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A protein involved in minichromosome maintenance in yeast binds a transcriptional enhancer conserved in eukaryotes
1Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
Abstract:
The Saccharomyces cerevisiae MCM1 gene product is a protein with multiple functions. It is a transcription factor necessary for expression of mating-type-specific genes and is also required for the maintenance of minichromosomes. MCM1 shows DNA-binding specificities similar to those of two previously reported DNA-binding factors, pheromone/receptor transcription factor (PRTF) and general regulator of mating type (GRM); like PRTF, its activity can be modulated by the alpha 1 protein. MCM1 binds to the dyad symmetry element 5'-CCTAATTAGG and related sequences, which we refer to as MCM1 control elements (MCEs). MCEs are found within the regulatory regions of a- and alpha-specific genes. Direct and indirect DNA binding assays suggest that a conserved 5'-ATTAGG in one-half of the dyad symmetry element is important for MCM1 binding whereas variants in the other half are tolerated. We have used a novel DNase I 'nicking interference' assay to investigate the interaction of MCM1 with its substrate. These data suggest that MCM1 binds as a dimer, interacting symmetrically with the ATTAGG residues in each half of the binding site. MCM1 contains striking homology to the DNA-binding domain of the human serum response factor (SRF) which mediates the transient transcriptional activation of growth-stimulated genes by binding to the serum response element (SRE). We have shown that MCM1 binds to the human c-fos SRE in vitro and, like other MCEs, the c-fos SRE exhibits MCM1-mediated upstream activating sequence (UAS) activity in vivo.
Insights
The Saccharomyces cerevisiae MCM1 protein acts as a transcription factor for mating-specific genes and maintains minichromosomes. It binds DNA sequences called MCM1 control elements (MCEs) as a dimer, similar to human serum response factor.
Area of Science:
- Molecular Biology
- Yeast Genetics
Background:
- The Saccharomyces cerevisiae MCM1 gene product is a multifunctional protein involved in gene expression and chromosome maintenance.
- MCM1 shares DNA-binding similarities with PRTF and GRM, and its activity is modulated by the alpha 1 protein.
Purpose of the Study:
- To characterize the DNA-binding specificities and interaction of the Saccharomyces cerevisiae MCM1 protein.
- To investigate the functional conservation between MCM1 and human serum response factor (SRF).
Main Methods:
- DNA-binding assays (direct and indirect) were used to determine MCM1 binding sites.
- A novel DNase I 'nicking interference' assay was employed to study MCM1-DNA interactions.
- In vitro binding assays and in vivo functional assays were performed using the human c-fos serum response element (SRE).
Main Results:
- MCM1 binds to dyad symmetry elements, termed MCM1 control elements (MCEs), in the regulatory regions of mating-type-specific genes.
- A conserved 5'-ATTAGG sequence is crucial for MCM1 binding, with variations tolerated in the other half of the binding site.
- MCM1 binds as a dimer, symmetrically interacting with the ATTAGG residues in each half of the MCE.
- MCM1 exhibits homology to human SRF and binds to the human c-fos SRE in vitro, conferring UAS activity in vivo.
Conclusions:
- MCM1 functions as a dimeric transcription factor that recognizes specific DNA sequences (MCEs).
- The study reveals functional conservation in DNA binding and transcriptional regulation between yeast MCM1 and human SRF.
- MCM1's role in regulating mating-type genes and maintaining minichromosomes is further elucidated.