Method to identify genomic targets of DNA binding proteins.

We have devised a cyclical immunoprecipitation protocol that can be used to identify and clone a specific DNA sequence that is recognized by a DNA binding protein, even if that sequence is present in only one copy in the genome of a mammal. As an example, we have used this procedure to purify mouse genomic sequences to which the simian virus 40 tumor (T) antigen binds.     

Two genes required for the binding of an essential Saccharomyces cerevisiae kinetochore complex to DNA.

Kinetochores are DNA-protein structures that assemble on centromeric DNA and attach chromosomes to spindle microtubules. Because of their simplicity, the 125-bp centromeres of Saccharomyces cerevisiae are particularly amenable to molecular analysis. Budding yeast centromeres contain three sequence elements of which centromere DNA sequence element III (CDEIII) appears to be particularly important. cis-acting mutations in CDEIII and trans-acting mutations in genes encoding subunits of the CDEIII-binding complex (CBF3) prevent correct chromosome transmission. Using temperature-sensitive mutations in CBF3 subunits, we show a strong correlation between DNA-binding activity measured in vitro and kinetochore activity in vivo. We extend previous findings by Goh and Kilmartin [Goh, P.-Y. & Kilmartin, J.V. (1993) J. Cell Biol. 121, 503-512] to argue that DNA-bound CBF3 may be involved in the operation of a mitotic checkpoint but that functional CBF3 is not required for the assembly of a bipolar spindle.     

A complex of nuclear proteins mediates SR protein binding to a purine-rich splicing enhancer.

A purine-rich splicing enhancer from a constitutive exon has been shown to shift the alternative splicing of calcitonin/CGRP pre-mRNA in vivo. Here, we demonstrate that the native repetitive GAA sequence comprises the optimal enhancer element and specifically binds a saturable complex of proteins required for general splicing in vitro. This complex contains a 37-kDa protein that directly binds the repetitive GAA sequence and SRp40, a member of the SR family of non-snRNP splicing factors. While purified SR proteins do not stably bind the repetitive GAA element, exogenous SR proteins become associated with the GAA element in the presence of nuclear extracts and stimulate GAA-dependent splicing. These results suggest that repetitive GAA sequences enhance splicing by binding a protein complex containing a sequence-specific RNA binding protein and a general splicing activator that, in turn, recruit additional SR proteins. This type of mechanism resembles the tra/tra-2-dependent recruitment of SR proteins to the Drosophila doublesex alternative splicing regulatory element.     

Macromolecular crowding increases binding of DNA polymerase to DNA: an adaptive effect.

Macromolecular crowding extends the range of ionic conditions supporting high DNA polymerase reaction rates. Reactions tested were nick-translation and gap-filling by DNA polymerase I of Escherichia coli, nuclease and polymerase activities of the large fragment of that polymerase, and polymerization by the T4 DNA polymerase. For all of these reactions, high concentrations of nonspecific polymers increased enzymatic activity under otherwise inhibitory conditions resulting from relatively high ionic strength. The primary mechanism of the polymer effect seems to be to increase the binding of polymerase to DNA. We suggest that this effect on protein-DNA complexes is only one example of a general "metabolic buffering" action of crowded solutions on a variety of macromolecular interactions.     

HD-Zip proteins: members of an Arabidopsis homeodomain protein superfamily.

Homeobox genes encode a large family of homeodomain proteins in animal systems. To test whether such genes are also abundant in higher plants, degenerate oligonucleotides complementary to sequences encoding the recognition helix (helix three) of the homeodomain were used to screen genomic and cDNA libraries from the plant Arabidopsis thaliana. Analysis of 8 of the 41 cDNAs isolated revealed that each encodes a presumptive homeodomain; interestingly, most of these clones also contain a leucine zipper motif tightly linked to the homeodomain. It is concluded that Arabidopsis encodes a large family of homeodomain proteins, including members that contain a homeodomain/leucine-zipper (HD-Zip) motif.     

Cyanobacterial protein with similarity to the chlorophyll a/b binding proteins of higher plants: evolution and regulation.

