Specific interaction between a Saccharomyces cerevisiae protein and a DNA element associated with certain autonomously replicating sequences.
We have isolated a protein from Saccharomyces cerevisiae that binds specifically to a nucleotide sequence associated with the autonomously replicating sequence (ARS) ARS120, located in the telomeric region of a yeast chromosome. "Footprinting" analysis revealed that a 26-base-pair DNA sequence, 5'-CAAGTGCCGTGCATAATGATGTGGGT-3', was protected by this protein from DNase I digestion. A plasmid containing 48 direct tandem repeats of this oligonucleotide was constructed and used to affinity-purify the binding activity. The purified protein, OBF1 (origin binding factor), showed specific binding to ARS120. The 26-base-pair OBF1-protected sequence was sufficient for the recognition and binding of the protein, since the mobility of a DNA fragment containing the synthetic binding site was retarded in agarose gels when incubated with OBF1. By performing competition experiments with a number of different ARSs, we showed that OBF1 binds tightly to some but not all ARSs. Interestingly, OBF1 does not appear to have a discernible affinity for ARS1 or the ARSs associated with mating type loci, HML alpha and HMRa, which are substrates for a DNA-binding activity reported by others. Since OBF1 appears to bind to DNA associated with a number of ARSs, we suggest that this protein may have a function related to ARS activity, perhaps in the initiation of DNA replication at selected ARSs.
Eisenberg
S
S
Department of Microbiology, School of Medicine, University of Connecticut Health Center, Farmington 06032.
Civalier
C
C
Tye
B K
BK
D004261:DNA Replication; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012093:Replicon; D012441:Saccharomyces cerevisiae / Q000032:analysis*
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins