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.     

Identification and characterization of Saccharomyces cerevisiae Cdc6 DNA-binding properties

Recent studies have shown that Cdc6 is an essential regulator in the formation of DNA replication complexes. However, the biochemical nature of the Cdc6 molecule is still largely unknown. In this report, we present evidence that the Saccharomyces cerevisiae Cdc6 protein is a double-stranded DNA-binding protein. First, we have demonstrated that the purified yeast Cdc6 can bind to double-stranded DNA (dissociation constant approximately 1 x 10(-7) M), not to single-stranded DNA, and that the Cdc6 molecule is a homodimer in its native form. Second, we show that GST-Cdc6 fusion proteins expressed in Escherichia coli bind DNA in an electrophoretic mobility shift assay. Cdc6 antibodies and GST antibodies, but not preimmune serum, induce supershifts of GST-Cdc6 and DNA complexes in these assays, which also showed that GST-Cdc6 binds to various DNA probes without apparent sequence specificity. Third, the minimal requirement for the binding of Cdc6 to DNA has been mapped within its N-terminal 47-amino acid sequence (the NP6 region). This minimal binding domain shows identical DNA-binding properties to those possessed by full-length Cdc6. Fourth, the GST-NP6 protein competes for DNA binding with distamycin A, an antibiotic that chelates DNA within the minor groove of the A+T-rich region. Finally, site-direct mutagenesis studies revealed that the (29)KRKK region of Cdc6 is essential for Cdc6 DNA-binding activity. To further elucidate the function of Cdc6 DNA binding in vivo, we demonstrated that a binding mutant of Cdc6 fails to complement either cdc6-1 temperature-sensitive mutant cells or Deltacdc6 null mutant cells at the nonpermissive temperature. The mutant gene also conferred growth impairments and increased the plasmid loss in its host, indicative of defects in DNA synthesis. Because the mutant defective in DNA binding also fails to stimulate Abf1 ARS1 DNA-binding activity, our results suggest that Cdc6 DNA-binding activity may play a pivotal role in the initiation of DNA replication.     

Crp1p,a new cruciform DNA-binding protein in the yeast Saccharomyces cerevisiae

A synthetic cruciform DNA (X-DNA) was used for screening cellular extracts of Saccharomyces cerevisiae for X-DNA-binding activity. Three X-DNA-binding proteins with apparent molecular mass of 28kDa, 26kDa and 24kDa, estimated by SDS-PAGE, were partially purified. They were identified as N-terminal fragments originating from the same putative protein, encoded by the open reading frame YHR146W, which we named CRP1 (cruciform DNA-recognising protein 1). Expression of CRP1 in Escherichia coli showed that Crp1p is subject to efficient proteolysis at one specific site. Cleavage leads to an N-terminal subpeptide of approximately 160 amino acid residues that is capable of binding specifically X-DNA with an estimated dissociation constant (K(d)) of 800nM, and a C-terminal subpeptide of approximately 305 residues without intrinsic X-DNA-binding activity. The N-terminal subpeptide is of a size similarly to that of the fragments identified in yeast, suggesting that the same cleavage process occurs in the yeast and the E.coli background. This makes the action of a site-specific protease unlikely and favours the possibility of an autoproteolytic activity of Crp1p. The DNA-binding domain of Crp1p was mapped to positions 120-141. This domain can act autonomously as an X-DNA-binding peptide and provides a new, lysine-rich DNA-binding domain different from those of known cruciform DNA-binding proteins (CBPs). As reported earlier for several other CBPs, Crp1p exerts an enhancing effect on the cleavage of X-DNA by endonuclease VII from bacteriophage T4.     

Identification of CBS2 as a mitochondrial protein in Saccharomyces cerevisiae

Summary The nuclear genome encoded yeast protein CBS2 is required for translational activation of mitochondrial cytochrome b RNA. Genetic studies have shown that the target sequence of the CBS2 protein is the 5 untranslated leader sequence of cytochrome b RNA. Here we report on the intracellular localization of CBS2. CBS2 protein, expressed in Escherichia coli and prepared from inclusion bodies, was used as an antigen to raise a polyclonal rabbit antiserum. Affinity-purified CBS2 antibodies detect a 45 kDa protein in mitochondrial lysates of wild-type cells, which is absent in a strain in which the CBS2 gene has been deleted. The protein is overexpressed in mitochondrial extracts of a transformant carrying the CBS2 gene on a high copy number plasmid, but undetectable in the post-mitochondrial supernatant. Intramitochondrial localization of CBS2 was verified by in vitro import of CBS2 protein that had been synthesized in a reticulocyte lysate programmed with CBS2 mRNA transcribed in vitro. Mitochondrial import of CBS2 is not accompanied by any detectable proteolytic processing.     

A common element involved in transcriptional regulation of two DNA alkylation repair genes (MAG and MGT1) of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae MAG gene encodes a 3-methyladenine DNA glycosylase that protects cells from killing by alkylating agents. MAG mRNA levels are induced not only by alkylating agents but also by DNA-damaging agents that do not produce alkylated DNA. We constructed a MAG-lacZ gene fusion to help identify the cis-acting promoter elements involved in regulating MAG expression. Deletion analysis defined the presence of one upstream activating sequence and one upstream repressing sequence (URS) and suggested the presence of a second URS. One of the MAG URS elements matches a decamer consensus sequence present in the promoters of 11 other S. cerevisiae DNA repair and metabolism genes, including the MGT1 gene, which encodes an O6-methylguanine DNA repair methyltransferase. Two proteins of 26 and 39 kDa bind specifically to the MAG and MGT1 URS elements. We suggest that the URS-binding proteins may play an important role in the coordinate regulation of these S. cerevisiae DNA repair genes.     

Interactions among the subunits of the G protein involved in Saccharomyces cerevisiae mating.

The SCG1 (GPA1), STE4, and STE18 genes of Saccharomyces cerevisiae encode mating-pathway components whose amino acid sequences are similar to those of the alpha, beta, and gamma subunits, respectively, of mammalian G proteins. Genetic evidence suggests that the STE4 and STE18 gene products interact. The mating defects of a set of ste4 mutants were partially suppressed by the overexpression of STE18, and, moreover, a combination of partially defective ste4 and ste18 alleles created a totally sterile phenotype, whereas such synthetic sterility was not observed when the ste18 allele was combined with a weakly sterile ste11 allele. Others have provided genetic evidence consistent with an interaction between the SCG1 (GPA1) and STE4 gene products. We have examined the physical interactions of these subunits by using an in vivo protein association assay. The STE4 and STE18 gene products associated with each other, and this association was disrupted by a mutation in the STE4 gene product whose phenotype was partially suppressed by overexpression of STE18. The STE4 and SCG1 (GPA1) gene products also interacted in the assay, whereas we detected no association of the SCG1 (GPA1) and STE18 gene products.