We have isolated, from the prokaryotic cyanobacterium Synechococcus sp. strain PCC 7942, a gene encoding a protein of 72 amino acids [designated high light inducible protein (HLIP)] with similarity to the extended family of eukaryotic chlorophyll a/b binding proteins (CABs). HLIP has a single membrane-spanning alpha-helix, whereas both the CABs and the related early light inducible proteins have three membrane-spanning helices. Hence, HLIP may represent an evolutionary progenitor of the eukaryotic members of the CAB extended family. We also show that the gene encoding HLIP is induced by high light and blue/UV-A radiation. The evolution, regulation, and potential function of HLIP are discussed.     

AtVPS34, a phosphatidylinositol 3-kinase of Arabidopsis thaliana, is an essential protein with homology to a calcium-dependent lipid binding domain.

The cDNA encoding phosphatidylinositol (PI) 3-kinase was cloned from Arabidopsis thaliana, and the derived amino acid sequence (AtVPS34) has a significantly higher homology to yeast PI 3-kinase (VPS34) than to the mammalian (p110). The protein has two conserved domains: a catalytic site with the ATP-binding site near the C terminus and a calcium-dependent lipid-binding domain near the N terminus. The plant cDNA does not rescue a yeast vps34 deletion mutant, but a chimeric gene in which the coding sequence for the C-terminal third of VPS34 is replaced by the corresponding sequence from the plant gene does rescue the yeast mutant. PI 3-kinase activity is detectable in extracts from plants that overexpress the plant PI 3-kinase. Expression of antisense constructs gives rise to second-generation transformed plants severely inhibited in growth and development.     

Determination of the orientation of a DNA binding motif in a protein-DNA complex by photocrosslinking.

We have developed a straightforward biochemical method to determine the orientation of the DNA binding motif of a sequence-specific DNA binding protein relative to the DNA site in the protein-DNA complex. The method involves incorporation of a photoactivatable crosslinking agent at a single site within the DNA binding motif of the sequence-specific DNA binding protein, formation of the derivatized protein-DNA complex, UV-irradiation of the derivatized protein-DNA complex, and determination of the nucleotide(s) at which crosslinking occurs. We have applied the method to catabolite gene activator protein (CAP). We have constructed and analyzed two derivatives of CAP: one having a phenyl azide photoactivatable crosslinking agent at amino acid 2 of the helix-turn-helix motif of CAP, and one having a phenyl azide photoactivatable crosslinking agent at amino acid 10 of the helix-turn-helix motif of CAP. The results indicate that amino acid 2 of the helix-turn-helix motif is close to the top-strand nucleotides of base pairs 3 and 4 of the DNA half site in the CAP-DNA complex, and that amino acid 10 of the helix-turn-helix motif is close to the bottom-strand nucleotide of base pair 10 of the DNA half site in the CAP-DNA complex. The results define unambiguously the orientation of the helix-turn-helix motif relative to the DNA half site in the CAP-DNA complex. Comparison of the results to the crystallographic structure of the CAP-DNA complex [Schultz, S., Shields, S. & Steitz, T. (1991) Science 253, 1001-1007] indicates that the method provides accurate, high-resolution proximity and orientation information.     

Use of a zinc-finger consensus sequence framework and specificity rules to design specific DNA binding proteins.

We have designed three zinc-finger proteins with different DNA binding specificities. The design strategy combines a consensus zinc-finger framework sequence with previously characterized recognition regions such that the specificity of each protein is predictable. The first protein consists of three identical zinc fingers, each of which was expected to recognize the subsite GCG. This protein binds specifically to the sequence 5'-GCG-GCG-GCG-3' with a dissociation constant of approximately 11 microM. The second protein has three zinc fingers with different predicted preferred subsites. This protein binds to the predicted recognition site 5'-GGG-GCG-GCT-3' with a dissociation constant of 2 nM. Furthermore, selection experiments indicate that this is the optimal binding site. A permuted version of the second protein was also constructed and shown to preferentially recognize the corresponding permuted site 5'-GGG-GCT-GCG-3' over the non-permuted site. These results indicate that earlier observations on the specificity of zinc fingers can be extended to generalized zinc-finger structures and realize the use of zinc fingers for the design of site-specific DNA binding proteins. This consensus-based design system provides a useful model system with which to study details of zinc-finger-DNA specificity.     

Cloning of an intracellular receptor for protein kinase C: a homolog of the beta subunit of G proteins.

