The yeast protein encoded by PUB1 binds T-rich single stranded DNA.

We have characterized binding activities in yeast which recognise the T-rich strand of the yeast ARS consensus element and have purified two of these to homogeneity. One (ACBP-60) is detectable in both nuclear and whole cell extracts, while the other (ACBP-67) is apparent only after fractionation of extracts by heparin-sepharose chromatography. The major binding activity detected in nuclear extracts was purified on a sequence-specific DNA affinity column as a single polypeptide with apparent mobility of 60kDa (ACBP-60). This protein co-fractionates with nuclei, is present at several thousand copies per cell and has a Kd for the T-rich single strand of the ARS consensus between 10(-9) and 10(-10) M. Competition studies with simple nucleic acid polymers show that ACBP-60 has marginally higher affinity for poly dT30 than for a 30 nt oligomer containing the T-rich strand of ARS 307, and approximately 10 fold higher affinity for poly rU. Internal sequence information of purified p60 reveals identity with the open reading frames of genes PUB1 and RNP1 which encode polyuridylate binding protein(s). The second binding activity, ACBP-67, also binds specifically to the T-rich single strand of the ARS consensus, but with considerably lower affinity than ACBP-60. Peptide sequence reveals that the 67kDa protein is identical to the major polyA binding protein in yeast, PAB1.     

Solubilization and Purification of an α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid Binding Protein from Bovine Brain

alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) is a selective ligand for an excitatory amino acid receptor subtype in mammalian brain. We have solubilized an AMPA binding protein from bovine brain membranes with 1% Triton X-100 in 0.5 M phosphate buffer and 20% glycerol at 37 degrees C and purified the stable binding sites using a series of chromatographic steps. Scatchard analysis of the purified preparation showed a curvilinear plot with dissociation constants of 10.6 and 323 nM and Bmax values of 670 and 1,073 pmol/mg of protein for the high- and low-affinity sites, respectively. Inhibition constants for several excitatory amino acid analogues were similar to those obtained for other membrane and solubilized preparations. Gel filtration of the soluble AMPA binding protein showed a single peak of [3H]AMPA binding activity at Mr approximately 500,000. With sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified AMPA binding protein showed a single major band at Mr = 110,000. Previously, we have shown that a monoclonal antibody (KAR-B1) against a frog brain kainate binding protein selectively recognizes an unknown protein in mammalian brain migrating at Mr approximately 100,000. We now show that this antibody recognizes the major component of the purified AMPA binding protein, supporting a structural similarity between the frog brain kainate binding protein and the mammalian AMPA binding protein.     

Identification and purification of DBF-A, a double-stranded DNA-binding protein from Saccharomyces cerevisiae.

Using oligonucleotide affinity chromatography with DNase I footprinting as an assay we have looked for proteins that interact with sequence elements within the yeast origin of replication, autonomously replicating sequence 1 (ARS1). In this work we describe a protein that binds with high affinity to DNA but displays only moderate sequence specificity. It is eluted at 0.7 M salt from an ARS1 oligonucleotide column. Footprinting analysis on ARS1 at a high protein concentration revealed at least three sites of protection flanking element A and its repeats. Element A itself is rendered hypersensitive to DNase I digestion upon protein binding. This pattern is also observed for the H4 and HMR-E ARSs, suggesting that the protein alters the DNA conformation at element A and its repeats. The affinity-purified fraction is also capable of supercoiling a relaxed, covalently closed plasmid in the presence of topoisomerase. Highly purified preparations of the protein are enriched in an 18-kDa polypeptide which can be renatured from a denaturing gel and shown to bind ARS1 DNA. We have designated this protein DBF-A, DNA-binding factor A.     

Purification and characterization of proteins that bind to yeast ARSs

Two proteins that bind to yeast ARS DNA have been purified using conventional and oligonucleotide affinity chromatography. One protein has been purified to homogeneity and has a mass of 135 kDa. Competitive binding studies and DNase I footprinting show that the protein binds to a sequence about 80 base pairs away from the core consensus in the region known as domain B. This region has previously been shown to be required for efficient replication of plasmids carrying ARS1 elements. To investigate further whether the protein might have a function related to the ability of ARSs to act as replicators, binding to another ARS was tested. The protein binds to the functional ARS adjacent to the silent mating type locus HMR, called the HMR-E ARS, about 60 base pairs from the core consensus sequence. Surprisingly, there is little homology between the binding site at the HMR-E ARS and the binding site at ARS1. The 135-kDa protein is probably the same as ABF-I (SBF I) (Shore, D., Stillman, D. J. Brand, A. H., and Nasmyth, K. A. (1987) EMBO J. 6, 461-467; Buchman, A. R., Kimmerly, W. J., Rine, J., and Kornberg, R. D. (1988) Mol. Cell. Biol. 8, 210-225). A second DNA-binding protein was separated from ABF-I during later stages of the purification. This protein, which we designate ABF-III, also binds specifically to the ARS1 sequence, as shown by DNase I footprinting, at a site adjacent to the ABF-I recognition site. Purification of these two ARS binding proteins should aid in our understanding of the complex mechanisms that regulate eukaryotic DNA replication.     

ARS binding factor I of the yeast Saccharomyces cerevisiae binds to sequences in telomeric and nontelomeric autonomously replicating sequences.

We have analyzed various autonomously replicating sequences (ARSs) in yeast nuclear extract with ARS-specific synthetic oligonucleotides. The EI oligonucleotide sequence, which is derived from HMRE-ARS, and the F1 oligonucleotide sequence, which is derived from telomeric ARS120, appeared to bind to the same cellular factor with high specificity. In addition, each of these oligonucleotides was a competitive inhibitor of the binding of the other. Binding of the ARS binding factor (ABF) to either of these oligonucleotides was inhibited strongly by plasmids containing ARS1 and telomeric TF1-ARS. DNase I footprinting analyses with yeast nuclear extract showed that EI and F1 oligonucleotides eliminated protection of the binding site of ARS binding factor I (ABFI) in domain B of ARS1. Sequence analyses of various telomeric (ARS120 and TF1-ARS) and nontelomeric ARSs (ARS1 and HMRE-ARS) showed the presence of consensus ABFI binding sites in the protein binding domains of all of these ARSs. Consequently, the ABFI and ABFI-like factors bind to these domain B-like sequences in a wide spectrum of ARSs, both telomeric and nontelomeric.     

Evidence for binding of at least two factors, including T-rich strand-binding factor(s) to the single-stranded ARS1 sequence in Saccharomyces cerevisiae.

Summary To study the mechanism of initiation of eukaryotic chromosomal replication, we examined protein factors interacting with the ARS1 region located near the centromere of chromosome IV in Saccharomyces cerevisiae. Using the gel shift assay, we found protein factor(s) which specifically bound to the T-rich strand of the region containing the core consensus and its flanking sequences in ARS1, but not to the opposite strand. We designated this factor ATS (ARS1, T-rich strand-binding factor(s)). Similar specific complexes were also detected with oligonucleotide probes specific for the H4 or C2G1 ARS. As we have previously identified another binding factor, we conclude that at least two factors bind to the single-stranded ARS1 sequence.     

A single-stranded DNA binding protein from S. cerevisiae specifically recognizes the T-rich strand of the core sequence of ARS elements and discriminates against mutant sequences.

A protein named ssARS-T binding protein has been purified from yeast that specifically binds to the T-rich strand of the consensus core sequence of yeast autonomously replicating sequence (ARS) elements. As assayed from gel mobility shift experiments the ssARS-T protein shows characteristics of a sequence specific single-stranded DNA binding protein. The complementary A-rich strand of the ARS core sequence is bound much more weakly and no binding can be detected for the double-stranded form of the core sequence. Three single base substitutions in the core sequence that are known to abolish ARS function in vivo also lead to weaker binding of the core sequence to the ssARS-T protein in vitro. The strong correlation between the binding of mutated sequences in vitro and the ARS properties of these sequences in vivo points to an essential function of the ssARS-T protein during replication initiation in yeast ARS elements.     

