The molecular and genetic characterization of the DAT1 gene product of Saccharomyces cerevisiae
Abstract
Non-alternating oligo(dA)$\cdot$oligo(dT) tracts (A$\cdot$T tracts) are common intergenic elements in a wide variety of organisms. In Saccharomyces cerevisiae A$\cdot$T tracts are abundant, occurring on average every 5 kbp, are found in the promoter region of approximately 25% of its sequenced genes, and have been shown to activate transcription. A$\cdot$T tracts (as compared to B-DNA) are characterized by a high degree of propeller twist resulting in a narrow minor groove, an unusual rigidness and an ability to exclude/destabilize nucleosomes. Exclusion of nucleosomes and the resulting potential to cause local perturbations in chromatin structure has been proposed to be a possible mechanism by which A$\cdot$T tracts promote transcription. DAT1 encodes a nonessential DNA binding protein (Dat1p) in Saccharomyces cerevisiae, that specifically recognizes A$\cdot$T tracts. This work addressed two major issues: (1) the molecular basis by which Dat1p recognizes and interacts with A$\cdot$T DNA and (2) Dat1p's biological function. Deletion analysis showed that the amino-terminal 36 amino acid residues of Dat1p are sufficient for specific binding activity, thus defining the DNA binding domain. Dat1p contains no obvious DNA binding motif (i.e. helix-turn-helix, zinc finger, etc.) within this region; however, the pentad Gly-Arg-Lys-Pro-Gly is repeated three times. Mutational analysis revealed that the arginine side chains are required for high-affinity binding, whereas the other amino acid side chains within this pentad are dispensable. Chemical interference experiments and competition studies using minor groove binding drugs showed that Dat1p interacts with the minor groove of A$\cdot$T tracts. However, the inability of oligo(dI)$\cdot$oligo(dC) DNA, which has the same minor groove constituents as A$\cdot$T tracts, to act as a specific competitor in binding assays demonstrated that A$\cdot$T DNA minor groove functional groups are not sufficient for high specificity DNA binding. The data suggest that the pentad arginines interact in a cooperative manner with a repeated minor groove feature of A$\cdot$T DNA to achieve high affinity recognition. We hypothesize that Dat1p's DNA binding domain is extended and flexible, allowing the peptide backbone to follow the minor groove path when complexed to A$\cdot$T DNA. Many other DNA binding proteins which recognize A+T-rich sequences contain amino acids regions that are similar to the Dat1p Gly-Arg-Lys-Pro-Gly motifs. These amino acid similarities with other binding proteins suggest that the Dat1p pentad represents a specialized example of a widespread motif used by proteins to recognize A$\cdot$T base pairs. Dat1p has been shown to be able to repress the transcription of a synthetic reporter construct containing an A$\cdot$T tract within its promoter region. Examination of endogenous yeast genes containing A$\cdot$T tracts within their promoters revealed that Dat1p has the capacity to act either as a repressor or activator. These studies indicate that Dat1p can bind A$\cdot$T tracts in vivo and that Dat1p can modulate A$\cdot$T tract promoter activity.
Subject Area
Biochemistry|Genetics|Molecular biology
Recommended Citation
Reardon, Brian James, "The molecular and genetic characterization of the DAT1 gene product of Saccharomyces cerevisiae" (1996). ProQuest ETD Collection - Thomas Jefferson University. AAI9703674.
https://jdc.jefferson.edu/dissertations/AAI9703674