Protein kinase C (PKC) translocates from the soluble to the cell particulate fraction on activation. Intracellular receptors that bind activated PKC in the particulate fraction have been implicated by a number of studies. Previous work identified 30- to 36-kDa proteins in the particulate fraction of heart and brain that bound activated PKC in a specific and saturable manner. These proteins were termed receptors for activated C-kinase, or RACKs. In the following study, we describe the cloning of a cDNA encoding a 36-kDa protein (RACK1) that fulfills the criteria for RACKs. (i) RACK1 bound PKC in the presence of PKC activators, but not in their absence. (ii) PKC binding to the recombinant RACK1 was not inhibited by a pseudosubstrate peptide or by a substrate peptide derived from the pseudosubstrate sequence, indicating that the binding did not reflect simply PKC association with its substrate. (iii) Binding of PKC to RACK1 was saturable and specific; two other protein kinases did not bind to RACK1. (iv) RACK1 contains two short sequences homologous to a PKC binding sequence previously identified in annexin I and in the brain PKC inhibitor KCIP. Peptides derived from these sequences inhibited PKC binding to RACK1. Finally, RACK1 is a homolog of the beta subunit of G proteins, which were recently implicated in membrane anchorage of the beta-adrenergic receptor kinase [Pitcher, J., Inglese, L., Higgins, J. B., Arriza, J. A., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M., Caron, M. G. & Lefkowitz, R. J. (1992) Science 257, 1264-1267]. Our in vitro data suggest a role for RACK1 in PKC-mediated signaling.     

Antisilencing role of the RNA-directed DNA methylation pathway and a histone acetyltransferase in Arabidopsis

REPRESSOR OF SILENCING 1 (ROS1) is a DNA demethylation enzyme that was previously identified during a genetic screen for the silencing of both RD29A-LUC and 35S-NPTII transgenes on a T-DNA construct. Here we performed a genetic screen to identify additional mutants in which the 35S-NPTII transgene is silenced. We identified several alleles of ros1 and of the following components of the RNA-directed DNA methylation (RdDM) pathway: NRPD1 (the largest subunit of polymerase IV), RDR2, NRPE1 (the largest subunit of polymerase V), NRPD2, AGO4, and DMS3. Our results show that the silencing of 35S-NPTII in the RdDM pathway mutants is due to the reduced expression of ROS1 in the mutants. We also identified a putative histone acetyltransferase (ROS4) from the genetic screen. The acetyltransferase contains a PHD-finger domain that binds to unmethylated histone H3K4. The mutation in ROS4 led to reduction of H3K18 and H3K23 acetylation levels. We show that the silencing of 35S-NPTII and some transposable element genes was released by the ddm1 mutation but that this also required ROS4. Our study identifies a unique antisilencing factor, and reveals that the RdDM pathway has an antisilencing function due to its role in maintaining ROS1 expression.     

Partial characterization of a neurotransmitter pathway regulating the in vivo release of prolactin.

Nicotinic cholinergic, opiate and serotonergic agonists as well as dopaminergic antagonists induce the release of pituitary prolactin. The purposes of the present studies were to determine if nicotine, morphine and the serotonin1A (5-HT1A) agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) utilize a common synaptic pathway to release prolactin and, if so, to establish the serial order of the receptors involved. We also sought to determine whether the pathway under investigation leads to the secretion of prolactin via a mechanism involving dopamine, the prolactin inhibitory factor. Male rats with indwelling jugular catheters were pretreated with saline, mecamylamine, naltrexone, methysergide or bromocriptine. In the saline-treated animals, administration of nicotine, morphine, 8-OH-DPAT and haloperidol resulted in significant increases in plasma prolactin levels. Mecamylamine pretreatment prevented the prolactin response to nicotine only. Naltrexone blocked the stimulation of prolactin release by morphine and by nicotine. Methysergide inhibited the effects of 8-OH-DPAT, morphine and nicotine but not haloperidol. Bromocriptine blocked the prolactin secretion induced by haloperidol as well as by each of the above agonists. Also, in dual-immunocytochemically stained sections, tyrosine hydroxylase-immunoreactive cells and serotonin-immunoreactive processes were detected in close anatomical proximity in the dorsomedial arcuate nucleus. These data indicate that nicotine, morphine and 8-OH-DPAT act to release prolactin via a common synaptic pathway expressing nicotinic cholinergic, opiate, and 5-HT1A receptors at synapses arranged serially in that functional order. Furthermore, the data indicate that the in vivo secretion of prolactin via this pathway may ultimately occur through the inhibition of dopamine release.     

