High-affinity DNA Binding Research Articles

Structural studies of the high mobility group globular domain and basic tail of HMG-D bound to disulfide cross-linked DNA.

HMG-D is an abundant high mobility group chromosomal protein present during early embryogenesis in Drosophila melanogaster. It is a non-sequence-specific member of a protein family that uses the HMG domain for binding to DNA in the minor groove. The highly charged C-terminal tail of HMG-D contains AK motifs that contribute to high-affinity non-sequence-specific DNA binding. To understand the interactions of the HMG domain and C-terminal tail of HMG-D with DNA in solution, a complex between a high-affinity truncated form of the protein and a disulfide cross-linked DNA fragment was studied using heteronuclear NMR techniques. Despite its relatively high affinity for the single "prebent" site on the DNA, K(d) = 1.4 nM, HMG-D forms a non-sequence-specific complex with the DNA as indicated by exchange broadening of the protein resonances at the DNA interface in solution. The secondary structural elements of the protein are preserved when the protein is complexed with the DNA, and the DNA-binding interface maps to the regions of the protein where the largest chemical shift differences occur. The C-terminal tail of HMG-D confers high-affinity DNA binding, has an undefined structure, and appears to make direct contacts in the major groove of DNA via residues that are potentially regulated by phosphorylation. We conclude that while the HMG domain of HMG-D recognizes DNA with a mode of binding similar to that used by the sequence-specific HMG domain transcription factors, there are noteworthy differences in the structure and interactions of the C-terminal end of the DNA-binding domain and the C-terminal tail.

Identification of the chromosomal replication origin from Thermus thermophilus and its interaction with the replication initiator DnaA

The chromosomal replication origin oriC and the gene encoding the replication initiator protein DnaA from Thermus thermophilus have been identified and cloned into an Escherichia coli vector system. The replication origin is composed of 13 characteristically arranged DnaA boxes, binding sites for the DnaA protein, and an AT-rich stretch, followed by the dnaN gene. The dnaA gene is located upstream of the origin and expresses a typical DnaA protein that follows the division into four domains, as with other members of the DnaA protein family. Here, we report the purification of Thermus-DnaA (Tth-DnaA) and characterize the interaction of the purified protein with the replication origin, with regard to the binding kinetics and stoichiometry of this interaction. Using gel retardation assays, surface plasmon resonance (SPR) and electron microscopy, we show that, unlike the E. coli DnaA, Tth-DnaA does not recognize a single DnaA box, instead a cluster of three tandemly repeated DnaA boxes is the minimal requirement for specific binding. The highest binding affinities are observed with full-length oriC or six clustered, tandemly repeated DnaA boxes. Furthermore, high-affinity DNA-binding of Tth-DnaA is dependent on the presence of ATP. The Thermus DnaA/oriC interaction will be compared with oriC complex formation generated by other DnaA proteins.

Mapping the DNA Binding Domain of the Zap1 Zinc-responsive Transcriptional Activator

The Zap1 transcriptional activator of Saccharomyces cerevisiae plays a major role in zinc homeostasis by inducing the expression of several genes under zinc-limited growth conditions. This activation of gene expression is mediated by binding of the protein to one or more zinc-responsive elements present in the promoters of its target genes. To better understand how Zap1 functions, we mapped its DNA binding domain using a combined in vivo and in vitro approach. Our results show that the Zap1 DNA binding domain maps to the carboxyl-terminal 194 amino acids of the protein; this region contains five of its seven potential zinc finger domains. Fusing this region to the Gal4 activation domain complemented a zap1Delta mutation for low zinc growth and also conferred high level expression on a zinc-responsive element-lacZ reporter. In vitro, the purified 194-residue fragment bound to DNA with a high affinity (dissociation constant in the low nanomolar range) similar to that of longer fragments of Zap1. Furthermore, by deletion and site-directed mutagenesis, we demonstrated that each of the five carboxyl-terminal zinc fingers are required for high affinity DNA binding.