A new topology of ACBP from Moniliophthora perniciosa

Acyl-CoA binding protein (ACBP) is a housekeeping protein and is an essential protein in human cell lines and in Trypanosoma brucei. The ACBP of Moniliophthora perniciosa is composed of 104 amino acids and is possibly a non-classic isoform exclusively from Basidiomycetes. The M. perniciosa acbp gene was cloned, and the protein was expressed and purified. Acyl-CoA ester binding was analyzed by isoelectric focusing, native gel electrophoresis and isothermal titration calorimetry. Our results suggest an increasing affinity of ACBP for longer acyl-CoA esters, such as myristoyl-CoA to arachidoyl-CoA, and best fit modeling indicates two binding sites. ACBP undergoes a shift from a monomeric to a dimeric state, as shown by dynamic light scattering, fluorescence anisotropy and native gel electrophoresis in the absence and presence of the ligand. The protein's structure was determined at 1.6 Å resolution and revealed a new topology for ACBP, containing five α-helices instead of four. α-helices 1, 2, 3 and 4 adopted a bundled arrangement that is unique from the previously determined four-helix folds of ACBP, while α-helices 1, 2, 4 and 5 formed a classical four-helix bundle. A MES molecule was found in the CoA binding site, suggesting that the CoA site could be a target for small compound screening.     

Purification and characterization of a nuclear factor which binds specifically to the upstream activation sequence of Saccharomyces cerevisiae enolase 1 gene

The nuclear factor which specifically binds to the upstream activation sequence (UAS) of the enolase 1 gene (ENO1) of yeast Saccharomyces cerevisiae was purified by sequence-specific affinity chromatography. The purified factor gave two closely migrated bands at 32 kDa on SDS/PAGE. The binding activities were eluted from a gel filtration column at molecular masses of 110 kDa and 60 kDa, suggesting a dimeric and a tetrameric assembly of the factor in the native form. The region protected by the purified factor against deoxyribonuclease I digestion contained the sequence ACCCAAACACC which is highly similar to the consensus sequence present in the 5'-flanking region of the ribosomal protein genes (RPG box). We also identified the other factor specific to the ENO1 UAS which gave a single peak at a molecular mass of 120 kDa in gel filtration. We suggest the existence of multiple binding to the ENO1 UAS by at least two factors: one is the factor which we purified with a molecular mass of 32 kDa on SDS/PAGE and the other is the factor like RAP1 protein which generally recognizes the RPG-box-like sequence.     

Identification of silencer binding proteins from yeast: possible roles in SIR control and DNA replication

The 'silent' yeast mating-type loci (HML and HMR) are repressed by sequences (HMLE and HMRE) located over 1 kb from their promoters which have properties opposite those of enhancers, and are called 'silencers'. Both silencers contain autonomously replicating sequences (ARS). Silencer activity requires four trans-acting genes called SIR (silent information regulator). We have identified two DNA binding factors , SBF-B and SBF-E, which bind to known regulatory elements at HMRE. SBF-B binds to a region involved in both the silencer and ARS functions of HMRE, but doesn not bind to HMLE. This factor also binds to the unlinked ARS1 element. SBF-E recognizes a sequence found at both silencers. These results suggest that the two silencers may be composed of different combinations of regulatory elements at least one of which is common to both. Neither factor appears to be a SIR gene product. Hence the SIR proteins may not directly interact with the silencer control sites.     

The chromatin structure of Saccharomyces cerevisiae autonomously replicating sequences changes during the cell division cycle.

The chromatin structures of two well-characterized autonomously replicating sequence (ARS) elements were examined at their chromosomal sites during the cell division cycle in Saccharomyces cerevisiae. The H4 ARS is located near one of the duplicate nonallelic histone H4 genes, while ARS1 is present near the TRP1 gene. Cells blocked in G1 either by alpha-factor arrest or by nitrogen starvation had two DNase I-hypersensitive sites of about equal intensity in the ARS element. This pattern of DNase I-hypersensitive sites was altered in synchronous cultures allowed to proceed into S phase. In addition to a general increase in DNase I sensitivity around the core consensus sequence, the DNase I-hypersensitive site closest to the core consensus became more nuclease sensitive than the distal site. This change in chromatin structure was restricted to the ARS region and depended on replication since cdc7 cells blocked near the time of replication initiation did not undergo the transition. Subsequent release of arrested cdc7 cells restored entry into S phase and was accompanied by the characteristic change in ARS chromatin structure.     

