terminal effector domain of KmMig1p mediates a seven- to nine-fold repression of the reporter gene, a five- to sixfold residual effect also exists that is independent of the C-terminal effector domain. Similar results were obtained when the corresponding mutations were made in ScMig1p. Moreover, we found that mutations in these residues affect the interaction between Mig1p and the general corepressor subunit Cyc8p (Ssn6p). Modeling of the C-terminal effector domain using a protein of known structure suggests that it may be folded into an alpha-helix.     

The high mobility group protein 1 enhances binding of the estrogen receptor DNA binding domain to the estrogen response element

We have examined the ability of the high-mobility group protein 1 (HMG1) to alter binding of the estrogen receptor DNA-binding domain (DBD) to the estrogen response element (ERE). HMG1 dramatically enhanced binding of purified, bacterially expressed DBD to the consensus vitellogenin A2 ERE in a dose-dependent manner. The ability of HMG1 to stabilize the DBD-ERE complex resulted in part from a decrease in the dissociation rate of the DBD from the ERE. Antibody supershift experiments demonstrated that HMG1 was also capable of forming a ternary complex with the ERE-bound DBD in the presence of HMG1-specific antibody. HMG1 did not substantially affect DBD-ERE contacts as assessed by methylation interference assays, nor did it alter the ability of the DBD to induce distortion in ERE-containing DNA fragments. Because HMG1 dramatically enhanced estrogen receptor DBD binding to the ERE, and the DBD is the most highly conserved region among the nuclear receptor superfamily members, HMG1 may function to enhance binding of other nuclear receptors to their respective response elements and act in concert with coactivator proteins to regulate expression of hormone-responsive genes.     

Datin, a yeast poly(dA:dT)-binding protein, behaves as an activator of the wild-type ILV1 promoter and interacts synergistically with Reb1p

We isolated a ganciclovir (GCV)-resistant human cytomegalovirus (HCMV) from a laboratory strain, AD169, and analysed the mutant. Attempts were also made to identify directly the mutated gene. The 50% inhibitory concentration (IC50) of GCV for the mutant strain was five times higher than that of the wild-type strain. The mutant strain showed similar sensitivity to phosphonoacetic acid and cidofovir as the wild-type strain. These data suggest mutation in the UL97 gene encoding for the phosphotransferase that phosphorylates GCV. Molecular analysis of the mutant strain revealed that a single base substitution of adenine by cytosine occurred at the 1796 nucleotide position of the UL97 gene region, resulting in the substitution of lysine by threonine at codon 599 in the UL97 gene product. Marker transfer experiment confirmed that this mutation conferred HCMV resistance to GCV. The mutation at codon 599 was easily identified by means of RsaI digestion of the selected PCR product.