MCM1 POINT MUTANTS DEFICIENT IN EXPRESSION OF ALPHA-SPECIFIC GENES - RESIDUES IMPORTANT FOR INTERACTION WITH ALPHA-1

Complexes formed between MCM1 and several coregulatory proteins-alpha1 , alpha2, and STE12-serve to govern transcription of the a- and alpha- specific gene sets in the yeast Saccharomyces cerevisiae. The N-termin al third of MCM1, MCM1(1-98), which includes a segment homologous to m ammalian serum response factor, is capable of performing all of the fu nctions necessary for cell-type-specific gene regulation, including DN A binding and interaction with coregulatory proteins. To explore the m echanisms by which MCM1(1-98) functions, we isolated point mutants tha t are specifically deficient in alpha-specific gene expression in vivo , anticipating that many of the mutants would be impaired for interact ion with alpha1. Indeed, in vitro DNA binding assays revealed that a s ubstantial number of the mutants were specifically defective in the ab ility to bind cooperatively with alpha1. Two other mutant classes were also found. One class, exemplified most clearly by substitutions at r esidues 22 and 27, exhibited a general defect in DNA binding. The seco nd class, exemplified by substitutions at residues 33 and 41, was prof icient at DNA binding and interaction with alpha1 in vitro, suggesting that these mutants may be defective in achieving an alpha1-mediated c onformational change required for transcription activation in vivo. Mo st of the mutants defective for interaction with alpha1 had substituti ons within residues 69 to 81, which correspond to a region of serum re sponse factor important for interaction with its coregulatory proteins . A subset of the mutants with changes in this region were also defect ive in the ability to bind with STE12 to DNA from an a-specific gene, suggesting that a common region of MCM1(1-98) mediates interaction wit h both alpha1 and STE12. This region of MCM1 does not seem to constitu te an independent domain of the protein, however, because some substit utions within this region affected DNA binding. Only two of the MCM1(1 -98) point mutants showed significant defects in the ability to form c omplexes with alpha2, suggesting that the mechanism by which MCM1 inte racts with alpha2 is distinct from that by which it interacts with alp ha1 and STE12.