Biophysical characterization of interactions between the core binding factor α and β subunits and DNA

Core binding factors (CBFs) play key roles in several developmental pathways and in human disease. CBFs consist of a DNA binding CBFα subunit and a non-DNA binding CBFβ subunit that increases the affinity of CBFα for DNA. We performed sedimentation equilibrium analyses to unequivocally establish the stoichiometry of the CBFα:β:DNA complex. Dissociation constants for all four equilibria involving the CBFα Runt domain, CBFβ, and DNA were defined. Conformational changes associated with interactions between CBFα, CBFβ, and DNA were monitored by nuclear magnetic resonance and circular dichroism spectroscopy. The data suggest that CBFβ ‘locks in’ a high affinity DNA binding conformation of the CBFα Runt domain.

Triple-helix formation induces recombination in mammalian cells via a nucleotide excision repair-dependent pathway.

The ability to stimulate recombination in a site-specific manner in mammalian cells may provide a useful tool for gene knockout and a valuable strategy for gene therapy. We previously demonstrated that psoralen adducts targeted by triple-helix-forming oligonucleotides (TFOs) could induce recombination between tandem repeats of a supF reporter gene in a simian virus 40 vector in monkey COS cells. Based on work showing that triple helices, even in the absence of associated psoralen adducts, are able to provoke DNA repair and cause mutations, we asked whether intermolecular triplexes could stimulate recombination. Here, we report that triple-helix formation itself is capable of promoting recombination and that this effect is dependent on a functional nucleotide excision repair (NER) pathway. Transfection of COS cells carrying the dual supF vector with a purine-rich TFO, AG30, designed to bind as a third strand to a region between the two mutant supF genes yielded recombinants at a frequency of 0.37%, fivefold above background, whereas a scrambled sequence control oligomer was ineffective. In human cells deficient in the NER factor XPA, the ability of AG30 to induce recombination was eliminated, but it was restored in a corrected subline expressing the XPA cDNA. In comparison, the ability of triplex-directed psoralen cross-links to induce recombination was only partially reduced in XPA-deficient cells, suggesting that NER is not the only pathway that can metabolize targeted psoralen photoadducts into recombinagenic intermediates. Interestingly, the triplex-induced recombination was unaffected in cells deficient in DNA mismatch repair, challenging our previous model of a heteroduplex intermediate and supporting a model based on end joining. This work demonstrates that oligonucleotide-mediated triplex formation can be recombinagenic, providing the basis for a potential strategy to direct genome modification by using high-affinity DNA binding ligands.

Specific Heterochromatic Banding of Metaphase Chromosomes Using Nuclear Yellow

The bis-benzimidazole compound nuclear yellow (NY) belongs to the same chemical family as the DNA binding fluorochromes Hoechst 33258 and Hoechst 33342. Spectroscopic studies of NY alone and in the presence of calf thymus DNA show high DNA binding affinity and behavior similar to the Hoechst fluorochromes above. Mitotic metaphase chromosomes from Balb/c mice stained with NY show C-banding and weak G/Q-banding, both of them disappearing after distamycin A (DA) or methyl green (MG) counterstaining. The same staining of human metaphase chromosomes from lymphocyte cultures, however, reveal only faint G/Q-banding (NY) and a characteristic DA-DAPI-like banding (NY-DA, NY-MG). Image analysis of NY stained human chromosomes, confirms that NY is suitable for studying polymorphisms affecting size in the pericentromeric hete-rochromatin of pairs 1, 9 and 16, and shows significant enhancement of NY fluorescence induced by DA in DA-DAPI heterochromatin. Our spectroscopic and cytological results show that NY, either alone or counterstained with DA or MG, can be used for DNA cytochemistry and chromosome banding. Possible mechanisms for the banding patterns induced by NY are discussed.

10th Conference on DNA Topoisomerases in therapy.