Interaction of the H4 autonomously replicating sequence core consensus sequence and its 3''-flanking domain.

Yeast autonomously replicating sequence (ARS) elements are composed of a conserved 11-base-pair (bp) core consensus sequence and a less well defined 3'-flanking region. We have investigated the relationship between the H4 ARS core consensus sequence and its 3'-flanking domain. The minimal sequences necessary and sufficient for function were determined by combining external 3' and 5' deletions to produce a nested set of ARS fragments. Sequences 5' of the core consensus were dispensable for function, but at least 66 bp of 3'-flanking domain DNA was required for full ARS function. The importance of the relative orientation of the core consensus element with respect to the 3'-flanking domain was tested by precisely inverting 14 bp of DNA including the core consensus sequence by oligonucleotide mutagenesis. This core inversion mutant was defective for all ARS function, showing that a fixed relative orientation of the core consensus and 3'-flanking domain is required for function. The 3'-flanking domain of the minimal functional H4 ARS fragment contains three sequences with a 9-of-11-bp match to the core consensus. The role of these near-match sequences was tested by directed mutagenesis. When all near-match sequences with an 8-of-11-bp match or better were simultaneously disrupted by point mutations, the resulting ARS construct retained full replication function. Therefore, multiple copies of a sequence closely related to the core consensus element are not required for H4 ARS function.     

Disruption of the acyl-CoA-binding protein gene delays hepatic adaptation to metabolic changes at weaning

The acyl-CoA-binding protein (ACBP)/diazepam binding inhibitor is an intracellular protein that binds C(14)-C(22) acyl-CoA esters and is thought to act as an acyl-CoA transporter. In vitro analyses have indicated that ACBP can transport acyl-CoA esters between different enzymatic systems; however, little is known about the in vivo function in mammalian cells. We have generated mice with targeted disruption of ACBP (ACBP(-/-)). These mice are viable and fertile and develop normally. However, around weaning, the ACBP(-/-) mice go through a crisis with overall weakness and a slightly decreased growth rate. Using microarray analysis, we show that the liver of ACBP(-/-) mice displays a significantly delayed adaptation to weaning with late induction of target genes of the sterol regulatory element-binding protein (SREBP) family. As a result, hepatic de novo cholesterogenesis is decreased at weaning. The delayed induction of SREBP target genes around weaning is caused by a compromised processing and decreased expression of SREBP precursors, leading to reduced binding of SREBP to target sites in chromatin. In conclusion, lack of ACBP interferes with the normal metabolic adaptation to weaning and leads to delayed induction of the lipogenic gene program in the liver.     

Sequence specificity of the human autonomously replicating sequence binding protein

We have previously found that the protein which bound to the essential region of human autonomously replicating sequences (ARS) could be the c-myc proto-oncogene product. Here we examined the binding specificity of human ARS binding protein purified from Burkitt's lymphoma Raji cells. Among several oligodeoxynucleotides, this protein bound to the fragments containing the sequence of 5'-CAPyCTCTNA-3'. Competition analysis revealed that the ARS binding protein could recognize not only the nucleotide sequences but also the high ordered structure.     

Single-strand-binding factor(s) which interact with ARS1 of Saccharomyces cerevisiae

Since plasmids containing autonomously replicating sequence(s) (ARS) can transform Saccharomyces cerevisiae cells at high frequency, ARS are considered to be the replication origins of chromosomes. To study the mechanism of initiation of eukaryotic chromosomal replication, we examined protein factors which interact with the ARS1 region located near the centromere of chromosome IV in S. cerevisiae. Using the gel-shift assay, we found protein factors which bound to a single-stranded, 97-bp fragment of the ARS1 region containing the core consensus. Competition experiments with various oligodeoxyribonucleotides (oligos) suggest that a site recognized by the factor(s) was within the element containing the core consensus and adjacent close matches to the core consensus of the minus strand. Indeed, when the oligo containing the minus strand of this element was used as a probe, two oligo-protein complexes were detected. Mutations in the core consensus reduced these binding activities. When the plus-strand oligo of the same region was used as a probe, a retarded band was also detected, but with less specific binding. Considering the fact that the core consensus and close matches to the core consensus are important for ARS function, these results imply that the protein factors detected in this experiment may participate in DNA replication.