A DNA replication enhancer in Saccharomyces cerevisiae.
We have dissected the autonomously replicating sequence ARS121 using site-directed in vitro mutagenesis. Three domains important for origin function were identified; one of these is essential and contains an 11-base-pair sequence resembling the canonical ARS core consensus; the second region, deletion of which affects the efficiency of the origin, is located 3' to the T-rich strand of the essential sequence and encompasses several elements with near matches to the ARS core consensus; the third region, containing two OBF1 DNA-binding sites and located 5' to the essential sequence, also affects the efficiency of the ARS. Here we demonstrate that a synthetic OBF1 DNA-binding site can substitute for the entire third domain in origin function. A dimer of the synthetic binding site, fused to a truncated origin containing only domains one and two, restored the origin activity to the levels of the wild-type ARS. The stimulation of origin function by the synthetic binding site was relatively orientation independent and could occur at distances as far as 1 kilobase upstream to the essential domain. Based on these results we conclude that the OBF1 DNA-binding site is an enhancer of DNA replication. We suggest that the DNA-binding site and the OBF1 protein are involved in the regulation of the activation of nuclear origins of replication in Saccharomyces cerevisiae.
Department of Microbiology, School of Medicine, University of Connecticut Health Center, Farmington 06032.
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Walker
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Francesconi
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Eisenberg
D001483:Base Sequence; D002872:Chromosome Deletion; D004261:DNA Replication; D004271:DNA, Fungal / Q000235:genetics; D004742:Enhancer Elements, Genetic; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D004271:DNA, Fungal