The 10th Conference on DNA Topoisomerases in Therapy 6-8 October 1999 in Amsterdam, The Netherlands) covered basic research on DNA topoisomerases and aspects of topoisomerase-directed therapy. The understanding of basic aspects of enzyme functions and structures was discussed throughout the meeting, as this knowledge is fundamental to further developments of new and more effective therapeutic approaches. Several new crystal structures were presented, and implications for function and interaction with DNA and drugs were discussed. Knock-out mice for various topoisomerase genes have been produced and genes have been shown to differ in importance for development and survival. The interaction of topoisomerases with other proteins involved in DNA metabolism, chromosome stability and physiology were discussed. The main focus for cancer therapy was on camptothecins (CPT) and related compounds stabilizing covalent DNA-intermediates of topoisomerase I. Reports on recent clinical trials of first-generation, water-soluble CPT-analogs (topotecan and irinotecan) confirmed earlier findings of activity in several solid tumors and hematological malignancies. Improvements in efficacy and toxicity profiles are being sought in orally absorbable compounds and other drug formulations (e.g. in liposomes). Several new CPT-analogs at preclinical stages of development might also provide a greater stability of the lactone ring, higher DNA-binding affinity, and improved water solubility. New drugs have also been developed from a number of new non-CPT compounds, which inhibit the activity of DNA-topoisomerases but do not stabilize the DNA-linked form of the enzymes. Another focus of the meeting was on therapeutic targeting of microbial DNA topoisomerases. The antibiotic potential of the quinolones has been extended to gram-positive pathogens, particularly Streptococcus pneumoniae. The cloning and biochemical characterization of the DNA-topoisomerases of eukaryotic parasites such as Plasmodium falciparum or Candida albicans have been completed and the search for specific inhibitors targeting these enzymes is under way. Copyright 1999 Harcourt Publishers Ltd.

An ATP/ADP-Dependent Molecular Switch Regulates the Stability of p53-DNA Complexes

Interaction with DNA is essential for the tumor suppressor functions of p53. We now show, for the first time, that the interaction of p53 with DNA can be stabilized by small molecules, such as ADP and dADP. Our results also indicate an ATP/ADP molecular switch mechanism which determines the off-on states for p53-DNA binding. This ATP/ADP molecular switch requires dimer-dimer interaction of the p53 tetramer. Dissociation of p53-DNA complexes by ATP is independent of ATP hydrolysis. Low-level ATPase activity is nonetheless associated with ATP-p53 interaction and may serve to regenerate ADP-p53, thus recycling the high-affinity DNA binding form of p53. The ATP/ADP regulatory mechanism applies to two distinct types of p53 interaction with DNA, namely, sequence-specific DNA binding (via the core domain of the p53 protein) and binding to sites of DNA damage (via the C-terminal domain). Further studies indicate that ADP not only stabilizes p53-DNA complexes but also renders the complexes susceptible to dissociation by specific p53 binding proteins. We propose a model in which the DNA binding functions of p53 are regulated by an ATP/ADP molecular switch, and we suggest that this mechanism may function during the cellular response to DNA damage.

Ku is associated with the telomere in mammals.

Telomeres are specialized DNA/protein complexes that comprise the ends of eukaryotic chromosomes. The highly expressed Ku heterodimer, composed of 70 and 80 K(d) subunits (Ku70 and Ku80), is the high-affinity DNA binding component of the DNA-dependent protein kinase. Ku is critical for nonhomologous DNA double-stranded break repair and site-specific recombination of V(D)J gene segments. Ku also plays an important role in telomere maintenance in yeast. Herein, we report, using an in vivo crosslinking method, that human and hamster telomeric DNAs specifically coimmunoprecipitate with human Ku80 after crosslinking. Localization of Ku to the telomere does not depend on the DNA-dependent protein kinase catalytic component. These findings suggest a direct link between Ku and the telomere in mammalian cells.