Theoretical aspects of translocation on DNA: adenosine triphosphatases and treadmilling binding proteins.

The basic kinetic and bioenergetic theory is outlined for two kinds of translocation on DNA: (i) helicases that use ATP to move along single-stranded DNA or to move on and invade double-stranded DNA at a replication fork; and (ii) DNA-binding proteins (not ATPases) that form bound aggregates on single-stranded DNA and facilitate replication by steady-state treadmilling of molecules between the ends of the aggregate. The respective resemblances to myosin--actin in muscle and to steady-state treadmilling in solution of actin or tubulin are pointed out.     

Discrimination between activators and nonactivators of the alternative pathway of complement: regulation via a sialic acid/polyanion binding site on factor H.

The alternative complement pathway is capable of discriminating human cells and tissues from a wide variety of potential pathogens. It has been recently demonstrated that attachment of complement component C3b to activator-derived molecules (e.g., small polysaccharides) restricts inactivation of C3b by factors H and I in a manner similar to activator surfaces. It is now shown that restriction is reversed by certain soluble polyanions (e.g., sialoglycopeptides, heparin, or dextran sulfate) that mimic the effects of sialic acid and glycosaminoglycans on human cells and tissues. Fluid-phase polyanions enhanced binding of factor H to C3b attached to activating particles, indicating that the effect resulted from increased affinity between C3b and factor H. The enhancement was specific for activator-bound C3b since no enhancement was observed on nonactivating particles. While several polyanions could cause this effect, some polyanions could not, indicating specificity. The active polyanions also inhibited lysis of cells via the alternative pathway. The binding site for sialic acid appears to reside on factor H, since factor H bound to heparin-agarose and to sialic acid-bearing fetuinagarose, whereas C3b bound to neither under the same conditions. These observations suggest that occupation of a specific site on factor H by polyanions induces an increase in the C3b-H affinity, resulting in discrimination of host cells and tissues from alternative pathway-activating foreign cells.     

Monomerization of RepA dimers by heat shock proteins activates binding to DNA replication origin.

DnaK is a major heat shock protein of Escherichia coli and the homolog of hsp70 in eukaryotes. We demonstrate the mechanism by which DnaK and another heat shock protein, DnaJ, render the plasmid P1 initiator RepA 100-fold more active for binding to the P1 origin of replication. Activation is the conversion of RepA dimers into monomers in an ATP-dependent reaction and the monomer form binds with high affinity to oriP1 DNA. Reversible chemical denaturants also convert RepA dimers to monomers and simultaneously activate oriP1 DNA binding. Increasing protein concentration converts monomers to dimers and deactivates RepA. Based on our data and previous work, we present a model for heat shock protein action under normal and stress conditions.     

DNA unwinding upon strand-displacement binding of a thymine-substituted polyamide to double-stranded DNA.

It was recently found that polyamide nucleic acid (PNA) analogues consisting of thymines attached to an aminoethylglycine backbone bind strongly and sequence-selectively to adenine sequences of oligonucleotides and double-stranded DNA [Nielsen, P. E., Egholm, M., Berg, R. H. & Buchardt, O. (1991) Science 254, 1497-1500]. It was concluded that the binding to double-stranded DNA was accomplished via strand displacement, in which the PNA bound to the Watson-Crick complementary adenine-containing strand, whereas the thymine-containing strand was extruded in a virtually single-stranded conformation. This model may provide a general way in which to obtain sequence-specific recognition of any sequence in double-stranded DNA by Watson-Crick hydrogen-bonding base-pair recognition, and it is thus paramount to rigorously establish this binding mode for synthetic DNA-binding ligands. We now report such results from electron microscopy. Furthermore, we show that binding of PNA to closed circular DNA results in unwinding of the double helix corresponding to approximately one turn of the double helix per 10 base pairs. The DNA.PNA complex, which is formed at low salt concentration (only a small portion of DNA molecules show complex formation at NaCl concentration higher than 40 mM), is exceptionally kinetically stable and cannot be dissociated by increasing salt concentration up to 500 mM.