Coregulatory proteins in steroid hormone receptor action:: The role of chromatin high mobility group proteins HMG-1 and -2

To directly activate specific gene expression, the progesterone receptor must bind to specific hormone response elements in target promoters. We have previously reported that progesterone receptor requires a nuclear factor, high mobility group 1 or 2 (HMG-1/-2) for high-affinity interaction with DNA in vitro and for full transcriptional activity in vivo. We have also observed that HMG-1/-2 selectively influences the activity of the steroid hormone class of nuclear receptors but does not affect other classes of nuclear receptors. This report is a summary of our published and unpublished studies to determine the effects of HMG-1/-2 on a broad range of nuclear receptor supergene family members and to define the mechanism for the specific effect of HMG-1/-2 on the steroid class of receptors. Our studies and available structural data suggest a model where the DNA binding domains of nonsteroid nuclear receptors contain a minor groove DNA interface, termed the C-terminal extension, that contributes to high-affinity DNA binding. Steroid receptors lack such a minor groove interface and therefore require an additional protein, HMG-1/-2, that functionally substitutes for the C-terminal extension to facilitate high-affinity interactions with DNA.

Mechanism of DNA replication in eukaryotic cells: cellular host factors stimulating adenovirus DNA replication.

Replication of adenovirus (Ad) DNA depends on interactions between three viral and three cellular proteins. Human transcription factors NFI and Oct-1 recruit the Ad DNA polymerase to the origin of DNA replication as a complex with the Ad protein primer pTP. High affinity and specificity DNA binding to recognition sites in this origin by the transcription factors stimulate and stabilize pre-initiation complex formation to compensate for the low binding specificity of the pTP/pol complex. In this review, we discuss the properties of NFI and Oct-1 and the mechanism by which they enhance initiation of DNA replication. We propose a model that describes the dynamics of initiation and elongation as well as the assembly and disassembly of the pre-initiation complex.

Differential DNA binding by the androgen and glucocorticoid receptors involves the second Zn-finger and a C-terminal extension of the DNA-binding domains

The androgen and glucocorticoid hormones evoke specific in vivo responses by activating different sets of responsive genes. Although the consensus sequences of the glucocorticoid and androgen response elements are very similar, this in vivo specificity can in some cases be explained by differences in DNA recognition between both receptors. This has clearly been demonstrated for the androgen response element PB-ARE-2 described in the promoter of the rat probasin gene. Swapping of different fragments between the androgen- and glucocorticoid-receptor DNA-binding domains demonstrates that (i) the first Zn-finger module is not involved in this sequence selectivity and (ii) that residues in the second Zn-finger as well as a C-terminal extension of the DNA-binding domain from the androgen receptor are required. For specific and high-affinity binding to response elements, the DNA-binding domains of the androgen and glucocorticoid receptors need a different C-terminal extension. The glucocorticoid receptor requires 12 C-terminal amino acids for high affinity DNA binding, while the androgen receptor only involves four residues. However, for specific recognition of the PB-ARE-2, the androgen receptor also requires 12 C-terminal residues. Our data demonstrate that the mechanism by which the androgen receptor binds selectively to the PB-ARE-2 is different from that used by the glucocorticoid receptor to bind a consensus response element. We would like to suggest that the androgen receptor recognizes response elements as a direct repeat rather than the classical inverted repeat.

Highly Specific DNA Recognition by a Designed Miniature Protein

Many DNA-binding proteins employ a short R-helix as their primary DNA recognition element.1-5 When removed from their natural contexts, however, most R-helices neither fold nor bind DNA with high affinity.6,7 Here we describe a strategy for the design of miniature proteins that present a solvent-exposed R-helix able to recognize DNA with high affinity and specificity. This strategy involves dissecting those R-helical residues required for DNA recognition from their native protein context and grafting them on a stable, miniature protein core (Figure 1). This strategy was used to design a 42-amino acid protein displaying the DNAcontact residues of a single basic region-leucine zipper (bZIP) protein. This miniature protein bound DNA in the low nanomolar concentration range under physiological solution conditions and exhibited greater sequence specificity than a natural bZIP protein. This strategy may represent a general approach to the design of small, folded proteins that recognize nucleic acid and protein targets with high affinity and specificity. Our design process began with avian pancreatic polypeptide (aPP), a small, well-folded protein of known structure consisting of a single R-helix stabilized by hydrophobic interaction with a type II polyproline helix (Figure 1).8-10 Since formation of the aPP core requires residues on only one face of the aPP R-helix (shown in blue), the opposite, solvent-exposed face of the R-helix is available for recognition of other macromolecular targets. By grafting various combinations of those thirteen residues (shown in pink) used by GCN4 to recognize the CRE half site (ATGAC, hsCRE)1,2 on the solvent-exposed R-helical face of aPP, we generated a series of polyproline helix-basic region (PPBRSR) molecules containing most or all of the DNA-contact residues of GCN4 and most or all of the folding residues of aPP (Figure 2). This procedure generated three positions of conflict, where essential DNA-contact and aPP-folding residues occupied a single position on the helix (Figure 2). No significant DNA binding was detected with peptides PPBR0SR, PPBR10SR, and PPBR11SR, which lacked one or more of these DNA-contact residues. Highaffinity DNA binding was observed with a peptide that contained these three residues: The equilibrium dissociation constant (Kd) of the PPBR2SR‚hsCRE complex was 5 nM under conditions of physiological ionic strength (Figure 3). DNA affinity was enhanced further by selective alanine substitutions that increased the overall R-helical propensity of the peptide, producing the PPBR4‚hsCRE24 complex whose Kd was 1.5 nM under identical conditions. Formation of the PPBR4‚hsCRE24 complex was unaffected by high concentrations of poly (dIdC)‚(dIdC) or a scrambled CRE site (NON), indicating that the high stability of PPBR4‚hsCRE24 was not due primarily to nonspecific ionic interactions. Circular dichroism experiments indicated that like bZIP peptides,6,11 PPBR4SR attained a fully R-helical conformation only in the presence of specific DNA.12 Although others have described monopartite DNA recognition by basic segment peptides, the affinities reported have been only moderate (60 nM-3 μM), and the complexes are stable only in very low ionic strength buffers.13,14 PPBR4SR represents the first example of high affinity, monopartite, major groove recognition at physiological ionic strength. To examine the contribution of hydrophobic core formation to PPBR4‚hsCRE24 complex stability, we studied peptides G27 * To whom correspondence should be addressed. (1) Ellenberger, T. E.; Brandl, C. J.; Struhl, K.; Harrison, S. C. Cell 1992, 71, 1223. (2) Konig, P.; Richmond, T. J. Mol. Biol. 1993, 233, 139. (3) Aggarwal, A. K.; Rodgers, D. W.; Drottar, M.; Ptashne, M.; Harrison, S. C. Science 1988, 242. (4) Ferre-D’Amare, A. R.; Prendergast, G. C.; Ziff, E. B.; Burley, S. K. Nature (London) 1993, 363, 38. (5) Kissinger, C. R.; Liu, B.; Martin-Blanco, E.; Kornberg, T. B.; Pabo, C. O. Cell 1990, 63, 579. (6) Weiss, M. A.; Ellenberger, T.; Wobbe, C. R.; Lee, J. P.; Harrison, S. C.; Struhl, K. Nature (London) 1990, 347, 575. (7) O’Neil, K. T.; Shuman, J. D.; Ampe, C.; DeGrado, W. F. Biochemistry 1991, 30, 9030. (8) Blundell, T. L.; Pitts, J. E.; Tickle, I. C.; Wood, S. P.; Wu, C.-W. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 4175. (9) Tonan, K.; Kawata, Y.; Hamaguchi, K. Biochemistry 1990, 29, 4424. (10) Hermans, J., Jr.; Scheraga, H. A. J. Am. Chem. Soc. 1961, 83, 3283. (11) O’Neil, K. T.; Hoess, R. H.; DeGrado, W. F. Science 1990, 249, 774. (12) The CD spectrum of PPBR4SR was unchanged between 1 and 20 μM, indicating that no detectable changes in secondary structure occurred in this range. Addition of hsCRE DNA significantly increased the R-helix content of PPBR4SR; smaller changes were observed upon addition of hsCEBP DNA (see Supporting Information). (13) Park, C.; Campbell, J. L.; Goddard, W. A., III J. Am. Chem. Soc. 1996, 118, 4235. (14) Morii, T.; Yamane, J.; Aizawa, Y.; Makino, K.; Sugiura, Y. J. Am. Chem. Soc. 1996, 118, 10011. Figure 1. Protein grafting strategy for the design of miniature DNAbinding proteins.

The role of HMG I(Y) in the assembly and function of the IFN-beta enhanceosome.

Transcriptional activation of the virus inducible enhancer of the human interferon-beta (IFN-beta) gene in response to virus infection requires the assembly of an enhanceosome, consisting of the transcriptional activators NF-kappaB, ATF-2/c-Jun, IRFs and the architectural protein of the mammalian high mobility group I(Y) [HMG I(Y)]. Here, we demonstrate that the first step in enhanceosome assembly, i.e. HMG I(Y)-dependent recruitment of NF-kappaB and ATF-2/c-Jun to the enhancer, is facilitated by discrete regions of HMG I and is mediated by allosteric changes induced in the DNA by HMG I(Y) and not by protein-protein interactions between HMG I(Y) and these proteins. However, we show that completion of the enhanceosome assembly process requires protein-protein interactions between HMG I(Y) and the activators. Finally, we demonstrate that once assembled, the IFN-beta enhanceosome is an unusually stable nucleoprotein structure that can activate transcription at high levels by promoting multiple rounds of reinitiation of transcription.

Protein-ligand interaction: grafting of the uridine-specific determinants from the CytR regulator of Salmonella typhimurium to Escherichia coli CytR

Members of the LacI family of transcriptional repressors respond to the presence of small effector molecules. The binding of the ligands affect the proteins ability to repress transcription by stabilizing a conformation that, in most cases, is unfavorable for high-affinity DNA binding. The CytR anti-activator diverges from the other family members by relying on the cooperative DNA binding with the global regulator CRP. The inducers of CytR do not affect CytR-DNA binding per se, but alleviate repression by interrupting protein-protein interactions between the two regulators. Here, we have studied of the CytR-inducer interaction by exploring a discrepancy in the inducer response observed for the homologous CytR regulators of Escherichia coli and Salmonella typhimurium. CytR of S. typhimurium (CytR St) appears to respond to the presence of both uridine and cytidine nucleosides, whereas E. coli CytR (CytR Ec) responds to cytidine only. We have used a combination of genetic and structural modeling studies to provide detailed information regarding the nature of this discrepancy. By analysis of hybrid CytR proteins followed by site-directed mutagenesis, we have successfully transferred the specificity determinants for uridine from CytR Stto CytR Ec, revealing that serine substitutions of only two residues (G131 and A152) in CytR Ec is required to make CytR Ec sensitive to uridine. In addition, by employing a genetic screen for induction of defective mutants, we have identified four amino acid residues in CytR St that appear to be important for the response to uridine. The implications of these findings for the understanding of the ligand binding and induction of CytR are discussed in the context of the structural knowledge of CytR and homologous protein-ligand complexes.

Transition from monomeric to homodimeric DNA binding by nuclear receptors: identification of RevErbAalpha determinants required for RORalpha homodimer complex formation.

Nuclear hormone receptors belong to a class of transcription factors that recognize specific DNA sequences either as monomers, homodimers, or heterodimers with the common partner retinoic X receptor. In vitro mutagenesis studies, as well as determination of the crystal structure of several complexes formed by the DNA-binding domain of receptors bound to their cognate response elements, have begun to explain the molecular basis for protein-DNA and protein-protein interactions essential for high-affinity and specific DNA binding by nuclear receptors. In this study, we have used the related orphan nuclear receptors, RORalpha and RevErbAalpha, to study the molecular determinants involved in the transition from monomeric to homodimeric modes of DNA binding by nuclear receptors. While both receptors bind DNA as monomers to a response element containing a core AGGTCA half-site preceded by a 5'-A/T-rich flanking sequence, RevErbAalpha also binds as a homodimer to an extended DR2 element. Gain-of-function experiments using point mutations and subdomain swaps between RORalpha and RevErbAalpha identify four amino acids within RevErbAalpha sufficient to confer RORalpha with the ability to form cooperative homodimer complexes on an extended DR2. This study reveals how the transition from monomer to homodimer DNA binding by members of the nuclear receptor superfamily could be achieved from relatively few amino acid substitutions.

Physical and genetic interactions between Alx4 and Cart1.

Alx4 and Cart1 are closely related members of the family of transcription factors that contain the paired-type homeodomain. In contrast to other types of homeodomains, the paired-type homeodomain has been shown to mediate high-affinity sequence-specific DNA binding to palindromic elements as either homodimers or as heterodimers with other family members. Alx4 and Cart1 are co-expressed at several sites during development, including the craniofacial mesenchyme, the mesenchymal derivatives of neural crest cells in the first branchial arch and the limb bud mesenchyme. Because of the molecular similarity and overlapping expression pattern, we have analyzed the functional and genetic relationships between Alx4 and Cart1. The two proteins have similar DNA-binding activity in vitro and can form DNA-binding heterodimers; furthermore, they activate transcription of reporter genes that contain high-affinity DNA-binding sites in cell culture in a similar manner. Therefore, at least by these criteria, the two proteins are functionally redundant. Analysis of double mutant animals reveals several genetic interactions. First, mutation of Cart1 exacerbates Alx4-dependent polydactyly in a manner that is dependent on gene dosage. Second, there are complex genetic interactions in the craniofacial region that reveal a role for both genes in the fusion of the nasal cartilages and proper patterning of the mandible, as well as other craniofacial structures. Third, double mutant mice show a split sternum that is not detected in mice with any other genotype. Interpreted in the context of the biochemical characterization, the genetic analysis suggests that Alx4 and Cart1 are indeed functionally redundant, and reveal both unique and redundant functions for these genes in development.

Sensitivity of Drosophila Heat Shock Transcription Factor to Low pH

The heat shock transcription factor (HSF) mediates the induction of heat shock gene expression. The activation of HSF involves heat shock-induced trimerization, binding to its cognate DNA sites, and the acquisition of transcriptional competence. In this study, the oligomeric properties of Drosophila HSF were analyzed by equilibrium analytical ultracentrifugation and gel filtration chromatography. Previous findings showed that trimerization of purified Drosophila HSF was directly sensitive to heat and oxidation (1). Here we report that low pH, in the physiological range, also directly induces HSF trimerization and DNA binding in vitro. Furthermore, the induction of HSF trimerization by low pH is synergistic with the actions of heat and oxidation. Since heat or chemical stress leads to a moderate decrease of intracellular pH, we suggest that intracellular acidification may contribute to activating the heat shock response in vivo.

ComEA is a DNA receptor for transformation of competent Bacillus subtilis.

Competent cells of Bacillus subtilis efficiently bind and internalize DNA. ComEA and the seven proteins encoded by the comG operon are required in vivo for the binding step. We show here that ComEA, a bitopic membrane protein, is itself capable of high-affinity DNA binding. A domain necessary for DNA binding is located at the C-terminus of ComEA. Proteins with similar 60-80 amino acid residue domains are widespread among bacteria and higher organisms. ComEA shows a marked preference for double-stranded DNA and can bind to oligomers as small as 22 bp in length. DNA binding by ComEA exhibits no apparent base sequence specificity. Using a membrane vesicle DNA-binding assay system we show that in the absence of cell wall, ComEA is still required for DNA binding, whereas the requirement for the ComG proteins is bypassed. We conclude that the ComG proteins are needed in vivo to provide access of the binding domain of ComEA to exogenous DNA. Possible specific roles for the ComG proteins are discussed.

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.